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This technical memorandum describes courses which have been monitored by the Project for Comprehensive Achievement Monitoring (Project CAM, formerly known as Project CRAM). The course structures range from very structured through differentially paced programs for blocks of students to individually paced programs. Project CAM has guided the development of monitors and schedules for each of these courses and is flexible enough to accommodate other variations. The nine courses described in this memorandum are but a sample of the 40 - odd courses which have used or are using CAM. (Author)

Vector-borne disease risk assessment is crucial to optimize surveillance, preventative measures (vector control), and resource allocation (medical supplies). High arthropod abundance and host interaction strongly correlate to vector-borne pathogen transmission. Increasing host density and movement increases the possibility of local and long-distance pathogen transmission. Therefore, we developed a risk-assessment framework using climate (average temperature and rainfall) and host demographic (host density and movement) data, particularly suitable for regions with unreported or underreported incidence data. This framework consisted of a spatiotemporal network-based approach coupled with a compartmental disease model and nonhomogeneous Gillespie algorithm. The correlation of climate data with vector abundance and host–vector interactions is expressed as vectorial capacity—a parameter that governs the spreading of infection from an infected host to a susceptible one via vectors. As an example, the framework is applied for dengue in Bangladesh. Vectorial capacity is inferred for each week throughout a year using average monthly temperature and rainfall data. Long-distance pathogen transmission is expressed with human movement data in the spatiotemporal network. We have identified the spatiotemporal suitability of dengue spreading in Bangladesh as well as the significant-incidence window and peak-incidence period. Analysis of yearly dengue data variation suggests the possibility of a significant outbreak with a new serotype introduction. The outcome of the framework comprised spatiotemporal suitability maps and probabilistic risk maps for spatial infection spreading. This framework is capable of vector-borne disease risk assessment without historical incidence data and can be a useful tool for preparedness with accurate human movement data.

Antagonistic coevolutionary relationships provide intense selection pressure which drive changes in the genotype. Predator-prey interactions have caused some venomous snakes and their predators/prey to evolve α-neurotoxin resistance through changes at the orthosteric site of nicotinic acetylcholine receptors. The presence of negatively charged amino acids at orthosteric site positions 191 and 195 is the ancestral state. These negatively charged amino acids have exerted a selection pressure for snake venom α-neurotoxins to evolve with strong positive charges on their molecular surface, with the opposite-charge attraction facilitating the binding by the neurotoxins. We aimed to test the effects of a series of mutations whereby one or both negatively charged amino acids are replaced by uncharged residues to ascertain if this was a novel form of reduced venom susceptibility in the varanid species. Using a biolayer interferometry assay, we tested the relative binding of α-neurotoxin-rich snake venoms against the orthosteric sites of V. giganteus (Perentie) and V. komodoensis (Komodo dragon), which both possess the negatively charged aspartic acid at position 191; V. mertensi (Merten’s water monitor), which also has aspartic acid at position 195; and Varanus exanthematicus (savannah monitor), which lacks negatively charged amino acids at both positions 191 and 195. The orthosteric sites of these species are otherwise identical. In order to complete the structure-function relationship examination, we also tested a mutant version with the negatively charged aspartic acid at both positions 191 and 195. It was demonstrated that the presence of a negatively charged amino acid at either position 191 or 195 is crucial for the successful binding of snake venom α-neurotoxins, with V. giganteus, V. komodoensis and V. mertensi all strongly bound. The mutant version containing a negatively charged amino acid at both positions was bound equipotently to the native forms of V. giganteus, V. komodoensis and V. mertensi. Thus, the presence of a negatively charged amino acid at both positions does not increase binding affinity. In contrast, Varanus exanthematicus, lacking a negatively charged amino acid at either position, displayed dramatically less sensitivity to neurotoxins compared with the other species. V. exanthematicus is distinguished from the other species examined in this study by being a small, terrestrial, slow-moving species living sympatrically with a high density of large cobra species that have neurotoxin-rich venoms. Thus, this vulnerable prey item seems to have evolved a novel form of reduced susceptibility to snake venom neurotoxins under a strong selection pressures from these neurotoxic predators. These results therefore contribute to the body of knowledge of predator/prey chemical arm races while providing novel insights into the structure-activity relationships of the orthosteric site of the nicotinic acetylcholine receptor alpha-subunit.

Evidence synthesis and knowledge translation are scientific methodological approaches used across different disciplines for combining results from individual studies, interpreting them based on the body of evidence and with the objective of supporting decision-making. Systematic, rapid, and scoping reviews have a direct application in human health and social care, but also in veterinary medicine, which are areas that can overlap with medical and veterinary entomology. As a scientific field, the objective of medical and veterinary entomology is to identify and quantify the role of insects as transmitters of important arthropod-borne pathogens, thus contributing to the study of human and veterinary issues of public health importance. The main objectives of this review are to introduce to entomologists the most common knowledge translation and synthesis methods described in the literature, to then review the use of systematic, rapid, and scoping reviews applied in the medical and veterinary entomology field, using a systematized review approach. We found 120 relevant articles in the literature addressing topics related to medical and veterinary entomology and using systematic reviews of the literature or other evidence synthesis methods, which is concurrent with the ongoing trend towards the use of these methodologies. The application of unbiased approaches to entomology in general, and to medical and veterinary entomology in particular, will strengthen science- and evidence-based conclusions to be used as a tool for informing policy, decisions, and interventions.

Thirty-four Escherichia coli isolates from 91 ready-to-eat lettuce packages, obtained from local supermarkets in Northern California, were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for β-lactamase genes. We found 15 distinct sequence types (STs). Six of these genotypes (ST1198, ST2625, ST2432, ST2819, ST4600, and ST5143) have been reported as pathogens found in human samples. Twenty-six (76%) E. coli isolates were resistant to ampicillin, 17 (50%) to ampicillin/sulbactam, 8 (23%) to cefoxitin, and 7 (20%) to cefuroxime. blaCTX-M was the most prevalent β-lactamase gene, identified in eight (23%) isolates. We identified a class A broad-spectrum β-lactamase SED-1 gene, blaSED, reported by others in Citrobacter sedlakii isolated from bile of a patient. This study found that fresh lettuce carries β-lactam drug-resistant E. coli, which might serve as a reservoir for drug-resistance genes that could potentially be transmitted to pathogens that cause human infections.

Rift Valley fever virus (RVFV) causes severe disease in domestic ungulates (cattle, goats, and sheep) and a febrile illness in humans (with ∼1% case fatality rate). This virus has been spreading geographically, and there is concern of it spreading to Europe or the Americas. Environmental temperature can significantly affect the ability of mosquitoes to transmit an arbovirus. However, these effects are not consistent among viruses or mosquito species. Therefore, we evaluated the effect of incubation temperatures ranging from 14°C to 30°C on infection and dissemination rates for Culex tarsalis and Aedes taeniorhynchus allowed to feed on hamsters infected with RVFV. Engorged mosquitoes were randomly allocated to cages and placed in incubators maintained at 14°C, 18°C, 22°C, 26°C, or 30°C. Although infection rates detected in Cx. tarsalis increased with increasing holding temperature, holding temperature had no effect on infection rates detected in Ae. taeniorhynchus. However, for both species, the percentage of mosquitoes with a disseminated infection after specific extrinsic incubation periods (4, 7, 10, 14, 17, or 21 days) increased with increasing incubation holding temperature, even after adjusting for the apparent increase in infection rate in Cx. tarsalis. The effects of environmental factors, such as ambient temperature, need to be taken into account when developing models for viral persistence and spread in nature.

Two cases with diagnosis of mesothelioma were referred to our laboratories with a request for tissue burden analysis in order to determine the presence of ferruginous bodies and uncoated elongated mineral particles in tissue samples. The individuals shared in common a past background of working in tile manufacturing facilities where industrial talc was used in the production of the products. Both were found to have ferruginous bodies in their lung tissues as well as elongated talc fibers/ribbons and elevated numbers of noncommercial amphiboles in their tissues. To our knowledge, this is the first report of tissue assessment for the presence of elongated mineral particles in individuals whose exposures to talc occurred were while working in the manufacture of tile products and who developed the fiber-related cancer - mesothelioma.

Escherichia coli recovered from poultry, and extraintestinal pathogenic E. coli (ExPEC), responsible for most cases of urinary tract infection (UTI) and bloodstream infection (BSI) in huma...

OBJECTIVES: To determine the contribution of specific uropathogenic Escherichia coli (UPEC) lineages, drug resistance genes, and plasmid incompatibility/replicon (Inc) groups to the prevalence of β-lactam-resistant urinary tract infections (UTIs) in a university community. METHODS: Urine samples were consecutively collected and cultured over a two-year period from patients presenting to a university health center with symptoms of UTI. Isolated UPEC were subtyped by MLST and fimH typing, and tested by PCR and sequencing for ß-lactamase genes and plasmid Inc groups. RESULTS: Among 273 UPEC isolates, 85 (31%) were ampicillin-resistant (AMP-R) and 188 (69%) were susceptible to all ß-lactam drugs (AMP-S). Six lineages accounted for two-thirds of the isolates: ST95 (21%), ST69 (11%), ST420 (11%), ST73 (10%), ST127 (8%), and ST404 (3%). ST69 and ST404 were associated with AMP-R (P=0.003, P=0.0005), while ST420 and ST127 were associated with AMPS (P

Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer (Odocoileus virginianus), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.

Extraintestinal pathogenicEscherichia coli(ExPEC) belonging to multilocus sequence type 95 (ST95) is one of the most geographically widespread ExPEC lineages causing bloodstream (BSI) and urinary tract infections (UTI). In contrast to other widespread ExPEC sequence types, a large proportion ST95 strains remains susceptible to all antimicrobial agents used to treat BSI or UTI. We aimed to identify genomic features of ST95 associated with its relatively high drug-susceptible frequency. We analyzed whole genomes of 1749 ST95 isolates, 80 from patients with BSI or UTI in Northern California, and 1669 isolates from the Enterobase database. We first compared whole-genome sequences (WGS) of 887 drug-susceptible strains and 862 strains resistant to one or more drugs (defined genotypically as strains harboring drug-resistance genes annotated in the ResFinder database) to identify genetic features associated with strains devoid of drug-resistance genes. We then conducted a pan-genome-wide association study on human clinical isolates of ST95, which included 553 UTI and BSI ST95 isolates. We found 44 accessory genes to be significantly associated with ST95 strains lacking drug resistance genes. Fifteen of these were not found in any of the WGSs of ST131 ExPEC strains, which are frequently multidrug-resistant. These genes were annotated to encode transporter or transfer systems and DNA repair polymerases. A large proportion of ST95 strains may have evolved to adapt to antibiotic-imposed stresses without acquiring drug-resistance genes.ImportanceDespite the increasing prevalence of antibiotic-resistantEscherichia colistrains that cause urinary tract and bloodstream infections, a major pandemic lineage of extraintestinal pathogenicE. coli(ExPEC) ST95 has a comparatively low frequency of drug resistance. We compared genomes of 1749 ST95 isolates to identify genetic features that may explain why most strains of ST95 resist becoming drug-resistant. Identification of such genomic features could contribute to the development of novel strategies to prevent the spread of antibiotic-resistant genes and devise new measures to control antibiotic-resistant infections.

Mosquito larvae encounter diverse assemblages of bacteria (i.e. ‘microbiota’) and fungi (i.e. ‘mycobiota’) in the aquatic environments they develop in. However, while a number of studies have addressed the diversity and function of microbiota in mosquito life history, relatively little is known about mosquito-mycobiota interactions outside of several key fungal entomopathogens. In this study, we used high-throughput sequencing of ITS2 gene amplicons to provide the first simultaneous characterization of the mycobiota in field-collected Aedes albopictus larvae and their associated aquatic environments. Our results reveal unprecedented variation in mycobiota among adjacent but discrete larval breeding habitats. Our results also reveal distinct mycobiota assembly in the mosquito gut versus other tissues, with gut-associated fungal communities being most similar to those present in the environment where larvae feed. Altogether, our results identify the environment as the dominant factor shaping mosquito mycobiota with no evidence of environmental filtering of the gut mycobiota. These results also identify mosquito feeding behavior and fungal mode of nutrition as potential drivers of tissue-specific mycobiota assembly after environmental acquisition.IMPORTANCEThe Asian tiger mosquito, Aedes albopictus, is the dominant mosquito species in the USA and an important vector of arboviruses of major public health concern. One aspect of mosquito control to curb mosquito-borne diseases has been the use of biological control agents such as fungal entomopathogens. Recent studies also demonstrate the impact of mosquito-associated microbial communities on various mosquito traits, including vector competence. However, while much research attention has been dedicated to understanding the diversity and function of mosquito-associated bacterial communities, relatively little is known about mosquito-associated fungal communities. A better understanding of the factors that drive mycobiota diversity and assembly in mosquitoes will be essential for future efforts to target mosquito micro- and mycobiomes for mosquito and mosquito-borne disease control.

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism., Nature Communications, 12 (1), ISSN:2041-1723

Sialyl-Lewis x (sLex, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLex expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLex+ and sLex- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts., Puan and San Luis et al. find that FUT6, encoding a fucosyltransferase, is required for the “rolling” behavior of certain white blood cells that enables them to move from blood vessels to tissues. They show that FUT6 deficiency leads to a loss of the tetrasaccharide sLex on the surface of basophils, resulting in cells that are less sticky and therefore less able to form the necessary adhesions for exiting the blood vessel to drive the allergic reaction.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested that these 24 mutants affect 24 different genes. We applied unsupervised clustering algorithms to classify the recovered mutants into three classes unbiasedly. Further computational analyses revealed that each of the recovered mutations in these three classes shows a unique effect on node subtype composition and connection property distribution of the intrahepatic biliary network. Besides, we found that most recovered mutations are viable. In those mutant fish, biliary network phenotypes persist into adulthood, which themselves are good animal models to study chronic cholestatic liver diseases. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Understanding features that fostered the persistence of Equisetum–Earth's oldest extant vascular plant genus–since Mesozoic times and through episodes of significant global environmental change, is of current interest in view of modern challenges to plant survival. In addition to known structural and physiological adaptations, we hypothesized that microscopy and shotgun metagenomic sequencing might reveal eukaryotic microorganisms such as fungi that may aid Equisetum survival. Here, we report evidence for several lineages of eukaryotic microbes associated with giant Equisetum xylochaetum, which dominates vegetation in saline streambeds of remote valleys in the hyper-arid Atacama Desert, Chile. Plant material was collected and field-preserved at two comparatively low-disturbance sites; DNA extracted in Chile using low-shear methods was later sequenced, 18S and 28S rDNA taxonomic marker sequences were selected for SILVAngs classification, allowing comparisons to eukaryotic microorganisms previously inferred for earlier-diverging plant lineages. SEM, fluorescence microscopy, and/or LM of toluidine blue-stained sections of roots indicated protists, epiphytic and endophytic fungi, and cortical nematodes. Eukaryotic genera inferred from 18S rDNA at >100X mean sequencing depth included the ciliate Engelmanniella, hyphal chytrid Monoblepharella, predatory ascomycete Cephaliophora, a salpingoecid choanoflagellate, and an annelid worm. 23S rDNA sequences indicated ascomycete Capnodiales fungi at one site and four types of Pezizomycotina fungi at the other. No evidence for vesicular-arbuscular mycorrhizal fungi was found, but we hypothesized that Equisetum may benefit from other types of fungal associations, some possibly inherited from ancestral plant lineages.

In 2017 Ontario experienced the largest mumps outbreak in the province in 8 years, at a time when multiple outbreaks were occurring across North America. Of 259 reported cases, 143 occurred in Toronto, primarily among young adults. Routine genotyping of the small hydrophobic gene indicated that the outbreak was due to mumps virus genotype G. We performed a retrospective study of whole genome sequencing of 26 mumps virus isolates from early in the outbreak, using a tiling amplicon method. Results indicated that two of the cases were genetically divergent, with the remaining 24 cases belonging to two major clades and one minor clade. Phylogeographic analysis confirmed circulation of virus from each clade between Toronto and other regions in Ontario. Comparison with other genotype G strains from North America suggested that the presence of co-circulating major clades may have been due to separate importation events from outbreaks in the United States. A transmission network analysis performed with the software program TransPhylo was compared with previously collected epidemiological data. The transmission tree correlated with known epidemiological links between nine patients and identified new potential clusters with no known epidemiological links.

The isolation of stool pathogens is difficult due to their fastidious nature and the rapid overgrowth of fecal flora. In this study, we evaluate the preservation and isolation of enteric pathogens from stool using the automated COPAN Walk-Away Specimen Processor (WASP®) in conjunction with FecalSwab™ and selenite media. Pathogen viability and fecal commensal abundance were stable in FecalSwab™ media under both room-temperature and refrigerated incubation conditions, resulting in a significantly increased number of well-isolated pathogen colonies observed when compared to samples incubated in modified Cary-Blair media. Isolation of individual pathogen colonies was improved via WASP® planting compared to those planted using the Isoplater system. Furthermore, preincubation using the newly formulated COPAN selenite media significantly enhanced the yield of Salmonella enterica serovar Typhimurium. Together, the automated WASP® system combined with FecalSwab™ and selenite media represents a rapid and efficient approach for the processing of stool specimens compared to standard methods.

The recent outbreaks of the insect-vectored Zika virus have demonstrated its potential to be sexually transmitted, which complicates modeling and our understanding of disease dynamics. Autochthonous outbreaks in the US mainland may be a consequence of both modes of transmission, which affect the outbreak size, duration, and virus persistence. We propose a novel individual-based interconnected network model that incorporates both insect-vectored and sexual transmission of this pathogen. This model interconnects a homogeneous mosquito vector population with a heterogeneous human host contact network. The model incorporates the seasonal variation of mosquito abundance and characterizes host dynamics based on age group and gender in order to produce realistic projections. We use a sexual contact network which is generated on the basis of real world sexual behavior data. Our findings suggest that for a high relative transmissibility of asymptomatic hosts, Zika virus shows a high probability of sustaining in the human population for up to 3 months without the presence of mosquito vectors. Zika outbreaks are strongly affected by the large proportion of asymptomatic individuals and their relative transmissibility. The outbreak size is also affected by the time of the year when the pathogen is introduced. Although sexual transmission has a relatively low contribution in determining the epidemic size, it plays a role in sustaining the epidemic and creating potential endemic scenarios.

Objective To better understand the clinical spectrum and course of congenital Zika syndrome (CZS) during the first 18 months of life of children whose mothers had rash during pregnancy. Methods This longitudinal observational study evaluated the clinical progress from birth until 18 months of life of children of mothers who developed rash during or up to 3 months before gestation. Maternal rash occurred from November 2015 to May 2017. The study subjects were divided into three groups: children whose mothers tested positive by RT-qPCR for Zika virus (ZIKV) (Group 1), children whose mothers tested negative by RT-qPCR for ZIKV (Group 2), and children whose mothers did not undergo any testing for ZIKV (Group 3) but tested negative for other congenital infections. Results Between April 2016 and July 2018, we studied 108 children: 43 in Group 1, 26 in Group 2 and 39 in Group 3. The majority of children were admitted into the study within 6 months of life. CZS was diagnosed in 26 children, equally distributed in Groups 1 and 3. Of 18 children with microcephaly, 6 were in Group 1 (1 postnatal) and 12 were in Group 3 (5 postnatal). Maternal rash frequency was 10 times higher during the first trimester than in the other trimesters (OR: 10.35; CI 95%: 3.52–30.41). CZS was diagnosed during the follow-up period in 14 (54%) cases. Developmental delays and motor abnormalities occurred in all children and persisted up to 18 months. Epilepsy occurred in 18 (69%) of the cases. Conclusions Infants born of mothers exposed to ZIKV during pregnancy showed progression of developmental, motor and neurologic abnormalities even if they were born asymptomatic. Continued postnatal monitoring of such newborns is necessary to preclude disability-associated complications.

The purpose of this risk assessment (RA) was to qualitatively estimate the risk of emergence of the Japanese encephalitis virus (JEV) in the United States (US). We followed the framework for RA of emerging vector-borne livestock diseases (de Vos et al. 2011), which consists of a structured questionnaire, whose answers to questions can be delivered in risk categories, descriptive statements, or yes or no type of answers, being supported by the literature. The most likely pathways of introduction of JEV identified were: (a) entry through infected vectors (by aircraft, cargo ships, tires, or wind); (b) import of infected viremic animals; (c) entry of viremic migratory birds; (d) import of infected biological materials; (e) import of infected animal products; (f) entry of infected humans; and (g) import/production of contaminated biological material (e.g., vaccines). From these pathways, the probability of introduction of JEV through infected adult mosquitoes via aircraft was considered very high and via ships/containers was deemed low to moderate. The probability of introduction via other pathways or modes of entry (vector eggs or larvae, hosts, and vaccines) was considered negligible. The probability of transmission of JEV was variable, ranging from low to high (in the presence of both competent vectors and hosts), depending on the area of introduction within the US. Lastly, the probability of establishment of JEV in the continental US was considered negligible. For that reason, we stopped the risk assessment at this point of the framework. This RA provides important information regarding the elements that contribute to the risk associated with the introduction of JEV in the US. This RA also indicates that infected mosquitoes transported in aircraft (and cargo ships) are the most likely pathway of JEV entry and therefore, mitigation strategies should be directed towards this pathway.

Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103–46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB). The six, anti-GNB antibodies were first screened against a panel of well-characterized clinical GNB isolates to optimize assay conditions and to determine individual antibody sensitivity and specificity. When tested against a diverse, blinded panel of 94 GNB clinical isolates, the I-ELISA exhibited the following sensitivity/specificity for each target: Enterobacteriaceae (94.4%/95%), E. coli (82.6%/88.7%), P. aeruginosa (83.3%/96%). An I–ELISA to detect and differentiate the most common GNB pathogens offers advantage in terms of simplicity over diagnostic tests currently used in most clinical settings.

The 28th Annual Latin American Symposium presented by the American Mosquito Control Association (AMCA) was held as part of the 84th Annual Meeting of the AMCA held in Kansas City, MO, in February 2018. The Latin American Symposium promotes the participation of vector control specialists, public health workers, and academic members from Latin America and the sharing of scientific data between continents. Generally, presentations are in Spanish with simultaneous translation into English. The majority of presentation slides are in English to facilitate communication among all meeting attendees. This publication includes summaries of 14 oral presentations by participants from Colombia, Mexico, and the United States of America. Topics addressed in the 3 sessions of the symposium included: larval habitats, insecticide resistance, new Aedes mosquito traps, blood meal analysis and fitness outcomes, vertical transmission of dengue, and transstadial composition of midgut microbiota. Control techniques discussed included sterile insect technique (SIT) with radiation, SIT by Wolbachia, thermal fogging, ultra-low volume pesticide applications, indoor residual spraying, Bacillus thuringiensis israelensis de Barjac (Bti), and Spinosad larval treatments. Presentations were also given on species composition and diversity of phlebotomine sand flies. Presentations were related to the vector species belonging to the mosquito genera, Anopheles, Aedes, and Culex as well as phlebotomine sand flies involved in the transmission of the causal agents of malaria, arboviruses (dengue, chikungunya, Zika), and leishmaniasis.

The level of gene flow between diverging lineages ultimately determines the outcome of a speciation event. If secondary contact occurs before this process is complete, reproductive isolation barriers must exist or evolve to prevent hybridization. The selective pressures facilitating and maintaining genetic divergence do not always involve an observable phenotypic response, thus cryptic species form. The inability to distinguish between sibling species can be a particularly serious problem in groups responsible for pathogen transmission. Culicoides biting midges occur almost world-wide and vector many disease-causing pathogens that affect wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a vector, and limited molecular and morphological differences have hindered vector surveillance. Here, genomic methods were used to investigate speciation and genetic structure within this complex. Single nucleotide polymorphism (SNP) data were generated using ddRAD sequencing for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses consistently suggest the occurrence of five putative species with significant differentiation occurring in both sympatric and allopatric populations. Evidence of hybridization was detected in three different species pairings, indicating a lack of pre-zygotic reproductive isolation within the complex. Mitochondrial genes were used to trace the hybrid parentage of these individuals, which illuminated discordance with the SNP data. In this study, we highlight the potential role of geographic, ecological, and behavioral isolation in speciation and in maintaining species boundaries, despite hybridization and long range dispersal.

Bluetongue virus (BTV) is a segmented RNA virus transmitted by Culicoides midges. Climatic factors, animal movement, vector species, and viral mutation and reassortment may all play a role in the occurrence of BTV outbreaks among susceptible ruminants. We used two enzootic strains of BTV (BTV-2 and BTV-10) to explore the potential for Culicoides sonorensis, a key North American vector, to be infected with these viruses, and identify the impact of temperature variations on virogenesis during infection. While BTV-10 replicated readily in C. sonorensis following an infectious blood meal, BTV-2 was less likely to result in productive infection at biologically relevant exposure levels. Moreover, when C. sonorensis were co-exposed to both viruses, we did not detect reassortment between the two viruses, despite previous in vitro findings indicating that BTV-2 and BTV-10 are able to reassort successfully. These results highlight that numerous factors, including vector species and exposure dose, may impact the in vivo replication of varying BTV strains, and underscore the complexities of BTV ecology in North America.

In the version of this article originally published, the e-mail address of corresponding author Patricia B. Munroe was incorrect. The error has been corrected in the HTML and PDF versions of the article.

A novel variant of SARS-CoV-2, B.1.1.7, originally discovered in the United Kingdom (UK), is rapidly overtaking other strains around the globe. In certain assays, absence of detection of the S-gene target, also known as S-gene target failure (SGTF) can be a sensitive surrogate of B.1.1.7. We analyzed daily counts of SGTF among samples from Dynacare Laboratory Ontario (which draws samples from the Greater Toronto Area) and resulted between December 16, 2020 and February 3, 2021. We identified 11,485 positive COVID-19 tests, of which 448 had SGTF (3.9%). The estimated prevalence of SGTF rose from 2.0% on December 16 to 15.2% on February 3 (1.8-fold weekly increase, 95%CI: 1.5, 2.2). The estimated reproduction number for SGTF cases was 1.17 (95%CI: 0.94 to 1.46), while the reproduction number for non-SGTF cases was 0.82 (95%CI: 0.65 to 1.01); the relative reproduction number was 1.44 (95%CI: 1.03, 1.99). 59 samples were sent for confirmatory testing, of which 53 (90%) were identified as B.1.1.7 using whole genome sequencing or found to have the N501Y mutation. In order to pre-emptively plan and implement public health measures to control COVID-19 now and through the spring, accurate and up-to-date early warning systems for new variants, including B.1.1.7, are essential across North America and the globe.

Objectives Bacterial antimicrobial resistance is a serious global public-health threat. Intestinal commensal drug-resistant bacteria have been suggested as an important reservoir of antimicrobial resistance genes (ARGs), which may be acquired via food. We aimed to identify risk factors associated with faecal carriage of drug-resistant commensal Escherichia coli among healthy adults focused on their dietary habits. Methods We conducted a cross-sectional study targeting healthy adult volunteers in a college community. Faecal samples and questionnaires were obtained from 113 volunteers. We conducted backward elimination logistic regression and least absolute shrinkage and selection operator (LASSO) methods to identify risk factors. Results We analysed responses from 81 of 113 volunteers who completed the questionnaire. The logistic regression and LASSO methods identified red meat consumption to be associated with an increased risk [OR = 6.13 (95% CI 1.83–24.2) and 1.82, respectively] and fish consumption with a reduced risk [OR = 0.27 (95% CI 0.08–0.85) and 0.81] for carriage of multidrug-resistant (MDR) E. coli, adjusted for biological sex, employment status, frequently used supermarket and previous travel. Conclusion Dietary habits are associated with risk of faecal carriage of MDR E. coli. This study supports the growing evidence that food may be an important source of ARGs present in human commensal E. coli.

Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches. Graphical Abstract

Escherichia coli is one of the most well-adapted and pathogenically versatile bacterial organisms. It causes a variety of human infections, including gastrointestinal illnesses and extraintestinal infections. It is also part of the intestinal commensal flora of humans and other mammals. Groups of E. coli that cause diarrhea are often described as intestinal pathogenic E. coli (IPEC), while those that cause infections outside of the gut are called extraintestinal pathogenic E. coli (ExPEC). IPEC can cause a variety of diarrheal illnesses as well as extraintestinal syndromes such as hemolytic-uremic syndrome. ExPEC cause urinary tract infections, bloodstream infection, sepsis, and neonatal meningitis. IPEC and ExPEC have thus come to be referred to as pathogenic variants of E. coli or pathovars. While IPEC can be distinguished from commensal E. coli based on their characteristic virulence factors responsible for their associated clinical manifestations, ExPEC cannot be so easily distinguished. IPEC most likely have reservoirs outside of the human intestine but it is unclear if ExPEC represent nothing more than commensal E. coli that breach a sterile barrier to cause extraintestinal infections. This question has become more complicated by the advent of whole genome sequencing (WGS) that has raised a new question about the taxonomic characterization of E. coli based on traditional clinical microbiologic and phylogenetic methods. This review discusses how molecular epidemiologic approaches have been used to address these questions, and how answers to these questions may contribute to our better understanding of the epidemiology of infections caused by E. coli . * This article is part of a curated collection.

The increasing prevalence of extended spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) β-lactamases among Enterobacterales threatens our ability to treat urinary tract infections (UTIs). These organisms are resistant to most β-lactam antibiotics and are frequently multidrug-resistant (MDR). Consequently, they are often resistant to antibiotics used to empirically treat UTIs. The lack of rapid diagnostic and antibiotic susceptibility tests (AST) makes clinical management of UTIs caused by such organisms difficult, as standard culture and susceptibility assays require several days. We have adapted a biochemical detection assay, termed dual-enzyme trigger-enabled cascade technology (DETECT) for rapid detection of resistance (time-to-result of 3 h) to other antibiotics commonly used in treatment of UTIs. DETECT is activated by the presence of CTX-M and pAmpC β-lactamases. In this proof-of-concept study, the adapted DETECT assay (AST-DETECT) has been performed on pure-cultures of Klebsiella pneumoniae and Escherichia coli (48 isolates) expressing ESBL or pAmpC β-lactamases to perform AST for ciprofloxacin (sensitivity 96.9%, specificity 100%, accuracy 97.9%) nitrofurantoin (sensitivity 95.7%, specificity 91.7%, accuracy 94%) and trimethoprim/sulfamethoxazole (sensitivity 83.3%, specificity 100%, accuracy 89.4%). These results suggest that AST-DETECT may be adapted as a potential diagnostic platform to rapidly detect multidrug-resistant E. coli and K. pneumoniae that cause UTI.

Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 Å shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding.

Vector-borne disease risk assessment is crucial to optimize surveillance, preventative measures (vector control), and resource allocation (medical supplies). High arthropod abundance and host interaction strongly correlate to vector-borne pathogen transmission. Increasing host density and movement increases the possibility of local and long-distance pathogen transmission. Therefore, we developed a risk assessment framework using climate (average temperature and rainfall) and host demographic (host density and movement) data, particularly suitable for regions with unreported or under-reported incidence data. This framework consisted of a spatiotemporal network-based approach coupled with a compartmental disease model and a non-homogeneous Gillespie algorithm. One-month and two-month lagged temperature and rainfall data have been used to develop the correlation of climate data with vector abundance and host-vector interactions. This correlation can be expressed as vectorial capacity— a parameter, which governs the spreading of infection from an infected host to a susceptible via vectors. As an example, the novel risk assessment framework is applied for dengue in Bangladesh. Vectorial capacity is inferred for each week throughout a year using average monthly temperature and rainfall data, while the whole country is divided into some spatial locations (upazilas). Long-distance pathogen transmission is expressed with human movement data in the spatiotemporal network. We have identified the spatiotemporal suitability of dengue spreading in Bangladesh as well as the significant-incidence window and peak incidence period. Analysis of yearly dengue data variation suggests the possibility of a significant outbreak with a new serotype introduction. The outcome of the framework comprises of weather-dependent spatiotemporal suitability maps and probabilistic risk maps for spatial infection spreading. This framework is capable of vector-borne disease risk assessment without historical incidence data and can be a useful tool for preparedness with accurate human movement data.

The administration of antibiotics while critical for treatment, can be accompanied by potentially severe complications. These include toxicities associated with the drugs themselves, the selection of resistant organisms and depletion of endogenous host microbiota. In addition, antibiotics may be associated with less well-recognized complications arising through changes in the pathogens themselves. Growing evidence suggests that organisms exposed to antibiotics can respond by altering the expression of toxins, invasins and adhesins, as well as biofilm, resistance and persistence factors. The clinical significance of these changes continues to be explored; however, it is possible that treatment with antibiotics may inadvertently precipitate a worsening of the clinical course of disease. Efforts are needed to adjust or augment antibiotic therapy to prevent the transition of pathogens to hypervirulent states. Better understanding the role of antibiotic-microbe interactions and how these can influence disease course is critical given the implications on prescription guidelines and antimicrobial stewardship policies.

Observational studies report that physical activity and metformin are associated with improved clinical outcome in patients with cancer. Inflammation is one biological mechanism hypothesized to mediate these associations. In this phase II, multicenter, 2 × 2 factorial trial, 139 patients with breast and colorectal cancer who completed standard therapy were randomized to one of four treatment groups for 12 weeks: exercise alone, metformin alone, exercise and metformin, or control. Inflammation outcomes included high-sensitivity C-reactive protein (hs-CRP), soluble tumor necrosis factor alpha receptor two (sTNFαR2), and IL6. The primary modeling strategy evaluated the trial product estimand that was quantified using a generalized linear mixed model. Compared with control, exercise alone reduced hs-CRP [−30.2%; 95% confidence interval (CI), −50.3, −1.0] and IL6 (−30.9%; 95% CI, −47.3, −9.5) but did not change sTNFαR2 (1.0%; 95% CI, −10.4, 13.9). Compared with control, metformin alone did not change hs-CRP (−13.9%; 95% CI, −40.0, 23.4), sTNFαR2 (−10.4%; 95% CI, −21.3, 2.0), or IL6 (−22.9%; 95% CI, −42.3, 2.0). Compared with control, exercise and metformin reduced sTNFαR2 (−13.1%; 95% CI, −22.9, −1.0) and IL6 (−38.7%; 95% CI, −52.3, −18.9) but did not change hs-CRP (−20.5%; 95% CI, −44.0, 12.7). The combination of exercise and metformin was not synergistic for hs-CRP, sTNFαR2, or IL6. In survivors of breast and colorectal cancer with low baseline physical activity and without type 2 diabetes, exercise and metformin reduced measures of inflammation that are associated with cancer recurrence and mortality.

Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria (GNB) are increasingly identified as the cause of both community and healthcare-associated urinary tract infections (UTIs), with CTX-Ms being the most common ESBLs identified. CTX-M-producing GNB are resistant to most β-lactam antibiotics and are frequently multidrug-resistant, which limits treatment options. Rapid diagnostic tests that can detect ESBL-producing GNB, particularly CTX-M producers, in the urine of patients with UTIs are needed. Results from such a test could direct the selection of appropriate antimicrobial therapy at the point-of-care (POC). In this study, we show that a chromogenic, dual enzyme-mediated amplification system (termed DETECT [dual-enzyme trigger-enabled cascade technology]) can identify CTX-M-producing GNB from unprocessed urine samples in 30 minutes. We first tested DETECT against a diverse set of recombinant β-lactamases and β-lactamase-producing clinical isolates to elucidate its selectivity. We then tested DETECT with 472 prospectively collected clinical urine samples submitted for urine culture to a hospital clinical microbiology laboratory. Of these, 118 (25%) were consistent with UTI, 13 (11%) of which contained ESBL-producing GNB. We compared DETECT results in urine against a standard phenotypic method to detect ESBLs, and polymerase chain reaction and sequencing for CTX-M genes. DETECT demonstrated 90.9% sensitivity and 97.6% specificity (AUC, 0.937; 95% confidence interval, 0.822-1.000), correctly identifying 10 of 11 urine samples containing a clinically significant concentration of CTX-M-producing GNB (including Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis). Our results demonstrate the clinical potential of DETECT to deliver diagnostic information at the POC, which could improve initial antibiotic selection.

The cell wall of wild-type (WT) Mycobacterium tuberculosis (Mtb), an etiologic agent of tuberculosis (TB) and a Mtb strain disrupted in a 13-gene operon mce1 (Δmce1) varies by more than 400 lipid species. Here, we examined Mtb lipid-induced response in murine macrophage, as well as in human T-cell subpopulations in order to gain an insight into how changes in cell wall lipid composition may modulate host immune response. Relative to WT Mtb cell wall lipids, the non-polar lipid extracts from Δmce1 enhanced the mRNA expression of lipid-sense nuclear receptors TR4 and PPAR-γ and dampened the macrophage expression of genes encoding TNF-α, IL-6, and IL-1β. Relative to untreated control, WT lipid-pre-stimulated macrophages from healthy individuals induced a higher level of CD4−CD8− double negative T-cells (DN T-cells) producing TNF-α. Conversely, compared to WT, stimulation with Δmce1 lipids induced higher mean fluorescence intensity (MFI) in IL-10-producing DN T cells. Mononuclear cells from TB patients stimulated with WT Mtb lipids induced an increased production of TNF-α by CD8+ lymphocytes. Taken together, these observations suggest that changes in mce1 operon expression during a course of infection may serve as a strategy by Mtb to evade the host pro-inflammatory responses.

Mechanisms underlying the acute respiratory distress syndrome (ARDS)-like clinical manifestations leading to deaths in patients who develop COVID-19 remain uncharacterized. While multiple factors could influence these clinical outcomes, we explored if differences in transmissibility and pathogenicity of SARS-CoV2 variants could contribute to these terminal clinical consequences of COVID-19. We analyzed 34,412 SARS-CoV2 sequences deposited in the Global Initiative for Sharing All Influenza Data (GISAID) SARS-CoV2 sequence database to determine if regional differences in circulating strain variants correlated with increased mortality in Europe, the United States, and California. We found two subclades descending from the Wuhan HU-1 strain that rapidly became dominant in Western Europe and the United States. These variants contained nonsynonymous nucleotide mutations in the Orf1ab segment encoding RNA-dependent RNA polymerase (C14408T), the spike protein gene (A23403G), and Orf1a (G25563T), which resulted in non-conservative amino acid substitutions P323L, D614G, and Q57H, respectively. In Western Europe, the A23403G-C14408T subclade dominated, while in the US, the A23403G-C14408T-G25563T mutant became the dominant strain in New York and parts of California. The high cumulative frequencies of both subclades showed inconsistent but significant association with high cumulative CFRs in some of the regions. When the frequencies of the subclades were analyzed by their 7-day moving averages across each epidemic, we found co-circulation of both subclades to temporally correlate with peak mortality periods. We postulate that in areas with high numbers of these co-circulating subclades, a person may get serially infected. The second infection may trigger a hyperinflammatory response similar to the antibody-dependent enhancement (ADE) response, which could explain the ARDS-like manifestations observed in people with co-morbidity, who may not mount sufficient levels of neutralizing antibodies against the first infection. Further studies are necessary but the implication of such a mechanism will need to be considered for all current COVID-19 vaccine designs.

We assessed the prevalence of antimicrobial resistance and screened for clinically relevant β-lactamase resistance determinants in Gram-negative bacteria from a large urbanized estuary. In contrast to the broad literature documenting potentially hazardous resistance determinants near wastewater treatment discharge points and other local sources of aquatic pollution, we employed a probabilistic survey design to examine ambient, near-shore sediments. We plated environmental samples from 40 intertidal and shallow subtidal areas around San Francisco Bay (California, USA) on drug-supplemented MacConkey agar, and we tested isolates for antimicrobial resistance and presence of clinically relevant β-lactamase resistance determinants. Of the 74 isolates identified, the most frequently recovered taxa were Vibrio spp. (40%), Shewanella spp. (36%), Pseudomonas spp. (11%), and Aeromonas spp. (4%). Of the 55 isolates tested for antimicrobial resistance, the Vibrio spp. showed the most notable resistance profiles. Most (96%) were resistant to ampicillin, and two isolates showed multidrug-resistant phenotypes: V. alginolyticus (cefotaxime, ampicillin, gentamicin, cefoxitin) and V. fluvialis (cefotaxime, ampicillin, cefoxitin). Targeted testing for class 1 integrons and presence of β-lactam-resistance gene variants TEM, SHV, OXA, CTX-M, and Klebsiella pneumonia carbapenemase (KPC) did not reveal any isolates harboring these resistance determinants. Thus, while drug-resistant, Gram-negative bacteria were recovered from ambient sediments, neither clinically relevant strains nor mobile β-lactam resistance determinants were found. This suggests that Gram-negative bacteria in this well-managed, urbanized estuary are unlikely to constitute a major human exposure hazard at this time.

Objective To evaluate auditory manifestations in children born to mothers who had exanthema during pregnancy, suspected to have been exposed to the Zika virus (ZIKV). Study design Prospective observational. Setting Tertiary referral center. Patients Children born in Rio de Janeiro, Brazil, between April 2016 and September 2017, who were referred for newborn hearing screening (NHS). Intervention The NHS was performed by the automated brainstem auditory-evoked potential test at an intensity of 30 dBHL (decibels Hearing Level) with the result presented as "PASS/FAIL." A follow-up test was performed 6 months after the first examination. Main outcome measures Hearing outcomes by audiological assessment. Results Ninety-eight children were recruited and 78 underwent the NHS test. In the first evaluation, the FAIL NHS result was observed in 4 of the 78 children. Three were diagnosed with sensorineural hearing loss and one had conductive loss. Including the first and second evaluation, the frequency of audiological alterations was 5.1%. Of the four children diagnosed with hearing loss, two were carriers of ZIKV, one had suspected ZIKV infection, and one was asymptomatic with confirmed exposure to the virus. There was no progression of hearing loss or other hearing abnormality in the children by the time of the second evaluation. The group of nonexposed children (negative quantitative reverse transcription polymerase chain reaction for ZIKV) showed no hearing loss. Conclusion Uni or bilateral sensorineural hearing loss was diagnosed in asymptomatic children at birth. These observations highlight the importance of periodic follow-up of patients with congenital Zika syndrome to better understand their long-term auditory clinical outcome.

The genome sequence of a uropathogenic Escherichia coli sequence type 420 strain isolated from a patient with urinary tract infection in northern California is described here. The draft genome sequence includes a 4.8-Mb chromosome, accompanied by a 114-kb plasmid containing IncFIB/IncFII/Col156 and a 35-kb plasmid containing IncN3.

Community‐acquired urinary tract infections (UTIs) are one of the most common bacterial infections worldwide. Escherichia coli is the most common cause of community‐acquired UTI. In general, UTI results from E. coli in the intestine that enters the bladder via the urethra. However, whether these E. coli strains that cause UTI represent members of the intestinal commensal E. coli or a distinct subgroup of pathogenic E. coli remains unestablished. Here, we analyzed E. coli isolates from fecal samples of healthy volunteers and urine samples of UTI patients obtained from a university‐affiliated health center. The E. coli isolates were genotyped by multilocus sequence typing (MLST). From May to October 2018, we analyzed 89 E. coli isolates from 76 (75%) rectal swabs from 113 unique healthy volunteers. We also analyzed 106 (27%) E. coli isolates from 398 unique urine samples collected between August and October 2018. Fecal and urine E. coli isolates each contained 31 distinct sequence types (STs). Nine STs were shared by fecal and urine E. coli isolates, which accounted for approximately 50% of urine isolates typed by MLST. Among the shared genotypes, ST10 and ST131 were significantly more frequently found in fecal samples, whereas ST95 and ST127 were significantly more frequently recovered from UTI samples. ST73 was found only among urine samples. These E. coli genotypes clustered and fluctuated over time. These observations suggest that E. coli genotypes found to cause UTI transiently colonize the intestine and that their primary reservoir may reside outside of the human intestine., We address an unresolved question regarding the source of Escherichia coli that causes community‐acquired urinary tract infection (CA‐UTI)—whether E. coli strains that cause UTI represent members of the intestinal commensal E. coli or a distinct subgroup of pathogenic E. coli. Our results based on multilocus sequence type (MLST) analysis of E. coli isolates from healthy volunteers versus urine isolates from patients with UTI indicate that uropathogenic E. coli has a source outside of the human intestine.

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free-living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Japanese encephalitis (JE) is a zoonotic, emerging disease transmitted by mosquito vectors infected with the Japanese encephalitis virus (JEV). Its potential for emergence into susceptible regions is high, including in the United States (US), and is a reason of economic concern among the agricultural community, and to public health due to high morbidity and mortality rates in humans. While exploring the complexities of interactions involved with viral transmission, we proposed a new outlook on the role of vectors, hosts and the environment under changing conditions. For instance, the role of feral pigs may have been underappreciated in our previous work, given research keeps pointing to the importance of susceptible populations of wild swine in naïve regions as key elements for the introduction of emergent vector-borne diseases. High risk of JEV introduction has been associated with the transportation of infected mosquitoes via aircraft. Nonetheless, no JEV outbreaks have been reported in the US to date and results from a qualitative risk assessment considered the risk of establishment to be negligible under the current conditions (environmental, vector, pathogen, and host). In this work, we discuss virus-vector-host interactions and ecological factors important for virus transmission and spread, review research on the risk of JEV introduction to the US considering the implications of risk dismissal as it relates to past experiences with similar arboviruses, and reflect on future directions, challenges, and implications of a JEV incursion.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with a febrile illness as well as severe complications, including microcephaly and Guillain-Barr&eacute, Syndrome. Antibody cross-reactivity between flaviviruses has been documented, and in regions where ZIKV is circulating, dengue virus (DENV) is also endemic, leaving the potential that previous exposure to DENV could alter clinical features of ZIKV infection. To investigate this, we performed a retrospective case-control study in which we compared Canadian travellers who had been infected with ZIKV and had serological findings indicating previous DENV or other flavivirus exposure (n = 16) to those without any previous exposure (n = 44). Patient samples were collected between February 2016 and September 2017 and submitted to Public Health Ontario for testing. ZIKV infection was determined using real-time RT-PCR and antibodies against DENV were identified by the plaque-reduction neutralization test. The mean time from symptom onset to sample collection was 5 days for both groups, the magnitude of viremia was not statistically different (Ct values: 35.6 vs. 34.9, p-value = 0.2). Clinical scores were also similar. Our findings indicate that previous DENV or other flavivirus exposure did not result in greater viremia or a higher illness score.

An assessment of the production, distribution and fate of highly branched isoprenoid (HBI) biomarkers produced by sea ice and pelagic diatoms is necessary to interpret their detection and proportions in the northern Bering and Chukchi Seas. HBIs measured in surface sediments collected from 2012 to 2017 were used to determine the distribution and seasonality of the biomarkers relative to sea ice patterns. A northward gradient of increasing ice algae deposition was observed with localized occurrences of elevated IP25 (sympagic HBI) concentrations from 68-70°N and consistently strong sympagic signatures from 71-72.5°N. A declining sympagic signature was observed from 2012 to 2017 in the northeast Chukchi Sea, coincident with declining sea ice concentrations. HBI fluxes were investigated on the northeast Chukchi shelf with a moored sediment trap deployed from August 2015 to July 2016. Fluxes of sea ice exclusive diatoms (Nitzschia frigida and Melosira arctica) and HBI-producing taxa (Pleurosigma, Haslea and Rhizosolenia spp.) were measured to confirm HBI sources and ice associations. IP25 was detected year-round, increasing in March 2016 (10 ng m-2 d-1) and reaching a maximum in July 2016 (1331 ng m-2 d-1). Snowmelt triggered the release of sea ice algae into the water column in May 2016, while under-ice pelagic production contributed to the diatom export in June and July 2016. Sea ice diatom fluxes were strongly correlated with the IP25 flux, however associations between pelagic diatoms and HBI fluxes were inconclusive. Bioturbation likely facilitates sustained burial of sympagic organic matter on the shelf despite the occurrence of pelagic diatom blooms. These results suggest that sympagic diatoms may sustain the food web through winter on the northeast Chukchi shelf. The reduced relative proportions of sympagic HBIs in the northern Bering Sea are likely driven by sea ice persistence in the region.

Background Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1–1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. Results Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. Conclusions Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure.

Genetic studies of blood pressure (BP) to date have mainly analyzed common variants (minor allele frequency > 0.05). In a meta-analysis of up to ~1.3 million participants, we discovered 106 new BP-associated genomic regions and 87 rare (minor allele frequency ≤ 0.01) variant BP associations (P