Low humidity enhances Zika virus infection and dissemination in Aedes aegypti mosquitoes.

As climate change alters Earth's biomes, it is expected the transmission dynamics of mosquito-borne viruses will change. While the effects of temperature changes on mosquito-virus interactions and spread of the pathogens have been elucidated over the last decade, the effects of relative humidity changes are still relatively unknown. To overcome this knowledge gap, we exposed Ae. aegypti females to various low humidity conditions and measured different components of vectorial capacity such as survival, blood-feeding rates, and changes in infection and dissemination of Zika virus. Survival decreased as the humidity level decreased, while infection rates increased as the humidity level decreased. Alternatively, blood feeding rates and dissemination rates peaked at the intermediate humidity level, but returned to the levels of the control at the lowest humidity treatment. These results provide empirical evidence that Ae. aegypti exposure to low humidity can enhance Zika virus infection in the mosquito, which has important implications in predicting how climate change will impact mosquito-borne viruses.

A high-throughput screen identifies RNA binding proteins that affect fertility in Caenorhabditis elegans and reveals a functional relationship between ADR-2 and SQD-1.

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Characterization of gross fertility defects, such as sterility, has identified RNA binding proteins that are critical regulators of germline gene expression; however, broader screens for RNA binding proteins involved in specific reproductive processes are lacking. Here, a reverse genetic screen was performed to identify RNA binding proteins that impact yolk and embryo production in Caenorhabditis elegans hermaphrodites. This screen makes use of animals expressing a vitellogenin (yolk protein) fusion with green fluorescent protein, in a genetic background that corrects for a previously identified fertility defect in this strain. From this screen, we identified 23 RNA binding proteins that regulate embryo production in Caenorhabditis elegans. This screen lays groundwork for future interrogations into the molecular roles of these proteins in yolk production and embryogenesis. Additionally, the screen uncovered a genetic interaction between ADR-2, a member of the Adenosine DeAminase Acting on RNA (ADAR) family, and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family. Transcriptome-wide assessment in animals depleted of sqd-1 revealed over 8000 misregulated transcripts, suggesting SQD-1 is a major regulator of gene expression. Consistent with this, microscopy and reproductive assays reveal that sqd-1 is essential for oogenesis. In the absence of adr-2, the effects of loss of sqd-1 on gene expression are attenuated, as well as the defects in oogenesis. Together, these data indicate that both ADR-2 and SQD-1 are important regulators of germline gene expression and oogenesis.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Dehydration induced AePer50 regulates midgut infection in Ae. aegypti.

In the face of climate change, mosquitoes will experience evolving climates including longer periods of drought. An important physiological response to dry environments is the protection against water loss or dehydration, here defined as desiccation tolerance. Various environmental factors including temperature are known to alter interactions between the mosquito, Aedes aegypti , and the arboviruses it transmits, but little is known about how low humidity impacts arboviral infection. Here, we report that a gene upregulated in response to desiccation is important for controlling midgut infection. We have identified two genetically diverse lines of Ae. aegypti with marked differences in desiccation tolerance. To understand if the genetic basis underlying desiccation tolerance is the same between the contrasting lines, we compared gene expression profiles between desiccant treated and non-desiccant treated individuals in both the desiccation tolerant and susceptible lines by RNAseq. Gene expression analysis demonstrates that different genes are differentially expressed in response to desiccation stress between desiccation tolerant and susceptible lines. The most highly expressed transcript under desiccation stress in the desiccation susceptible line encodes a peritrophin protein, Ae Per50. Peritrophins play a crucial role in peritrophic matrix formation after a bloodmeal. Gene silencing of Ae Per50 by RNAi demonstrates that expression of Ae Per50 is required for survival of the desiccation susceptible line under desiccation stress, but not for the desiccation tolerant line. Moreover, the knockdown of Ae Per50 results in higher infection rates and viral replication rates of ZIKV and higher infection rates of CHIKV. Finally, following a bloodmeal, the desiccation susceptible line develops a thicker peritrophic matrix than the desiccation tolerant line. Together these results provide a functional link between the protection against desiccation and midgut infection which has important implications in predicting how climate change will impact mosquito-borne viruses.

Gut microbiome perturbation, antibiotic resistance, and Escherichia coli strain dynamics associated with international travel: a metagenomic analysis.

BACKGROUND: Culture-based studies have shown that acquisition of extended-spectrum ß-lactamase-producing Enterobacterales is common during international travel; however, little is known about the role of the gut microbiome before and during travel, nor about acquisition of other antimicrobial-resistant organisms. We aimed to identify (1) whether the gut microbiome provided colonisation resistance against antimicrobial-resistant organism acquisition, (2) the effect of travel and travel behaviours on the gut microbiome, and (3) the scale and global heterogeneity of antimicrobial-resistant organism acquisition. METHODS: In this metagenomic analysis, participants were recruited at three US travel clinics (Boston, MA; New York, NY; and Salt Lake City, UT) before international travel. Participants had to travel internationally between Dec 8, 2017, and April 30, 2019, and have DNA extractions for stool samples both before and after travel for inclusion. Participants were excluded if they had at least one low coverage sample (<1 million read pairs). Stool samples were collected at home before and after travel, sent to a clinical microbiology laboratory to be screened for three target antimicrobial-resistant organisms (extended-spectrum ß-lactamase-producing Enterobacterales, carbapenem-resistant Enterobacterales, and mcr-mediated colistin-resistant Enterobacterales), and underwent DNA extraction and shotgun metagenomic sequencing. We profiled metagenomes for taxonomic composition, antibiotic-resistant gene content, and characterised the Escherichia coli population at the strain level. We analysed pre-travel samples to identify the gut microbiome risk factors associated with acquisition of the three targeted antimicrobial resistant organisms. Pre-travel and post-travel samples were compared to identify microbiome and resistome perturbation and E coli strain acquisition associated with travel. FINDINGS: A total of 368 individuals travelled between the required dates, and 296 had DNA extractions available for both before and after travel. 29 travellers were excluded as they had at least one low coverage sample, leaving a final group of 267 participants. We observed a perturbation of the gut microbiota, characterised by a significant depletion of microbial diversity and enrichment of the Enterobacteriaceae family. Metagenomic strain tracking confirmed that 67% of travellers acquired new strains of E coli during travel that were phylogenetically distinct from their pre-travel strains. We observed widespread enrichment of antibiotic-resistant genes in the gut, with a median 15% (95% CI 10-20, p<1 × 10-10) increase in burden (reads per kilobase per million reads). This increase included antibiotic-resistant genes previously classified as threats to public health, which were 56% (95% CI 36-91, p=2 × 10-11) higher in abundance after travel than before. Fluoroquinolone antibiotic-resistant genes were aquired by 97 (54%) of 181 travellers with no detected pre-travel carriage. Although we found that visiting friends or relatives, travel to south Asia, and eating uncooked vegetables were risk factors for acquisition of the three targeted antimicrobial resistant organisms, we did not observe an association between the pre-travel microbiome structure and travel-related antimicrobial-resistant organism acquisition. INTERPRETATION: This work highlights a scale of E coli and antimicrobial-resistant organism acquisition by US travellers not apparent from previous culture-based studies, and suggests that strategies to control antimicrobial-resistant organisms addressing international traveller behaviour, rather than modulating the gut microbiome, could be worthwhile. FUNDING: US Centers for Disease Control and Prevention and National Institute of Allergy and Infectious Diseases.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can influence adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we show that similarities in bacterial community structures when given identical microbiomes between different genetic backgrounds of Ae. aegypti was dependent on the source microbiome, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti . We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Intrinsic Resistance to Colistin in the Genus Hafnia.

A bacterial species is considered to be intrinsically resistant to an antimicrobial when nearly all of the wild-type isolates (i.e., those without acquired resistance) exhibit minimum inhibitory concentration (MIC) values that are sufficiently high such that susceptibility testing is unnecessary, and that the antimicrobial should not be considered for therapy. Accordingly, knowledge of intrinsic resistance influences both the selection of treatment regimens and the approach to susceptibility testing in the clinical laboratory, where unexpected results also facilitate the recognition of microbial identification or susceptibility testing errors. Previously, limited data have suggested that Hafnia spp. may be intrinsically resistant to colistin. We evaluated the in vitro activity of colistin against 119 Hafniaceae that were isolated from human samples: 75 (63%) from routine clinical cultures and 44 (37%) from stool samples of travelers undergoing screening for antimicrobial resistant organisms. Broth microdilution colistin MICs were ≥4 µg/mL for 117 of 119 (98%) isolates. Whole-genome sequencing of 96 of the isolates demonstrated that the colistin-resistant phenotype was not lineage-specific. 2 of the 96 (2%) isolates harbored mobile colistin resistance genes. Compared to whole-genome sequencing, VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and VITEK 2 GN ID failed to consistently distinguish between Hafnia alvei, Hafnia paralvei, and Obesumbacterium proteus. In conclusion, using a reference antimicrobial susceptibility testing method and a genetically diverse collection of isolates, we found Hafnia spp. to be intrinsically resistant to colistin. The recognition of this phenotype will help inform rational approaches by which to perform antimicrobial susceptibility testing and therapy for patients with infections that are caused by Hafnia spp.

Parents' math anxiety and mathematics performance of pre-kindergarten children.

Prior research demonstrates that individuals' math anxiety may be negatively related to their mathematics performance. However, little research has examined how caregivers' math anxiety is associated with children's mathematics performance prior to kindergarten. The purpose of this study was to investigate the relation between parents' math anxiety and the change in children's mathematics performance during the preschool year. Participants were 310 preschool-age children (155 female; 4.12-5.78 years of age, M = 5.20 years, SD = 0.29). Structural equation modeling results demonstrated that parents' math anxiety was significantly negatively related to change in children's mathematics performance during the pre-kindergarten year when controlling for fall mathematics performance and demographics. Moreover, multigroup path analyses revealed that this association did not differ for male versus female children.

Influence of Affect on Physical Activity: An Integrative Review.

Affective states, such as feelings of anger and excitement, are linked to health outcomes and behaviors. The benefits of physical activity for individual affect is known; however, how affect influences physical activity participation is less understood. Using Whittemore and Knafl's framework, this integrative review examines the influence of affect on adult physical activity. using six databases, 19 articles published between 1997 and 2019. Themes found include support for the influence of positive affect on increased physical activity, a temporal aspect of affect, a variety of measurement tools, and varying uses of theoretical frameworks across studies. Advanced practice nurses and registered nurses may improve patient health behaviors, such as physical activity, by incorporating affect-focused assessments. Review findings support consideration of affect in physical activity counseling. Further research using theory-driven methods and consistent affect assessments is needed to test the complex relationship between affect and physical activity.

The effect of transport temperature and time on the recovery of antimicrobial-resistant Enterobacterales in stool.

Surveillance for antibiotic-resistant (AR) bacteria is challenging. We evaluated AR Enterobacterales survival in stool over various transport conditions. Stool in Cary-Blair medium was spiked with AR Enterobacterales, held at 3 °C, 20 °C, or 37 °C, and cultured on days 3, 8, and 15. Stool from US international travelers sent through the US mail was also screened. We compared recovery rates using Fisher's exact tests and linear regression models. AR Enterobacterales recovery reduced with time (86% versus 75% versus 61% at days 3, 8, and 15; Beta for linear trend=-0.02, r2=0.99, P=0.02) and colder temperatures [56% (3 °C) versus 89% (20 °C) versus 86% (37 °C); P=0.003]. Traveler sample recovery also reduced with transport time (Beta for linear trend=-0.03, r2=0.70, P=0.01) but not with season [20% (cold) versus 22% (warm), P=0.7]. AR Enterobacterales are found over variable transport conditions, providing rationale for expanding surveillance sample processing timelines.

Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients.

We measured plasma and/or serum antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in 343 North American patients infected with SARS-CoV-2 (of which 93% required hospitalization) up to 122 days after symptom onset and compared them to responses in 1548 individuals whose blood samples were obtained prior to the pandemic. After setting seropositivity thresholds for perfect specificity (100%), we estimated sensitivities of 95% for IgG, 90% for IgA, and 81% for IgM for detecting infected individuals between 15 and 28 days after symptom onset. While the median time to seroconversion was nearly 12 days across all three isotypes tested, IgA and IgM antibodies against RBD were short-lived with median times to seroreversion of 71 and 49 days after symptom onset. In contrast, anti-RBD IgG responses decayed slowly through 90 days with only 3 seropositive individuals seroreverting within this time period. IgG antibodies to SARS-CoV-2 RBD were strongly correlated with anti-S neutralizing antibody titers, which demonstrated little to no decrease over 75 days since symptom onset. We observed no cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies with other widely circulating coronaviruses (HKU1, 229 E, OC43, NL63). These data suggest that RBD-targeted antibodies are excellent markers of previous and recent infection, that differential isotype measurements can help distinguish between recent and older infections, and that IgG responses persist over the first few months after infection and are highly correlated with neutralizing antibodies.

Dynamics and significance of the antibody response to SARS-CoV-2 infection.

BACKGROUND: Characterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence. METHODS: We measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic. RESULTS: Between 14-28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63. CONCLUSIONS: Among symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity.

Acquisition and Long-term Carriage of Multidrug-Resistant Organisms in US International Travelers.

We performed prospective screening of stool for multidrug-resistant organisms from 608 US international travelers and identified an acquisition rate of 38% following travel. Carriage rates remained significantly elevated for at least 6 months post-travel. Travel-related diarrhea was a risk factor for acquisition, as well as for long-term carriage upon return.

Evaluation of a Screening Method for the Detection of Colistin-Resistant Enterobacteriaceae in Stool.

Emergence of mobile colistin resistance (mcr)-containing Enterobacteriaceae is a public health threat, prompting enhanced surveillance through the Centers for Disease Control and Prevention. We evaluated a selective culture medium for the isolation of Enterobacteriaceae with non-wild-type colistin minimum inhibitory concentrations, including those with mcr-1 genes, in spiked stool samples.

A Simplified Sample Preparation Method for the Assessment of Plasma Ascorbic Acid in Clinical Settings.

BACKGROUND: Vitamin C deficiency is difficult to diagnose on the basis of clinical presentation alone and requires plasma levels for confirmation. Reference laboratories typically specify shipment of plasma on dry ice. This requirement may complicate clinic work flow and delay vitamin C measurement. Additionally, patients with vitamin C deficiency may experience unnecessary testing and increased health-care costs, as other diagnoses are often considered first. We examined an alternative, more practical shipping method. METHODS: Plasma was collected from 17 healthy volunteers by use of heparin tubes with gel separators, and all tubes were centrifuged immediately to separate the plasma layer from the cells. Baseline vitamin C was measured in plasma obtained immediately after specimen collection. Remaining sample tubes were held in Styrofoam containers with cold packs for 30 h or 48 h, followed by vitamin C measurement. Additional samples were exposed to conditions that simulated harsher shipping conditions. RESULTS: Mean plasma vitamin C was 69.6 µmol/L (SD = 21.5 µmol/L). Vitamin C losses were 5.4% at 30 h (SD = 5.55%, P < 0.05) and 7.6% at 48 h (SD = 5.56%, P < 0.05), which is slightly more than freeze-and-thaw treatment (average loss of 1.4%, SD = 6.9%, NS). The vitamin C method had an intraday variation of 1.88%. Vigorous shaking of 2 samples for 24 h resulted in a -1.9% change in 1 sample, and a +4.1% change in another sample. Exposure of the shipping container to elevated temperature (35 °C for 30 h) did not change the internal temperature of the container. CONCLUSIONS: The shipping procedure uses routine sample handling, standard vacutainers, and can be replicated by health-care centers seeking to evaluate patient vitamin C status.

Better access, quality, and cost for clinically complex veterans with home-based primary care.

In successfully reducing healthcare expenditures, patient goals must be met and savings differentiated from cost shifting. Although the Department of Veterans Affairs (VA) Home Based Primary Care (HBPC) program for chronically ill individuals has resulted in cost reduction for the VA, it is unknown whether cost reduction results from restricting services or shifting costs to Medicare and whether HBPC meets patient goals. Cost projection using a hierarchical condition category (HCC) model adapted to the VA was used to determine VA plus Medicare projected costs for 9,425 newly enrolled HBPC recipients. Projected annual costs were compared with observed annualized costs before and during HBPC. To assess patient perspectives of care, 31 veterans and caregivers were interviewed from three representative programs. During HBPC, Medicare costs were 10.8% lower than projected, VA plus Medicare costs were 11.7% lower than projected, and combined hospitalizations were 25.5% lower than during the period without HBPC. Patients reported high satisfaction with HBPC team access, education, and continuity of care, which they felt contributed to fewer exacerbations, emergency visits, and hospitalizations. HBPC improves access while reducing hospitalizations and total cost. Medicare is currently testing the HBPC approach through the Independence at Home demonstration.

Knowledge, attitudes, and practices of US travelers to Asia regarding seasonal influenza and H5N1 avian influenza prevention measures.

BACKGROUND: International travel is a potential risk factor for the spread of influenza. In the United States, approximately 5%-20% of the population develops an influenza-like illness annually. The purpose of this study was to describe the knowledge, attitude, and practices of US travelers to Asia regarding seasonal influenza and H5N1 avian influenza (AI) prevention measures. METHODS: We surveyed travelers to Asia waiting at the departure lounges of 38 selected flights at four international airports in New York, Chicago, Los Angeles, and San Francisco. Of the 1,301 travelers who completed the pre-travel survey, 337 also completed a post-travel survey. Univariate and multivariate logistic regression were used to calculate prevalence odds ratios (with 95% CI) to compare foreign-born (FB) to US-born travelers for various levels of knowledge and behaviors. RESULTS: Although the majority of participants were aware of influenza prevention measures, only 41% reported receiving the influenza vaccine during the previous season. Forty-three percent of participants reported seeking at least one type of pre-travel health advice, which was significantly higher among US-born, Caucasians, traveling for purposes other than visiting friends and relatives, travelers who received the influenza vaccine during the previous season, and those traveling with a companion. Our study also showed that Asians, FB travelers, and those working in occupations other than health care/animal care were less likely to recognize H5N1 AI transmission risk factors. CONCLUSION: The basic public health messages for preventing influenza appear to be well understood, but the uptake of influenza vaccine was low. Clinicians should ensure that all patients receive influenza vaccine prior to travel. Tailored communication messages should be developed to motivate Asians, FB travelers, those visiting friends and relatives, and those traveling alone to seek pre-travel health advice as well as to orient them with H5N1 AI risk factors.

Neisseria meningitidis serogroup W-135 carriage among US travelers to the 2001 Hajj.

In 2000, a large international outbreak of meningococcal disease caused by Neisseria meningitidis serogroup W-135 was identified among pilgrims returning from the Hajj in Saudi Arabia. To assess ongoing risk, we evaluated N. meningitidis carriage among US travelers to the 2001 Hajj. Of 25 N. meningitidis isolates obtained, 15 (60%) were nongroupable and 8 (32%) were serogroup W-135 when tested by standard slide-agglutination techniques. Two additional nongroupable isolates were characterized as serogroup W-135 when tested by polymerase chain reaction. Nine of 10 serogroup W-135 isolates were indistinguishable from the Hajj-2000 clone. None of the departing, but 9 (1.3%) of the returning, pilgrims carried serogroup W-135 (P=.01); all carriers reported previous vaccination. Carriage of N. meningitidis serogroup W-135 increased significantly in pilgrims returning from the Hajj. Although the risk of disease to pilgrims appears to be low, the risk of spread to others of this pathogenic strain remains a concern.