Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Parasite manipulation of host phenotypes inferred from transcriptional analyses in a trematode-amphipod system.

Manipulation of host phenotypes by parasites is hypothesized to be an adaptive strategy enhancing parasite transmission across hosts and generations. Characterizing the molecular mechanisms of manipulation is important to advance our understanding of host-parasite coevolution. The trematode (Levinseniella byrdi) is known to alter the colour and behaviour of its amphipod host (Orchestia grillus) presumably increasing predation of amphipods which enhances trematode transmission through its life cycle. We sampled 24 infected and 24 uninfected amphipods from a salt marsh in Massachusetts to perform differential gene expression analysis. In addition, we constructed novel genomic tools for O. grillus including a de novo genome and transcriptome. We discovered that trematode infection results in upregulation of amphipod transcripts associated with pigmentation and detection of external stimuli, and downregulation of multiple amphipod transcripts implicated in invertebrate immune responses, such as vacuolar ATPase genes. We hypothesize that suppression of immune genes and the altered expression of genes associated with coloration and behaviour may allow the trematode to persist in the amphipod and engage in further biochemical manipulation that promotes transmission. The genomic tools and transcriptomic analyses reported provide new opportunities to discover how parasites alter diverse pathways underlying host phenotypic changes in natural populations.

Discovery and structure-activity relationships of a novel oxazolidinone class of bacterial type II topoisomerase inhibitors.

There is an increasingly urgent and unmet medical need for novel antibiotic drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. Novel bacterial type II topoisomerase inhibitors (NBTIs) are of high interest due to limited cross-resistance with fluoroquinolones, however analogues with Gram-negative activity often suffer from hERG channel inhibition. A novel series of bicyclic-oxazolidinone inhibitors of bacterial type II topoisomerase were identified which display potent broad-spectrum anti-bacterial activity, including against MDR strains, along with an encouraging in vitro safety profile. In vivo proof of concept was achieved in a A. baumannii mouse thigh infection model.

A Comparison of Less Invasive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Diagnostic Specimens in Nursing Home Residents-Arkansas, June-August 2020.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing remains essential for early identification and clinical management of cases. We compared the diagnostic performance of 3 specimen types for characterizing SARS-CoV-2 in infected nursing home residents. METHODS: A convenience sample of 17 residents were enrolled within 15 days of first positive SARS-CoV-2 result by real-time reverse transcription polymerase chain reaction (RT-PCR) and prospectively followed for 42 days. Anterior nasal swabs (AN), oropharyngeal swabs (OP), and saliva specimens (SA) were collected on the day of enrollment, every 3 days for the first 21 days, and then weekly for 21 days. Specimens were tested for presence of SARS-CoV-2 RNA using RT-PCR and replication-competent virus by viral culture. RESULTS: Comparing the 3 specimen types collected from each participant at each time point, the concordance of paired RT-PCR results ranged from 80% to 88%. After the first positive result, SA and OP were RT-PCR-positive for ≤48 days; AN were RT-PCR-positive for ≤33 days. AN had the highest percentage of RT-PCR-positive results (21/26 [81%]) when collected ≤10 days of participants' first positive result. Eleven specimens were positive by viral culture: 9 AN collected ≤19 days following first positive result and 2 OP collected ≤5 days following first positive result. CONCLUSIONS: AN, OP, and SA were effective methods for repeated testing in this population. More AN than OP were positive by viral culture. SA and OP remained RT-PCR-positive longer than AN, which could lead to unnecessary interventions if RT-PCR detection occurred after viral shedding has likely ceased.

Initial and Repeated Point Prevalence Surveys to Inform SARS-CoV-2 Infection Prevention in 26 Skilled Nursing Facilities - Detroit, Michigan, March-May 2020.

Skilled nursing facilities (SNFs) are focal points of the coronavirus disease 2019 (COVID-19) pandemic, and asymptomatic infections with SARS-CoV-2, the virus that causes COVID-19, among SNF residents and health care personnel have been described (1-3). Repeated point prevalence surveys (serial testing of all residents and health care personnel at a health care facility irrespective of symptoms) have been used to identify asymptomatic infections and have reduced SARS-CoV-2 transmission during SNF outbreaks (1,3). During March 2020, the Detroit Health Department and area hospitals detected a sharp increase in COVID-19 diagnoses, hospitalizations, and associated deaths among SNF residents. The Detroit Health Department collaborated with local government, academic, and health care system partners and a CDC field team to rapidly expand SARS-CoV-2 testing and implement infection prevention and control (IPC) activities in all Detroit-area SNFs. During March 7-May 8, among 2,773 residents of 26 Detroit SNFs, 1,207 laboratory-confirmed cases of COVID-19 were identified during three periods: before (March 7-April 7) and after two point prevalence surveys (April 8-25 and April 30-May 8): the overall attack rate was 44%. Within 21 days of receiving their first positive test results, 446 (37%) of 1,207 COVID-19 patients were hospitalized, and 287 (24%) died. Among facilities participating in both surveys (n = 12), the percentage of positive test results declined from 35% to 18%. Repeated point prevalence surveys in SNFs identified asymptomatic COVID-19 cases, informed cohorting and IPC practices aimed at reducing transmission, and guided prioritization of health department resources for facilities experiencing high levels of SARS-CoV-2 transmission. With the increased availability of SARS-CoV-2 testing, repeated point prevalence surveys and enhanced and expanded IPC support should be standard tools for interrupting and preventing COVID-19 outbreaks in SNFs.

Survival, Persistence, and Isolation of the Emerging Multidrug-Resistant Pathogenic Yeast Candida auris on a Plastic Health Care Surface.

The emerging multidrug-resistant pathogenic yeast Candida auris represents a serious threat to global health. Unlike most other Candida species, this organism appears to be commonly transmitted within health care facilities and causes health care-associated outbreaks. To better understand the epidemiology of this emerging pathogen, we investigated the ability of C. auris to persist on plastic surfaces common in health care settings compared with that of Candida parapsilosis, a species known to colonize the skin and plastics. Specifically, we compiled comparative and quantitative data essential to understanding the vehicles of spread and the ability of both species to survive and persist on plastic surfaces under controlled conditions (25°C and 57% relative humidity), such as those found in health care settings. When a test suspension of 104 cells was applied and dried on plastic surfaces, C. auris remained viable for at least 14 days and C. parapsilosis for at least 28 days, as measured by CFU. However, survival measured by esterase activity was higher for C. auris than C. parapsilosis throughout the 28-day study. Given the notable length of time Candida species survive and persist outside their host, we developed methods to more effectively culture C. auris from patients and their environment. Using our enrichment protocol, public health laboratories and researchers can now readily isolate C. auris from complex microbial communities (such as patient skin, nasopharynx, and stool) as well as environmental biofilms, in order to better understand and prevent C. auris colonization and transmission.

Effects of Simulated Low Vision on Postural Adjustment to Changes in Center of Mass in Older Adults.

This study examined how instrumental activities of daily living (IADL) performed by older adults under low-vision simulation conditions affect postural adjustments to changes in center of mass (COM). Ten participants with normal vision performed seven activities under two conditions, normal vision, and simulated macular degeneration (MD). Postural adjustment to changes in COM and time to complete activities were recorded. Low vision was compared to normal vision using Wilcoxon signed rank and t tests. Differences between the two conditions were statistically significant for postural adjustments to change in COM and time. Postural adjustments and time to perform IADLs are greater under simulated low vision conditions versus normal vision. These preliminary findings support research with older adults with MD, who may be at risk when making movement transitions like descending or ascending stairs, stepping in and out of a tub, stooping, or reaching from one surface to another.

Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position.

We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging.

Episodic foresight and schizophrenia.

OBJECTIVE: People with schizophrenia have difficulty engaging in specific future-directed thoughts and behaviours, such as generating phenomenological characteristics of future events (a component of episodic foresight), and executing directed preparatory behaviours (a component of prospective memory). However, it remains unclear whether they also exhibit difficulties using episodic foresight to appropriately guide future-directed behaviours. METHOD: People with schizophrenia and non-clinical controls were administered a behavioural measure that met strict criteria for assessing episodic foresight. In keeping with our focus on the functional application of foresight, this measure required participants to identify a problem, self-generate a resolution, and execute the appropriate future-directed intention. RESULTS: Relative to controls, people with schizophrenia were less likely to spontaneously acquire items that would later allow a problem to be solved, and were also less likely to subsequently use these items to solve the problems. There was no interaction between group and task, indicating that these two components of foresight were disrupted to an equivalent degree. In the clinical (but not the control) group, item acquisition and item use were correlated with general cognitive capacity. No significant associations with clinical variables emerged. CONCLUSION: The capacity to apply episodic foresight in a functionally adaptive way is disrupted in schizophrenia and may at least partially reflect broader cognitive dysfunction. Future work is now needed to clarify the implications of these difficulties in everyday life, as well as how these difficulties might be remediated. PRACTITIONER POINTS: People with schizophrenia have known difficulties with episodic foresight, and it now appears that those difficulties extend to the performance of foresightful preparatory behaviours. Because preparatory behaviours are central to routine and adaptive planning, difficulties with episodic foresight may contribute to or be a result of some of the functional difficulties experienced by people with schizophrenia. Further research is needed to determine whether interventions might be developed for people with reduced episodic foresight. Interventions may include the use of remedial tools that support and encourage the performance of foresightful behaviour, or cognitive training programs that actively improve the ability and propensity to exercise foresight independently.

Characterization of major histocompatibility complex class I loci of the lark sparrow (Chondestes grammacus) and insights into avian MHC evolution.

The major histocompatibilty complex (MHC) has become increasingly important in the study of the immunocapabilities of non-model vertebrates due to its direct involvement in the immune response. The characterization of MHC class I loci in the lark sparrow (Chondestes grammacus) revealed multiple MHC class I loci with elevated genetic diversity at exon 3, evidence of differential selection between the peptide binding region (PBR) and non-PBR, and the presence of multiple pseudogenes with limited divergence. The minimum number of functional MHC class I loci was estimated at four. Sequence analysis revealed d N /d S ratios significantly less than one at non-PBR sites, indicative of negative selection, whereas PBR sites associated with antigen recognition showed ratios greater than 1 but non-significant. GenBank surveys and phylogenetic analyses of previously reported avian MHC class I sequences revealed variable signatures of evolutionary processes acting upon this gene family, including gene duplication and potential concerted evolution. An increase in the number of class I loci across species coincided with an increase in pseudogene prevalence, revealing the importance of gene duplication in the expansion of multigene families and the creation of pseudogenes.

Distribution, prevalence and host specificity of avian malaria parasites across the breeding range of the migratory lark sparrow (Chondestes grammacus).

The lark sparrow (Chondestes grammacus) is a ground-nesting passerine that breeds across much of the central North American steppe and sand barrens. Through genotyping and sequencing of avian malaria parasites we examined levels of malaria prevalence and determined the distribution of Haemoproteus and Plasmodium lineages across the breeding range of the lark sparrow. Analysis of 365 birds collected from five breeding locations revealed relatively high levels of malaria prevalence in adults (80 %) and juveniles (46 %), with infections being primarily of Haemoproteus (91 % of sequenced samples). Levels of genetic diversity and genetic structure of malaria parasites with respect to the avian host populations revealed distinct patterns for Haemoproteus and Plasmodium, most likely as a result of their distinct life histories, host specificity, and transmission vectors. With the exception of one common Haemoproteus haplotype detected in all populations, all other haplotypes were either population-specific or shared by two to three populations. A hierarchical analysis of molecular variance of Haemoproteus sequences revealed that 15-18 % of the genetic variation can be explained by differences among host populations/locations (p < 0.001). In contrast to the regional patterns of genetic differentiation detected for the lark sparrow populations, Haemoproteus parasites showed high levels of population-specific variation and no significant differences among regions, which suggests that the population dynamics of the parasites may be driven by evolutionary processes operating at small spatial scales (e.g., at the level of host populations). These results highlight the potential effects of host population structure on the demographic and evolutionary dynamics of parasites.

In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

Recovery efficiency of two glove-sampling methods.

Two methods to sample pathogens from gloved hands were compared: direct imprint onto agar and a sponge-wipe method. The sponge method was significantly better at recovering Clostridiodes difficile spores, and no difference was observed between the methods at 101 inoculum for carbapenemase-producing KPC+ Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Acinetobacter baumannii.

Factors influencing environmental sampling recovery of healthcare pathogens from non-porous surfaces with cellulose sponges.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield's buffer or ATS, deposited on 323cm2 steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from <1% (KPC) to almost 60% (CD) and was more dependent upon the organism's characteristics and presence of ATS than on surface type. KPC persistence as determined by culture also declined by >1 log10 within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield's buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

Sampling efficiency of Candida auris from healthcare surfaces: culture and nonculture detection methods.

Sponges and swabs were evaluated for their ability to recover Candida auris dried 1 hour on steel and plastic surfaces. Culture recovery ranged from <0.1% (sponges) to 8.4% (swabs), and cells detected with an esterase activity assay revealed >50% recovery (swabs), indicating that cells may enter a viable but nonculturable state.

Descriptive evaluation of antibody responses to severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection in plasma and gingival crevicular fluid in a nursing home cohort-Arkansas, June-August 2020.

OBJECTIVE: To characterize and compare severe acute respiratory coronavirus virus 2 (SARS-CoV-2)-specific immune responses in plasma and gingival crevicular fluid (GCF) from nursing home residents during and after natural infection. DESIGN: Prospective cohort. SETTING: Nursing home. PARTICIPANTS: SARS-CoV-2-infected nursing home residents. METHODS: A convenience sample of 14 SARS-CoV-2-infected nursing home residents, enrolled 4-13 days after real-time reverse transcription polymerase chain reaction diagnosis, were followed for 42 days. After diagnosis, plasma SARS-CoV-2-specific pan-Immunoglobulin (Ig), IgG, IgA, IgM, and neutralizing antibodies were measured at 5 time points, and GCF SARS-CoV-2-specific IgG and IgA were measured at 4 time points. RESULTS: All participants demonstrated immune responses to SARS-CoV-2 infection. Among 12 phlebotomized participants, plasma was positive for pan-Ig and IgG in all 12 participants. Neutralizing antibodies were positive in 11 participants; IgM was positive in 10 participants, and IgA was positive in 9 participants. Among 14 participants with GCF specimens, GCF was positive for IgG in 13 participants and for IgA in 12 participants. Immunoglobulin responses in plasma and GCF had similar kinetics; median times to peak antibody response were similar across specimen types (4 weeks for IgG; 3 weeks for IgA). Participants with pan-Ig, IgG, and IgA detected in plasma and GCF IgG remained positive throughout this evaluation, 46-55 days after diagnosis. All participants were viral-culture negative by the first detection of antibodies. CONCLUSIONS: Nursing home residents had detectable SARS-CoV-2 antibodies in plasma and GCF after infection. Kinetics of antibodies detected in GCF mirrored those from plasma. Noninvasive GCF may be useful for detecting and monitoring immunologic responses in populations unable or unwilling to be phlebotomized.

Optimisation of pharmacokinetic properties to afford an orally bioavailable and selective V1A receptor antagonist.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.

Structure based evolution of a novel series of positive modulators of the AMPA receptor.

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.