Monitoring SARS-CoV-2 RNA in wastewater from a shared septic system and sub-sewershed sites to expand COVID-19 disease surveillance.

Wastewater-based epidemiology has expanded as a tool for collecting COVID-19 surveillance data, but there is limited information on the feasibility of this form of surveillance within decentralized wastewater systems (e.g., septic systems). This study assessed SARS-CoV-2 RNA concentrations in wastewater samples from a septic system servicing a mobile home park (66 households) and from two pumping stations serving a similarly sized (71 households) and a larger (1,000 households) neighborhood within a nearby sewershed over 35 weeks in 2020. Also, raw wastewater from a hospital in the same sewershed was sampled. The mobile home park samples had the highest detection frequency (39/39 days) and mean concentration of SARS-CoV-2 RNA (2.7 × 107 gene copies/person/day for the N1) among the four sampling sites. N1 gene and N2 gene copies were highly correlated across mobile home park samples (Pearson's r = 0.93, p < 0.0001). In the larger neighborhood, new COVID-19 cases were reported every week during the sampling period; however, we detected SARS-CoV-2 RNA in 12% of the corresponding wastewater samples. The results of this study suggest that sampling from decentralized wastewater infrastructure can be used for continuous monitoring of SARS-CoV-2 infections.

Is detection of enteropathogens and human or animal faecal markers in the environment associated with subsequent child enteric infections and growth: an individual participant data meta-analysis.

BACKGROUND: Quantifying contributions of environmental faecal contamination to child diarrhoea and growth faltering can illuminate causal mechanisms behind modest health benefits in recent water, sanitation, and hygiene (WASH) trials. We aimed to assess associations between environmental detection of enteropathogens and human or animal microbial source tracking markers (MSTM) and subsequent child health outcomes. METHODS: In this individual participant data meta-analysis we searched we searched PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus for WASH intervention studies with a prospective design and concurrent control that measured enteropathogens or MSTM in environmental samples, or both, and subsequently measured enteric infections, diarrhoea, or height-for-age Z-scores (HAZ) in children younger than 5 years. We excluded studies that only measured faecal indicator bacteria. The initial search was done on Jan 19, 2021, and updated on March 22, 2023. One reviewer (AM) screened abstracts, and two independent reviewers (AM and RT) examined the full texts of short-listed articles. All included studies include at least one author that also contributed as an author to the present Article. Our primary outcomes were the 7-day prevalence of caregiver-reported diarrhoea and HAZ in children. For specific enteropathogens in the environment, primary outcomes also included subsequent child infection with the same pathogen ascertained by stool testing. We estimated associations using covariate-adjusted regressions and pooled estimates across studies. FINDINGS: Data from nine published reports from five interventions studies, which included 8603 children (4302 girls and 4301 boys), were included in the meta-analysis. Environmental pathogen detection was associated with increased infection prevalence with the same pathogen and lower HAZ (ΔHAZ -0·09 [95% CI -0·17 to -0·01]) but not diarrhoea (prevalence ratio 1·22 [95% CI 0·95 to 1·58]), except during wet seasons. Detection of MSTM was not associated with diarrhoea (no pooled estimate) or HAZ (ΔHAZ -0·01 [-0·13 to 0·11] for human markers and ΔHAZ -0·02 [-0·24 to 0·21] for animal markers). Soil, children's hands, and stored drinking water were major transmission pathways. INTERPRETATION: Our findings support a causal chain from pathogens in the environment to infection to growth faltering, indicating that the lack of WASH intervention effects on child growth might stem from insufficient reductions in environmental pathogen prevalence. Studies measuring enteropathogens in the environment should subsequently measure the same pathogens in stool to further examine theories of change between WASH, faecal contamination, and health. Given that environmental pathogen detection was predictive of infection, programmes targeting specific pathogens (eg, vaccinations and elimination efforts) can environmentally monitor the pathogens of interest for population-level surveillance instead of collecting individual biospecimens. FUNDING: The Bill & Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.

Long-term impacts of an urban sanitation intervention on enteric pathogens in children in Maputo city, Mozambique: study protocol for a cross-sectional follow-up to the Maputo Sanitation (MapSan) trial 5 years postintervention.

INTRODUCTION: We previously assessed the effect of an onsite sanitation intervention in informal neighbourhoods of urban Maputo, Mozambique on enteric pathogen detection in children after 2 years of follow-up (Maputo Sanitation (MapSan) trial, ClinicalTrials.gov: NCT02362932). We found significant reductions in Shigella and Trichuris prevalence but only among children born after the intervention was delivered. In this study, we assess the health impacts of the sanitation intervention after 5 years among children born into study households postintervention. METHODS AND ANALYSIS: We are conducting a cross-sectional household study of enteric pathogen detection in child stool and the environment at compounds (household clusters sharing sanitation and outdoor living space) that received the pour-flush toilet and septic tank intervention at least 5 years prior or meet the original criteria for trial control sites. We are enrolling at least 400 children (ages 29 days to 60 months) in each treatment arm. Our primary outcome is the prevalence of 22 bacterial, protozoan, and soil transmitted helminth enteric pathogens in child stool using the pooled prevalence ratio across the outcome set to assess the overall intervention effect. Secondary outcomes include the individual pathogen detection prevalence and gene copy density of 27 enteric pathogens (including viruses); mean height-for-age, weight-for-age, and weight-for-height z-scores; prevalence of stunting, underweight, and wasting; and the 7-day period prevalence of caregiver-reported diarrhoea. All analyses are adjusted for prespecified covariates and examined for effect measure modification by age. Environmental samples from study households and the public domain are assessed for pathogens and faecal indicators to explore environmental exposures and monitor disease transmission. ETHICS AND DISSEMINATION: Study protocols have been reviewed and approved by human subjects review boards at the Ministry of Health, Republic of Mozambique and the University of North Carolina at Chapel Hill. Deidentified study data will be deposited at https://osf.io/e7pvk/. TRIAL REGISTRATION NUMBER: ISRCTN86084138.

Long-term impacts of an urban sanitation intervention on enteric pathogens in children in Maputo city, Mozambique: study protocol for a cross-sectional follow-up to the Maputo Sanitation (MapSan) trial 5 years postintervention

Introduction We previously assessed the effect of an onsite sanitation intervention in informal neighbourhoods of urban Maputo, Mozambique on enteric pathogen detection in children after 2 years of follow-up (Maputo Sanitation (MapSan) trial, ClinicalTrials.gov: NCT02362932). We found significant reductions in Shigella and Trichuris prevalence but only among children born after the intervention was delivered. In this study, we assess the health impacts of the sanitation intervention after 5 years among children born into study households postintervention. Methods and analysis We are conducting a crosssectional household study of enteric pathogen detection in child stool and the environment at compounds (household clusters sharing sanitation and outdoor living space) that received the pour-flush toilet and septic tank intervention at least 5 years prior or meet the original criteria for trial control sites. We are enrolling at least 400 children (ages 29 days to 60 months) in each treatment arm. Our primary outcome is the prevalence of 22 bacterial, protozoan, and soil transmitted helminth enteric pathogens in child stool using the pooled prevalence ratio across the outcome set to assess the overall intervention effect. Secondary outcomes include the individual pathogen detection prevalence and gene copy density of 27 enteric pathogens (including viruses); mean height-for-age, weight-for-age, and weight-for-height z-scores; prevalence of stunting, underweight, and wasting; and the 7-day period prevalence of caregiver-reported diarrhoea. All analyses are adjusted for prespecified covariates and examined for effect measure modification by age. Environmental samples from study households and the public domain are assessed for pathogens and faecal indicators to explore environmental exposures and monitor disease transmission.

Effects of water, sanitation, and hygiene interventions on detection of enteropathogens and host-specific faecal markers in the environment: a systematic review and individual participant data meta-analysis.

BACKGROUND: Water, sanitation, and hygiene (WASH) improvements are promoted to reduce diarrhoea in low-income countries. However, trials from the past 5 years have found mixed effects of household-level and community-level WASH interventions on child health. Measuring pathogens and host-specific faecal markers in the environment can help investigate causal pathways between WASH and health by quantifying whether and by how much interventions reduce environmental exposure to enteric pathogens and faecal contamination from human and different animal sources. We aimed to assess the effects of WASH interventions on enteropathogens and microbial source tracking (MST) markers in environmental samples. METHODS: We did a systematic review and individual participant data meta-analysis, which included searches from Jan 1, 2000, to Jan 5, 2023, from PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus, of prospective studies with water, sanitation, or hygiene interventions and concurrent control group that measured pathogens or MST markers in environmental samples and measured child anthropometry, diarrhoea, or pathogen-specific infections. We used covariate-adjusted regression models with robust standard errors to estimate study-specific intervention effects and pooled effect estimates across studies using random-effects models. FINDINGS: Few trials have measured the effect of sanitation interventions on pathogens and MST markers in the environment and they mostly focused on onsite sanitation. We extracted individual participant data on nine environmental assessments from five eligible trials. Environmental sampling included drinking water, hand rinses, soil, and flies. Interventions were consistently associated with reduced pathogen detection in the environment but effect estimates in most individual studies could not be distinguished from chance. Pooled across studies, we found a small reduction in the prevalence of any pathogen in any sample type (pooled prevalence ratio [PR] 0·94 [95% CI 0·90-0·99]). Interventions had no effect on the prevalence of MST markers from humans (pooled PR 1·00 [95% CI 0·88-1·13]) or animals (pooled PR 1·00 [95% CI 0·97-1·03]). INTERPRETATION: The small effect of these sanitation interventions on pathogen detection and absence of effects on human or animal faecal markers are consistent with the small or null health effects previously reported in these trials. Our findings suggest that the basic sanitation interventions implemented in these studies did not contain human waste and did not adequately reduce exposure to enteropathogens in the environment. FUNDING: Bill and Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.

Detection of SARS-CoV-2 RNA in wastewater and comparison to COVID-19 cases in two sewersheds, North Carolina, USA.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be useful for monitoring population-wide coronavirus disease 2019 (COVID-19) infections, especially given asymptomatic infections and limitations in diagnostic testing. We aimed to detect SARS-CoV-2 RNA in wastewater and compare viral concentrations to COVID-19 case numbers in the respective counties and sewersheds. Influent 24-hour composite wastewater samples were collected from July to December 2020 from two municipal wastewater treatment plants serving different population sizes in Orange and Chatham Counties in North Carolina. After a concentration step via HA filtration, SARS-CoV-2 RNA was detected and quantified by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) and quantitative PCR (RT-qPCR), targeting the N1 and N2 nucleocapsid genes. SARS-CoV-2 RNA was detected by RT-ddPCR in 100 % (24/24) and 79 % (19/24) of influent wastewater samples from the larger and smaller plants, respectively. In comparison, viral RNA was detected by RT-qPCR in 41.7 % (10/24) and 8.3 % (2/24) of samples from the larger and smaller plants, respectively. Positivity rates and method agreement further increased for the RT-qPCR assay when samples with positive signals below the limit of detection were counted as positive. The wastewater data from the larger plant generally correlated (⍴ ~0.5, p < 0.05) with, and even anticipated, the trends in reported COVID-19 cases, with a notable spike in measured viral RNA preceding a spike in cases when students returned to a college campus in the Orange County sewershed. Correlations were generally higher when using estimates of sewershed-level case data rather than county-level data. This work supports use of wastewater surveillance for tracking COVID-19 disease trends, especially in identifying spikes in cases. Wastewater-based epidemiology can be a valuable resource for tracking disease trends, allocating resources, and evaluating policy in the fight against current and future pandemics.

Timing and Trends for Municipal Wastewater, Lab-Confirmed Case, and Syndromic Case Surveillance of COVID-19 in Raleigh, North Carolina.

Objectives. To compare 4 COVID-19 surveillance metrics in a major metropolitan area. Methods. We analyzed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater influent and primary solids in Raleigh, North Carolina, from April 10 through December 13, 2020. We compared wastewater results with lab-confirmed COVID-19 cases and syndromic COVID-like illness (CLI) cases to answer 3 questions: (1) Did they correlate? (2) What was the temporal alignment of the different surveillance systems? (3) Did periods of significant change (i.e., trends) align? Results. In the Raleigh sewershed, wastewater influent, wastewater primary solids, lab-confirmed cases, and CLI were strongly or moderately correlated. Trends in lab-confirmed cases and wastewater influent were observed earlier, followed by CLI and, lastly, wastewater primary solids. All 4 metrics showed sustained increases in COVID-19 in June, July, and November 2020 and sustained decreases in August and September 2020. Conclusions. In a major metropolitan area in 2020, the timing of and trends in municipal wastewater, lab-confirmed case, and syndromic case surveillance of COVID-19 were in general agreement. Public Health Implications. Our results provide evidence for investment in SARS-CoV-2 wastewater and CLI surveillance to complement information provided through lab-confirmed cases. (Am J Public Health. 2023;113(1):79-88. https://doi.org/10.2105/AJPH.2022.307108).

Protocol to identify host-viral protein interactions between coagulation-related proteins and their genetic variants with SARS-CoV-2 proteins.

Here, we describe a bioinformatics pipeline that evaluates the interactions between coagulation-related proteins and genetic variants with SARS-CoV-2 proteins. This pipeline searches for host proteins that may bind to viral protein and identifies and scores the protein genetic variants to predict the disease pathogenesis in specific subpopulations. Additionally, it is able to find structurally similar motifs and identify potential binding sites within the host-viral protein complexes to unveil viral impact on regulated biological processes and/or host-protein impact on viral invasion or reproduction. For complete details on the use and execution of this protocol, please refer to Holcomb et al. (2021).

Exposure to industrial hog and poultry operations and urinary tract infections in North Carolina, USA.

An increasing share of urinary tract infections (UTIs) are caused by extraintestinal pathogenic Escherichia coli (ExPEC) lineages that have also been identified in poultry and hogs with high genetic similarity to human clinical isolates. We investigated industrial food animal production as a source of uropathogen transmission by examining relationships of hog and poultry density with emergency department (ED) visits for UTIs in North Carolina (NC). ED visits for UTI in 2016-2019 were identified by ICD-10 code from NC's ZIP code-level syndromic surveillance system and livestock counts were obtained from permit data and aerial imagery. We calculated separate hog and poultry spatial densities (animals/km2) by Census block with a 5 km buffer on the block perimeter and weighted by block population to estimate mean ZIP code densities. Associations between livestock density and UTI incidence were estimated using a reparameterized Besag-York-Mollié (BYM2) model with ZIP code population offsets to account for spatial autocorrelation. We excluded metropolitan and offshore ZIP codes and assessed effect measure modification by calendar year, ZIP code rurality, and patient sex, age, race/ethnicity, and health insurance status. In single-animal models, hog exposure was associated with increased UTI incidence (rate ratio [RR]: 1.21, 95 % CI: 1.07-1.37 in the highest hog-density tertile), but poultry exposure was associated with reduced UTI rates (RR: 0.86, 95 % CI: 0.81-0.91). However, the reference group for single-animal poultry models included ZIP codes with only hogs, which had some of the highest UTI rates; when compared with ZIP codes without any hogs or poultry, there was no association between poultry exposure and UTI incidence. Hog exposure was associated with increased UTI incidence in areas that also had medium to high poultry density, but not in areas with low poultry density, suggesting that intense hog production may contribute to increased UTI incidence in neighboring communities.

Synonymous ADAMTS13 variants impact molecular characteristics and contribute to variability in active protein abundance.

The effects of synonymous single nucleotide variants (sSNVs) are often neglected because they do not alter protein primary structure. Nevertheless, there is growing evidence that synonymous variations may affect messenger RNA (mRNA) expression and protein conformation and activity, which may lead to protein deficiency and disease manifestations. Because there are >21 million possible sSNVs affecting the human genome, it is not feasible to experimentally validate the effect of each sSNV. Here, we report a comprehensive series of in silico analyses assessing sSNV impact on a specific gene. ADAMTS13 was chosen as a model for its large size, many previously reported sSNVs, and associated coagulopathy thrombotic thrombocytopenic purpura. Using various prediction tools of biomolecular characteristics, we evaluated all ADAMTS13 sSNVs registered in the National Center for Biotechnology Information database of single nucleotide polymorphisms, including 357 neutral sSNVs and 19 sSNVs identified in patients with thrombotic thrombocytopenic purpura. We showed that some sSNVs change mRNA-folding energy/stability, disrupt mRNA splicing, disturb microRNA-binding sites, and alter synonymous codon or codon pair usage. Our findings highlight the importance of considering sSNVs when assessing the complex effects of ADAMTS13 alleles, and our approach provides a generalizable framework to characterize sSNV impact in other genes and diseases.

Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Exposure to industrial hog operations and gastrointestinal illness in North Carolina, USA.

With 9 million hogs, North Carolina (NC) is the second leading hog producer in the United States. Most hogs are housed at concentrated animal feeding operations (CAFOs), where millions of tons of hog waste can pollute air and water with fecal pathogens that can cause diarrhea, vomiting, and/or nausea (known as acute gastrointestinal illness (AGI)). We used NC's ZIP code-level emergency department (ED) data to calculate rates of AGI ED visits (2016-2019) and swine permit data to estimate hog exposure. Case exposure was estimated as the inverse distances from each hog CAFO to census block centroids, weighting with Gaussian decay and by manure amount per CAFO, then aggregated to ZIP code using population weights. We compared ZIP codes in the upper quartile of hog exposure ("high hog exposed") to those without hog exposure. Using inverse probability of treatment weighting, we created a control with similar demographics to the high hog exposed population and calculated rate ratios using quasi-Poisson models. We examined effect measure modification of rurality and race using adjusted models. In high hog exposed areas compared to areas without hog exposure, we observed a 11% increase (95% CI: 1.06, 1.17) in AGI rate and 21% increase specifically in rural areas (95% CI: 0.98, 1.43). When restricted to rural areas, we found an increased AGI rate among American Indian (RR = 4.29, 95% CI: 3.69, 4.88) and Black (RR = 1.45, 95% CI: 0.98, 1.91) residents. The association was stronger during the week after heavy rain (RR = 1.41, 95% CI: 1.19, 1.62) and in areas with both poultry and swine CAFOs (RR = 1.52, 95% CI: 1.48, 1.57). Residing near CAFOs may increase rates of AGI ED visits. Hog CAFOs are disproportionally built near rural Black and American Indian communities in NC and are associated with increased AGI most strongly in these populations.

Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: A six-month long study in Charlotte, North Carolina.

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus to be the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to 105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases when lagging 5 to 12 days (ρ = 0.52-0.92, p < 0.001-0.02). A major finding of this study is that RT-qPCR and RT-ddPCR generated SARS-CoV-2 data that was positively correlated (ρ = 0.569, p < 0.0001) and can be successfully used to monitor SARS-CoV-2 signals across the WWTP of different sizes and metropolitan service functions without significant anomalies.

Impacts of an Urban Sanitation Intervention on Fecal Indicators and the Prevalence of Human Fecal Contamination in Mozambique.

Fecal source tracking (FST) may be useful to assess pathways of fecal contamination in domestic environments and to estimate the impacts of water, sanitation, and hygiene (WASH) interventions in low-income settings. We measured two nonspecific and two human-associated fecal indicators in water, soil, and surfaces before and after a shared latrine intervention from low-income households in Maputo, Mozambique, participating in the Maputo Sanitation (MapSan) trial. Up to a quarter of households were impacted by human fecal contamination, but trends were unaffected by improvements to shared sanitation facilities. The intervention reduced Escherichia coli gene concentrations in soil but did not impact culturable E. coli or the prevalence of human FST markers in a difference-in-differences analysis. Using a novel Bayesian hierarchical modeling approach to account for human marker diagnostic sensitivity and specificity, we revealed a high amount of uncertainty associated with human FST measurements and intervention effect estimates. The field of microbial source tracking would benefit from adding measures of diagnostic accuracy to better interpret findings, particularly when FST analyses convey insufficient information for robust inference. With improved measures, FST could help identify dominant pathways of human and animal fecal contamination in communities and guide the implementation of effective interventions to safeguard health.

A conserved epitope III on hepatitis C virus E2 protein has alternate conformations facilitating cell binding or virus neutralization.

Epitope III, a highly conserved amino acid motif of 524APTYSW529 on the hepatitis C virus (HCV) E2 glycoprotein, resides in the critical loop that binds to the host receptor CD81, thus making it one of the most important antibody targets for blocking HCV infections. Here, we have determined the X-ray crystal structure of epitope III at a 2.0-Å resolution when it was captured by a site-specific neutralizing antibody, monoclonal antibody 1H8 (mAb1H8). The snapshot of this complex revealed that epitope III has a relatively rigid structure when confined in the binding grooves of mAb1H8, which confers the residue specificity at both ends of the epitope. Such a high shape complementarity is reminiscent of the "lock and key" mode of action, which is reinforced by the incompatibility of an antibody binding with an epitope bearing specific mutations. By subtly positioning the side chains on the three residues of Tyr527, Ser528, and Trp529 while preserving the spatial rigidity of the rest, epitope III in this cocrystal complex adopts a unique conformation that is different from previously described E2 structures. With further analyses of molecular docking and phage display-based peptide interactions, we recognized that it is the arrangements of two separate sets of residues within epitope III that create these discrete conformations for the epitope to interact selectively with either mAb1H8 or CD81. These observations thus raise the possibility that local epitope III conformational dynamics, in conjunction with sequence variations, may act as a regulatory mechanism to coordinate "mAb1H8-like" antibody-mediated immune defenses with CD81-initiated HCV infections.

New approaches to predict the effect of co-occurring variants on protein characteristics.

Predicting the effect of a mutated gene before the onset of symptoms of genetic diseases would greatly facilitate diagnosis and potentiate early intervention. There have been myriad attempts to predict the effects of single-nucleotide variants. However, the applicability of these efforts does not scale to co-occurring variants. Furthermore, an increasing number of protein therapeutics contain co-occurring nucleotide variations, adding uncertainty during development to the safety and efficiency of these drugs. Co-occurring nucleotide variants may often have synergistic, additive, or antagonistic effects on protein attributes, further complicating the task of outcome prediction. We tested four models based on the cooperative and antagonistic effects of co-occurring variants to predict pathogenicity and effectiveness of protein therapeutics. A total of 30 attributes, including amino acid and nucleotide features, as well as existing single-variant effect prediction tools, were considered on the basis of previous studies on single-nucleotide variants. Importantly, the effects of synonymous variants, often seen in protein therapeutics, were also included in our models. We used 12 datasets of people with monogenic diseases and controls with co-occurring genetic variants to evaluate the accuracy of our models, accomplishing a degree of accuracy comparable to that of prediction tools for single-nucleotide variants. More importantly, our framework is generalizable to new, well-curated datasets of monogenic diseases and new variant scoring tools. This approach successfully assists in addressing the challenging task of predicting the effect of co-occurring variants on pathogenicity and protein effectiveness and is applicable for a wide range of protein therapeutics and genetic diseases.

Impact of an Urban Sanitation Intervention on Enteric Pathogen Detection in Soils.

Environmental fecal contamination is common in many low-income cities, contributing to a high burden of enteric infections and associated negative sequelae. To evaluate the impact of a shared onsite sanitation intervention in Maputo, Mozambique on enteric pathogens in the domestic environment, we collected 179 soil samples at shared latrine entrances from intervention (n = 49) and control (n = 51) compounds during baseline (preintervention) and after 24 months (postintervention) as part of the Maputo Sanitation Trial. We tested soils for the presence of nucleic acids associated with 18 enteric pathogens using a multiplex reverse transcription qPCR platform. We detected at least one pathogen-associated gene target in 91% (163/179) of soils and a median of 3 (IQR = 1, 5) pathogens. Using a difference-in-difference analysis and adjusting for compound population, visibly wet soil, sun exposure, wealth, temperature, animal presence, and visible feces, we estimate the intervention reduced the probability of detecting ≥1 pathogen gene by 15% (adjusted prevalence ratio, aPR = 0.85; 95% CI: 0.70, 1.0) and the total number of pathogens by 35% (aPR = 0.65; 0.44, 0.95) in soil 24 months following the intervention. These results suggest that the intervention reduced the presence of some fecal contamination in the domestic environment, but pathogen detection remained prevalent 24 months following the introduction of new latrines.

In Silico Evaluation of Cyclophilin Inhibitors as Potential Treatment for SARS-CoV-2.

BACKGROUND: The advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provoked researchers to propose multiple antiviral strategies to improve patients' outcomes. Studies provide evidence that cyclosporine A (CsA) decreases SARS-CoV-2 replication in vitro and decreases mortality rates of coronavirus disease 2019 (COVID-19) patients. CsA binds cyclophilins, which isomerize prolines, affecting viral protein activity. METHODS: We investigated the proline composition from various coronavirus proteomes to identify proteins that may critically rely on cyclophilin's peptidyl-proline isomerase activity and found that the nucleocapsid (N) protein significantly depends on cyclophilin A (CyPA). We modeled CyPA and N protein interactions to demonstrate the N protein as a potential indirect therapeutic target of CsA, which we propose may impede coronavirus replication by obstructing nucleocapsid folding. RESULTS: Finally, we analyzed the literature and protein-protein interactions, finding evidence that, by inhibiting CyPA, CsA may impact coagulation proteins and hemostasis. CONCLUSIONS: Despite CsA's promising antiviral characteristics, the interactions between cyclophilins and coagulation factors emphasize risk stratification for COVID patients with thrombosis dispositions.

Effects of an urban sanitation intervention on childhood enteric infection and diarrhea in Maputo, Mozambique: A controlled before-and-after trial.

We conducted a controlled before-and-after trial to evaluate the impact of an onsite urban sanitation intervention on the prevalence of enteric infection, soil transmitted helminth re-infection, and diarrhea among children in Maputo, Mozambique. A non-governmental organization replaced existing poor-quality latrines with pour-flush toilets with septic tanks serving household clusters. We enrolled children aged 1-48 months at baseline and measured outcomes before and 12 and 24 months after the intervention, with concurrent measurement among children in a comparable control arm. Despite nearly exclusive use, we found no evidence that intervention affected the prevalence of any measured outcome after 12 or 24 months of exposure. Among children born into study sites after intervention, we observed a reduced prevalence of Trichuris and Shigella infection relative to the same age group at baseline (<2 years old). Protection from birth may be important to reduce exposure to and infection with enteric pathogens in this setting.

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.