Background: Glucagon-like peptide-1 (GLP-1) is an incretin hormone that plays an important role in glucose homeostasis and food intake. In people, GLP-1 receptor agonists (GLP-1RAs) are commonly used for the treatment of type 2 diabetes mellitus (DM) and obesity; however, non-adherence to injectable medications is common. OKV-119 is an investigational drug delivery system intended for subdermal implantation and delivery of the GLP-1RA exenatide for up to 6 months.Hypothesis/Objectives: Develop protocols for the subcutaneous (SC) insertion and removal of OKV-119 and to evaluate its tolerability, in vivo drug-releasing characteristics, and weight-loss effects in cats.Animals: Two cadaveric and 19 purpose-bred cats.Methods: In cadavers, OKV-119 insertion protocol and imaging were performed at three SC locations. The safety and tolerability of OKV-119 implants were assessed in a small (n = 4 cats) 62-day study. Weekly plasma exenatide concentrations and body weight were measured in a 42-day proof-of-concept study designed to evaluate OKV-119 prototypes implanted in cats (n = 15).Results: In anesthetized cats, the duration of insertion and removal procedures was 1–2 min. OKV-119 was easily identified on radiographs, and well-tolerated without any apparent implant site reactions. Following implantation, exanatide plasma concentrations were observed for up to 35 days. Plasma exenatide concentrations were correlated to weight loss.Conclusion and clinical importance: Our findings suggest that OKV-119 could be easily inserted and removed during a routine clinic visit and can be used to safely and effectively deliver exenatide. Future studies of OKV-119, configured to release exenatide for a longer extended months-long duration, are warranted to determine whether the combination of metabolic improvements and beneficial weight-loss, coupled with minimal impact on pet-owner's lifestyle, lead to improved outcomes for obese cats and feline DM patients. Glucagon-like peptide-1 (GLP-1), an important incretin hormone that is secreted from the intestines during a meal and participates in the regulation of systemic metabolism, food intake, gastrointestinal motility, and more , with exAlthough limited, data in cats demonstrate that GLP-1RAs generally have similar effects to those seen in human patients and might be useful in treating feline DM –16. FeliIn human medicine, non-adherence to antidiabetic medications is common and results in poor long-term glycemic control and the development of diabetic complications –23. MediLong-term extended-release (ER) technologies that minimize injection burden have been shown to be well-tolerated and improve self-reported quality of life in human diabetic patients , 29. Givin vivo drug releasing properties of OKV-119 prototypes over a 42-day period. Therefore, weekly exenatide plasma concentrations and corresponding weight-loss effects were measured in healthy cats implanted with OKV-119 prototype systems.Herein, we report our initial experiences with an investigational drug delivery system OKV-119;  that wilThe overall design and primary working principle of the OKV-119 systems used herein were based on the prototype depicted previously , 34. MadThe rate of exenatide released from the OKV-119 implant is controlled by adjusting pore size and the exposed number of nanotubes  33, 34), 3433, 3An initial study was performed in two cat cadavers to develop insertion and removal techniques of OKV-119 and to evaluate radiopacity and echogenicity of the implant following implantation . OKV-119 implants were placed in three subcutaneous locations in each cat: the medial brachium, dorsal lumbar, and lateral crus. Imaging was performed using digital radiography and ultrasound of each location to determine the most suitable modality for locating the implant before removal in live animals. Implantation sites were compared for ease of insertion and removal in this species.n = 4) were anesthetized with 2 mg/Kg xylazine (20 mg/mL) and 1 mg/Kg ketamine (100 mg/mL) followed by administration of inhaled 4% isoflurane. A 4 × 4 cm area was clipped and aseptically prepared on the dorsal lumbar area. A 12 g needle was loaded with OKV-119 through its beveled tip until it was entirely within its lumen. A K-wire was then inserted into the opposite side of the needle while tilting the needle upwards and avoiding expulsion of the implant through the beveled tip of the needle. Next, the skin was incised (1 mm) with a scalpel. The skin was held with forceps close to the incision and the 12 g needle (and the and K-wire in it) was inserted about 3 cm into the subcutaneous tissue through the incision. The needle was then retracted over the K-wire while stabilizing the K-wire, causing the OKV-119 implant to be pushed out of the needle and into the space that was created by the retracted needle. The K-wire was removed, the skin edges were apposed, and the incision closed with a drop of tissue glue. Finally, the anesthesia was discontinued and the cat was monitored until fully awake.In a subsequent safety and tolerability study, cats (n = 15) were anesthetized and the implant site was prepared as described above. The skin was incised (2–3 mm) with a scalpel. The scalpel was then used to gently create a small subcutaneous cavity. The OKV-119 implant was inserted using Brown-Adson tissue forceps to hold the implant at its center. After insertion, the skin edges were apposed, and the incision closed with a drop of tissue glue.Lastly, in a OKV-119 proof-of-concept study, cats .n = 2) and dorsal lumbar (n = 2).The safety and tolerability of OKV-119 was initially assessed over an 8-week period. In this part of the study, prototype OKV-119 implants configured to release supratherapeutic exenatide doses were inserted subcutaneously in healthy, purpose-bred, domestic short hair cats in the lateral crus .Cats were considered healthy at time of enrollment based on physical examination. They were acclimated for 7 days prior to treatment in the room where they were to be housed during the study. During the course of the study, cats were housed separately in pens in compliance with current recommendations for the Guide for the Care and Use of Laboratory Animals and under the standard operating procedures of the testing facility. Cats were fed a standardized pelleted feline diet A 6-week study, in which weekly exenatide plasma concentrations were measured, was performed to evaluate OKV-119 implants configured to release exenatide at three different rates. Cats in Group 1 were implanted with OKV-119 configured with the largest nanopores , while the implants used for Group 3 were configured using the thickest ALD profile . The selection of these prototype OKV-119 configurations was informed by prior studies performed in rats (unpublished data).n = 3), Group 2 (n = 6), or Group 3 (n = 6). Cats were individually housed and monitored throughout the study. Body weight and exenatide plasma measurements were collected weekly. Implants were removed from the cats on a pre-determined weekly schedule  and then weekly for the duration of each study. In the proof-of-concept study, all samples were collected after an overnight fast.Blood samples were collected from the jugular vein and placed in pre-chilled EDTA tubes that were immediately placed on ice. Samples were then centrifuged , plasma was collected, divided into 4 aliquots and stored at −80°C until analysis.R2 = 0.997 (P < 0.0001), slope = 1.0 ± 0.02 (CI = 0.96–1.07) and a Y intercept = −0.73 ± 0.91. Recovery of quality control material was 106.6% at 3 ng/mL and 103.0% at 75 ng/mL. When plasma exenatide concentrations were below the level of detection (0.5 ng/mL), a value of 0 ng/mL was assigned.A qualified LC-MS/MS method for the determination of exenatide concentrations in feline plasma was developed at an independent laboratoryp ≤ 0.05.Statistical analysis was performed using a commercially available computer softwareAfter insertion in the SC space, the implant is palpable. To prepare for the possibility of migration over time and the need to locate the implant anywhere in the body, radiographs were obtained after inserting implants at different locations in 2 cat cadavers. The implant is radiopaque and was easily identified on radiographs at all insertion sites including when overlaid by bone . The echIn anesthetized cats, the duration of insertion and removal procedures was 1–2 min.Cats appeared to tolerate the implants well. Cats did not lick or scratch the implant site and no visible evidence of inflammation was observed in the skin overlying the implant . There wIn all cats studied, plasma exenatide concentrations were below the level of detection at baseline. In the safety and tolerability study, there was no difference in body weight between day −7 and day 0 (median [range] difference = 35 g [−55 to +107 g]). Following insertion of OKV-119 systems configured to release supratherapeutic doses of exenatide, mean exenatide plasma concentrations peaked at week 1 (median [range] = 9.3 ng/mL [4.7–10.5]), and were maintained above 2 ng/mL in all cats for 28 days . Weight In the subsequent OKV-119 drug-releasing concept study, average Day 7 plasma exenatide levels were 2.45 ng/ml (range: 1.65–3.02 ng/ml) and 2.65 ng/ml (range: 0.33–4.33 ng/ml) in Groups 1 and 2, respectively . In Group < 0.0001) and the larger study evaluating drug release of OKV-119 prototypes . Consistent weight loss was observed in all three groups  that is under development for the treatment of feline obesity and diabetes mellitus. We present our initial experiences with the implant insertion and removal procedures, as well as preliminary tolerability and safety observations. The proof-of-concept study demonstrates that exenatide was released for a 35-day period and suggests that the OKV-119 drug delivery system is tunable and can be configured for cats. Additional study of OKV-119 implants will be needed to verify that constant target drug delivery rates can be maintained for longer, months long duration.The subcutaneous insertion and removal of the implant in cats was found to be quick and simple, suggesting that these procedures may be performed during routine clinic visits under short anesthesia (for example with a dexmedetomidine injection that is then reversed with atipamezole). No implant-site AEs were observed, but larger and longer studies will be needed to verify that the OKV-119 implant is biocompatible and will be well-tolerated by cats over months and years. In particular, although feline injection site sarcomas are believed to be exceedingly rare and have not been linked to daily insulin injections or to microchip implants –37, thisFeline obesity is a significant health concern with current weight management strategies largely limited to restricting caloric intake , 39; howStudies show that overweight people administered exenatide generally experience clinically beneficial weight loss within the first weeks of treatment, with body weight stabilizing after a few months, and then remaining relatively unchanged over years of treatment , 43, 44.Following extended exposure to exenatide, anti-exenatide antibody formation has been detected in rats, monkeys, and humans. The presence of antibodies does not appear to have neutralizing activity in these species, although antibody titers were observed to influence pharmacokinetic parameters [NDA #22-200]. It is suspected that higher anti-drug antibody (ADA) titers may lead to an apparent increase in exposure because of the perturbation of the renal clearance of exenatide in the presence of ADA [NDA #22-200]. In future studies, anti-exenatide antibody titers will need to be measured to determine whether they have neutralizing effects in felines and/or whether they affect drug exposure.In cats, anorexia and negative energy balance that results in excessive weight loss are clinical hallmarks of hepatic lipidosis (HL); however, the pathophysiology is incompletely understood and it remains unknown why some cats develop HL while other cats with a similar negative energy balance do not –48. ExpeFollowing the classification schemes used in human medicine, diabetic cats generally suffer from a type 2-like diabetes mellitus that may be phenotypically characterized by a combination of impaired insulin action in liver, muscle, and adipose tissue (insulin resistance), and beta-cell failure . Given tHuman clinical studies performed under real world conditions have identified lack of adherence as a significant factor associated with poorer glycemic control and other diabetes associated complications , 54–59. Injection concerns  are believed to negatively impact pet-owner adherence rates and may partly account for the 10% euthanasia rate reported for newly diagnosed diabetic pets . Treatmein vivo exenatide plasma concentrations relative to OKV-119 configurations . The implant insertion and removal procedures also need to be refined before the protocols can be incorporated into routine clinic visits. Ultimately, clinical studies in client-owned obese and diabetic feline patients will be needed to evaluate both the clinical and logistical benefits of the “hassle-free” long-term delivery of exenatide.To address real-world adherence and persistence challenges, OKV-119 delivery systems designed to provide continuous subcutaneous delivery of exenatide for a months-long duration are under development. Pharmacokinetic studies, in cats, using future OKV-119 prototype systems will be needed to better target The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by University of Florida Institutional Animal Care and Use Committee and by the AAALAC accredited and USDA registered testing facility Institutional Animal Care and Use Committee (IACUC).MK, WA, and CG contributed to conception and design of the study. CA contributed to the development of the insertion and removal procedures. All authors contributed to manuscript revision, read, and approved the submitted version.MK and WA are Okava Pharmaceuticals shareholders. CG is a consultant to Okava Pharmaceuticals. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The COVID-19 lockdown has been associated with physical inactivity. We prospectively evaluated changes in moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) among 1565 cardiovascular disease (CVD) patients using validated questionnaires at 5 weeks after lockdown initiation  and at every 4 subsequent weeks, until July 2020. Multivariate mixed model analyses were performed to identify whether age, sex, CVD-subtype, lockdown adherence and mental health factors impacted changes in physical (in)activity. Patients were 67 (interquartile range: 60–73) years and primarily diagnosed with coronary artery disease. Time spent in MVPA was 143 min/day  137; 148) at baseline. Female sex, heart-failure, fear of COVID-19 infection and limited possibilities for physical activity were independently associated with lower levels of MVPA across time. After adjusting for confounders, overall MVPA did not change. ST was 567  min/day at baseline. Lack of social contact, limited possibilities for physical activity and younger age were independently associated with higher levels of ST. After adjusting for confounders, ST progressively increased following 8 ) and 12 weeks  min/day) of lockdown. Despite a phased relaxation of the lockdown, CVD patients progressively increased ST and reported no change in MVPA. This highlights the need to target physical inactivity during and beyond the COVID-19 pandemic. To limit the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), lockdown restrictions have been instituted worldwide. These measures have had a major impact on everyday life activities, including physical activity and sedentary behaviour in the home environment, in relation to commuting and during leisure time activities ,2. The mCardiovascular disease (CVD) patients are not only at increased risk for severe COVID-19 complications , but givIn this study, we prospectively evaluated changes in physical activity and sedentary behaviour among chronic CVD patients during the first-wave COVID-19 lockdown in the Netherlands and aimed to identify patient and disease characteristics associated with physical inactivity. Given the gradual relaxation of restrictions across the study period (April–July 2020), but also due to seasonal changes in physical activity patterns, we hypothesized an increase in physical activity levels and attenuation of sedentary behaviour in CVD patients during the first-wave COVID-19 lockdown in the Netherlands.n = 2178) [We invited CVD patients who were included in an ongoing multicenter study ( = 2178)  via e-maPhysical activity and sedentary behaviour were assessed using online questionnaires at four different timepoints. Baseline data were collected between 17 and 24 April 2020 (Q1), i.e., 36 days after initiation of the COVID-19 lockdown in the Netherlands. Data on follow-up measurements were collected every subsequent four weeks, i.e., 15–22 May 2020 (Q2); 12–19 June 2020 (Q3) and 10–17 July 2020 (Q4). Dutch lockdown restrictions varied within the study period and for each timepoint of data collection, the applicable measures are summarized in Daily amount of moderate-to-vigorous intensity physical activity (MVPA) was assessed by the Short Questionnaire to Assess Health-Enhancing Physical Activity (SQUASH) . We calcSedentary behaviour was assessed using the Sedentary Behaviour Questionnaire (SBQ) , determiWe determined age, sex and CVD subtype at baseline. Adherence to the lockdown restrictions was assessed by asking to what extent patients adhered to the applicable COVID-19 lockdown restrictions at the four different timepoints. We used a 10-point Likert scale with 0 representing no adherence and 10 representing complete adherence. Additionally, we repeatedly evaluated the extent to which patients were impeded in their everyday life by a lack of social contact; fear of COVID-19 infection; possibilities for physical activity and stress due to financial consequences of the COVID-19 pandemic. The extent of experiencing these limitations was categorized into low, moderate or high. p < 0.05. Analyses were performed with IBM SPSS Statistics-25 . Original individual data are available upon request.Data were reported as number (%) for categorical variables, mean (±standard deviation) for normally distributed continuous variables and median (interquartile range) for non-normally distributed continuous variables. Changes in physical activity and time spent sedentary were analyzed with mixed models analyses using random intercepts. Time was described as categorical variable for baseline (Q1), follow-up after 4 weeks (Q2), after 8 weeks (Q3) and after 12 weeks (Q4). Furthermore, multivariate mixed model analyses were performed using forward stepwise selection to identify whether age, sex, CVD subtype, lockdown adherence and mental health factors impacted changes in physical activity and sedentary behaviour over time. This approach allowed us to take the variation within sub-populations into account and to correct for these confounding factors in the adjusted model. All statistical tests were two-sided and significance was set at In total, 1565 patients participated in this longitudinal cohort study (response-rate: 72%). At baseline and 4, 8 and 12 weeks of follow-up, questionnaires regarding physical activity were completed by 1565; 1004; 919 and 871 CVD patients, and for sedentary behaviour by 1490; 1000; 918 and 870 CVD patients, respectively. n = 609, data not shown). Adherence to lockdown restrictions was very high (9 (8–10)) at baseline, but gradually decreased across the various phases of COVID-19 lockdown  −0.8; −0.6) after 12 weeks). The extent of being impeded due to a lack of social contact was high in the majority of our patients (60%). To a lesser extent, they were impeded by fear of a COVID-19 infection  and limited possibilities for physical activity . A minority was highly limited because of stress due to financial consequences  years old, mostly male (73%) and diagnosed with myocardial infarction (48%), angina pectoris (18%), heart valve disease (9%), heart failure (8%) or other CVD 17%; . These cDaily time spent in MVPA was 143 min  at baseline (Q1), with a minor increase  min/day) at 8 weeks (Q3), but not at 4 (Q2) or 12 weeks (Q4) A. ChangeMost of the time in MVPA was spent during leisure time activities, with 152  min/day in the adjusted model at baseline . No signp = 0.049) after 12 weeks of COVID-19 lockdown (Q4)  min/day at baseline and progressively increased , own (Q4) B. Changeown (Q4) . Adjusteown (Q4) D. There Most of the time spent sedentary was work-related, with 405  min/day in the adjusted model at baseline, which did not change during follow-up . The incThe purpose of this study was to assess changes in physical activity and sedentary behaviour in CVD patients across the various phases of COVID-19 lockdown in the Netherlands. During 12 weeks of follow-up, COVID-19 lockdown restrictions were gradually lifted. Nonetheless, physical activity levels did not significantly change, while time spent sedentary progressively increased. Factors that limited engagement in physical activity were female sex, heart failure, fear of a COVID-19 infection and limited possibilities for physical activity. Fewer possibilities for physical activity, but also relatively younger age and lack of social contact, provoked sedentary behaviour. The lack of improvement in physical activity levels and further increase in sedentary behaviour, despite easing COVID-19 lockdown restrictions, is worrisome and could lead to detrimental health effects in the mid- to long-term ,11.To the best of our knowledge, this is the first prospective cohort study that assessed longitudinal changes in physical activity and sedentary time during the COVID-19 pandemic in chronic CVD patients. We found that daily time spent in MVPA did not change during follow-up, despite partial lifting of lockdown restrictions. These observations are contradictory to a previous study in the general population, showing that smartphone-based daily step counts gradually increased with prolongation of the lockdown and/or relaxing restrictions . There aAfter 12 weeks of lockdown, sedentary time increased by 25 min/day, adjusted for age and changes in the extent of being impeded by lack of social contact and limited possibilities for physical activity. This substantial increase in sedentary behaviour was primarily due to an accumulation of time spent sitting during leisure time activities, specifically during reading, eating and drinking. These observations have important implications, since a large majority of CVD patients were already highly sedentary before the lockdown ,13,22. WThe pathways that underlie these sitting-induced health risks are still not fully understood, but probably act through multiple biological systems including vascular function, blood pressure, carbohydrate metabolism and cerebral blood flow . This coThe impact of the COVID-19 lockdown on physical activity and sedentary behaviour in our study were, at least partly, influenced by patient- and disease characteristics, but not by lockdown adherence or stress due to the financial consequences of the COVID-19 pandemic. The type of CVD diagnosis, sex and the extent of being impeded in daily life by limited possibilities for physical activity and fear of a COVID-19 infection impacted physical activity across time. Specifically, MVPA levels were lower in patients with heart failure compared to the other CVD groups during the COVID-19 lockdown. This is in line with previous research that described lower habitual physical activity levels in heart failure patients , with a A large amount of chronic CVD patients reported that they were impeded in their everyday life by a lack of social contact, fear of a COVID-19 infection and limited possibilities for physical activity. With the reopening of sports clubs, patients again had full access to sports facilities and therefore the opportunity to re-engage in (supervised) physical activity. However, many patients still experienced limited possibilities for physical activity, presumably due to a persistently high level of fear of a COVID-19 infection in this vulnerable population. Possibly, loosening lockdown restrictions actually increased the risk perception in patients with regard to a COVID-19 infection. Last year, Lippi and colleagues  already Certain limitations apply to our study, including its observational design, which cannot confirm causality, but only describes statically significant changes in physical activity patterns during different phases of the COVID-19 lockdown and independent associations between observed changes and patient characteristics. Unfortunately, data on follow-up measures were not completed in all participants. We limited the impact of missing data due to use of mixed model analyses, but acknowledge that selection bias could not be completely ruled out. Additionally, we specifically focused on the Dutch situation and our findings may not be directly extrapolated to other countries with different types of lockdowns or with cultural differences. Furthermore, self-reported physical activity seems relatively high in our cohort, but this is mainly driven by time spent doing odd jobs and walking, while time spent during exercise was only minor. The use of subjective measurements could have led to over- and underestimation of physical activity and sedentary behaviour, respectively. Nevertheless, we performed longitudinal measurements and since the used questionnaires have good reproducibility , our stuDespite the gradual relaxation of lockdown restrictions across the study period, physical activity levels did not improve, while sedentary time progressively increased in chronic CVD patients during 12 weeks of COVID-19 lockdown. Female sex, relatively younger age, heart failure diagnosis and an increasing extent of being impeded by limited possibilities for physical activity and by a lack of social contact were associated with physical inactivity. Since deterioration in physical activity patterns is strongly associated with medium to long-term detrimental health effects, targeting physical inactivity should be a top priority in order to limit the collateral damage of COVID-19 lockdown restrictions. Our findings indicate that simply lifting lockdown measures does not automatically recover physical activity patterns in CVD patients, highlighting the need to (1) frequently monitor activity levels, (2) raise awareness concerning the health risks of a physically inactive lifestyle and (3) to develop  solutions to effectively promote physical activity in these patients.


Emergency Departments (EDs) can serve as surveillance sites for infectious diseases. Our purpose was to determine the burden of SARS-CoV-2 infection and prevalence of vaccination against COVID-19 among patients attending an urban ED in Baltimore City.Using 1914 samples of known exposure status, we developed an algorithm to differentiate previously infected, vaccinated, and unexposed individuals using a combination of antibody assays. We applied this testing algorithm to 4360 samples ED patients obtained in the springs of 2020 and 2021. Using multinomial logistic regression, we determined factors associated with infection and vaccination.For the algorithm, sensitivity and specificity for identifying vaccinated individuals was 100% and 99%, respectively, and 84% and 100% for naturally infected individuals. Among the ED subjects, seroprevalence to SARS-CoV-2 increased from 2% to 24% between April 2020 and March 2021. Vaccination prevalence rose to 11% by mid-March 2021. Marked differences in burden of disease and vaccination coverage were seen by sex, race, and ethnicity. Hispanic patients, though 7% of the study population, had the highest relative burden of disease  but similar vaccination rates. Women and White individuals were more likely to be vaccinated than men or Black individuals .Individuals previously infected with SARS-CoV-2 can be differentiated from vaccinated individuals using a serologic testing algorithm. SARS-CoV-2 exposure and vaccination uptake frequencies reflect gender, race and ethnic health disparities in this urban context. Using an antibody testing algorithm, we distinguished between immune responses from SARS-CoV-2-infected and vaccinated individuals. When applied to blood samples from an emergency department in Baltimore, disparities in disease burden and vaccine uptake by sex, race, and ethnicity were identified. The United States has recorded more than 700,000 deaths and documented infections in over 10% of the population. Within the U.S., Black and Latino individuals have experienced higher rates of infection and mortality, relative to White individuals, since the onset of the pandemic3. Rooted in long-standing racial and structural injustice, these disparate health outcomes reflect the disproportionate effects of social determinants of health among U.S. racial and ethnic minority groups5.As of October 2021, over 238 million cases of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), have been reported globally6. The authorized vaccines from Pfizer, Moderna, and Johnson & Johnson, each elicit an immune response against the spike protein of the SARS-CoV-2 virion9. As of the 20th of September 2021, 74.6% of persons aged ≥ 12 years have received at least one dose of a COVID-19 vaccine in the United States10. This uptake, however, has varied dramatically by race, socioeconomic status, and geographic location. Several studies have described the potential for vaccine hesitancy among Black and Hispanic Americans13. These issues related vaccine hesitancy and access could result in disparate rates of vaccine uptake among racial and ethnic minority groups.Currently, three vaccines for COVID-19 have been authorization by the U.S. Food and Drug Administration14. By comparing the results of serologic assays that either detect antibodies to spike (S1), the spike glycoprotein receptor binding domain (RBD), or the nucleocapsid (N), it should be possible to distinguish between SARS-CoV-2 naturally infected , vaccinated (with no evidence of prior infection), and uninfected individuals.In contrast to vaccinated individuals, naturally infected patients create antibodies to several parts of the virus, including the spike and nucleocapsid proteins17. Although case-reporting can provide an estimate of population-level seroprevalence, relying on case-reporting alone may underestimate the burden of infection, emphasizing the need for accurate serologic assessment of seroprevalence18.By providing data on symptomatic infection rates, emergency departments (EDs) have historically played a critical role in prior epidemics and pandemics and thus present a rich opportunity for conducting SARS-CoV-2 serosurveillanceThis study used samples from parent studies approved by The Johns Hopkins University School of Medicine Institutional Review Board . The Moderna vaccine trial was part of the Division of Microbiology and Infectious Diseases Protocol Number: 20–0003. For those studies, all individuals provided written informed consent. The JHU School of Medicine Institutional Review Board  approved the de-identified serosurvey performed on waste material. All studies were conducted according to the ethics standards of the Helsinki Declaration of the World Medical Association.8 (n=68) and vaccinated health care professionals 21. The 494 samples from individuals known to have been infected by SARS-CoV-2 were drawn from three cohorts: convalescent plasma donors 22; and Clinical Characterization Protocol for Severe Infectious Diseases 23, and HCP (n=4)24. All samples were from individuals with a known positive SARS-CoV-2 RT-PCR test result. The majority of the CCP donors had mild disease, with 9% of this cohort reporting hospitalization. Among the CCPSEI, 14% received oxygen therapy, 33% received ventilation, and 13% died. All HCP had mild disease. Additionally, there were 46 HCP who were infected and subsequently vaccinated, 28 with confirmed a PCR date and 18 who were suspected as having prior infection . Specificity of the testing algorithm was then assessed using 992 samples from pre-pandemic remnant CBC samples collected from Johns Hopkins Hospital Emergency Department (JHH ED) patients collected between December 2015 and January 201625.Three sample sets with known previous infection and vaccination to SAR-CoV-2 were used to validate the antibody testing algorithm . Samples26, remnant CBC blood samples from ED patients aged >17 years were collected during the study period. Each sample was assigned a unique study code, processed, and stored at −80°C. Basic patient demographic characteristics  were abstracted from the ED administrative database, and all identifiers and protected health information removed from the dataset. Data regarding COVID-19 vaccination status was not available. Laboratory testing was then performed on stored specimens after delinking the demographic dataset. Using the unique study code, SARS-CoV-2 serostatus and demographic data were then merged.The testing algorithm was subsequently applied to two serosurveys conducted among patients attending the JHH ED from 16 March to 30 April 2020 and from 11 January to 10 March 2021. As previously described in an identity-unlinked seroprevalence studyThe testing algorithm required three serologic assays that could differentiate serologic reactivity to SARS-CoV-2 S1, RBD and nucleocapsid. These assays were limited to standard ELISA and point of care assays, as we did not have access to chemiluminescent detection equipment. We selected ELISA based technologies for the initial high throughput screening, thought confirmation testing could include point of care assays. Additional information on the assays used is available in 28, ease-of-use characteristics  and availability. The Euroimmun ELISA measures IgG responses to the S1 protein of SARS-CoV-2, whereas the Bio-Rad ELISA measures total antibodies to nucleocapsid. Both ELISA assays generate a ratio of the optical density of the sample divided by the control . For the Euroimmun and Bio-Rad ELISAs, an S/C ≥ 0.8 was considered a positive result. The CoronaCHEK™ lateral flow assay (LFA) tests for the presence of both IgM and IgG antibodies to the receptor-binding domain (RBD) of the spike protein. Any visual band was considered a positive result. Each assay was performed according to the manufacturer’s instructions.Plasma and serum samples were analyzed using three commercially available serologic assays: the Euroimmun Anti-SARS-CoV-2 ELISA , the CoronaCHEK™ COVID-19 IgG/IgM Rapid Test Cassette (Hangzhou Biotest Biotech Co Ltd), and the Bio-Rad Platelia SARS-CoV-2 Total Antibody ELISA . Each assay was selected for previously determined performanceAn algorithm composed of the Euroimmun, Bio-Rad, and CoronaCHEK assays was used to differentiate samples into three groups: naturally infected (who may or may not subsequently be vaccinated); vaccinated (who were never infected); and unexposed . All samTo evaluate the diagnostic accuracy of the testing algorithm for a particular state , the sensitivity for each state was determined from samples with that known status , unvaccinated individuals with PCR confirmed natural infection (n=494), and those seen in the ED pre-pandemic (n=992) were tested on all three assays . The EurThere were significant differences between SARS-CoV-2 serostatus and the level of antibody reactivity to spike and nucleocapsid among the cohorts used for algorithm validation . For vacUsing the testing algorithm, 1536 JHH ED 2020 samples and 2824 JHH ED 2021 samples were evaluated. During the two collection periods, combined seroprevalence of antibodies to SARS-CoV-2 from 1.6%  to 23.8%  . During The prevalence of antibodies to SARS-CoV-2 indicating previous infection or vaccination is presented in In the 2021 survey, there were no statistically different rates of infection between age groups . In contWe then disaggregated the data by sex, race and ethnicity. In terms of natural infection , individThis study describes a method for distinguishing between SARS-CoV-2 vaccinated, naturally infected, and uninfected individuals using commercially available serologic assays when no previous vaccination or infection history is available. The algorithm utilized in this study indicates a 10-fold increase in seropositivity to SARS-CoV-2 infection in the Baltimore metropolitan area from April 2020 to March 2021. Furthermore, this study highlights disparities based on sex and race/ethnicity in SARS-CoV-2 prevalence and vaccine distribution within metropolitan Baltimore during the spring of 2021.29, in the ability to differentiate vaccinated from infected individuals based on antibody responses to the S1 and N proteins of SARS-CoV-2. In contrast to the Suhandynata et al. study which utilized chemiluminescent assays (Roche Elecsys Anti-SARS-CoV-2 S- and N-antibody)29, we applied more commonly available ELISA and LFA methods in our study. We further expanded their work by incorporating larger validation cohorts and applying the algorithm to population level surveillance. Our study also confirms previous reports of high burden of COVID-19 among the Baltimore Hispanic population30. Additionally, the discrepancies in vaccine uptake among racial and ethnic minority groups are clearly demonstrated. While recent data suggest that racial and ethnic gaps in vaccination have narrowed10, our data from early 2021 suggests that disparities in vaccination were present in the initial stages of the vaccine rollout. Surprisingly, despite prioritizing older Americans during the vaccine rollout31, patients older than 60 were as likely to be vaccinated as those between the ages of 18–44.Our study follows the work of Suhandynata et al.32. In contrast, the half-life of anti-spike IgG antibodies could not be measured, as 94% of healthcare workers did not exhibit significant loss during follow-up32. Additionally, anti-nucleocapsid antibody decline was more rapid in younger patients and those with milder symptoms. Thus, in the proposed algorithm, a proportion of naturally infected individuals will be misclassified as vaccinated as anti-nucleocapsid antibodies wane with time. This effect will be differential by age and initial symptomology. The time to seroreversion of spike and nucleocapsid antibodies in SARS-CoV-2 infected patients is significantly affected by both disease severity and assay platform. The effect of these variations should be considered when interpreting the results of serosurveillance studies.Our method demonstrated 100% sensitivity in identifying individuals who were fully vaccinated with both the Pfizer and Moderna vaccines. Further studies are needed to determine if other authorized vaccines have similar performance characteristics. One critical potential limitation to the use of anti-spike and anti-nucleocapsid antibody testing to differentiate naturally infected from vaccinated individuals is differential loss of antibody reactivity to these two targets. In a cohort of 3276 UK healthcare workers, Lumley et al. estimated that anti-nucleocapsid IgG antibodies exhibit a half-life of 85 days from the maximum titer 35. Using our testing algorithm, 16% of individuals with a previous positive RT-PCR test were seronegative by our algorithm. Similarly, Self et al. found that in a convenience sample of 156 mildly infected frontline healthcare personnel, 93.6% experienced a decline in antibody response and 28.2% seroreverted within 60 days36. These studies illustrate the difficulty of identifying infected persons several months after infection, especially in cases of mild infection. It should be noted that antibody reactivity is also dependent on the assay used, especially at 6 months after SARS-CoV-2 infection37.Our study has several additional limitations. We did not test any individuals with known breakthrough infection (vaccinated then infected), nor could we distinguish between naturally infected individuals who were and were not subsequently vaccinated. Furthermore, a lack of seroreactivity occurred in a minority of naturally infected individuals. The lack of seroconversion in infected individuals has been observed in other studies, and occurs most frequently in individuals with asymptomatic infection 38, we demonstrated that a serosurvey can be performed to differentiate vaccinated, naturally infected and at-risk unexposed individuals in a population when vaccination or infection history is not available. This information provides evidence for targeted public health intervention in preparation for the continued spread of endemic SARS-CoV-2 infections.Although the correlates of antibody protection for naturally infected individuals have not been well-established1


Untreated patients had a lower risk of death . The risk of death was higher for older patients and those suffering from cardiac failure . Age, CLL-directed treatment, and cardiac failure were significant risk factors of OS. Untreated patients had a better chance of survival than those on treatment or recently treated.Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to Coronavirus disease 2019 (COVID-19) due to age, disease, and treatment-related immunosuppression. We aimed to assess risk factors of outcome and elucidate the impact of CLL-directed treatments on the course of COVID-19. We conducted a retrospective, international study, collectively including 941 patients with CLL and confirmed COVID-19. Data from the beginning of the pandemic until March 16, 2021, were collected from 91 centers. The risk factors of case fatality rate (CFR), disease severity, and overall survival (OS) were investigated. OS analysis was restricted to patients with severe COVID-19 . CFR in patients with severe COVID-19 was 38.4%. OS was inferior for patients in all treatment categories compared to untreated ( The Coronavirus disease 2019 (COVID-19) has cost the lives of more than 3 million people worldwide, since the first cases were reported in December 2019 in Wuhan, China [Patients with hematological malignancies often experience severe disease and have high case fatality (CFR) and mortality rates. A meta-analysis of 3240 adult patients with a hematological malignancy reported a risk of death of 34% . In a si+ lymphopenia, innate immune dysfunction, and neutropenia [Chronic lymphocytic leukemia (CLL) is the most frequent adult hematological malignancy in the West, affecting particularly the elderly, with a median age at diagnosis of 72 years [tropenia . Antileutropenia . CLL immtropenia .Early in the pandemic, reports from hospitalized patients showed that those with hematological malignancies were more susceptible to severe infection from SARS-CoV-2 and had higher CFR compared to the general population , 12. In Mato et al. reported the outcomes of 198 patients with COVID-19 and CLL mainly from the USA . The oveThe ERIC and US cohorts were also analyzed together with a Spanish cohort in a joint effort. The CFR was 30–34%. Age was the only risk factor of fatality in both cohorts, while the effect of CLL treatment on OS was inconsistent across cohorts. None of the CLL-directed treatments affected OS .As SARS-CoV-2 is still surging across the globe, we significantly expanded our retrospective international multicenter cohort of patients with CLL and COVID-19. We aimed at reassessing the risk factors for COVID-19-related fatality and elucidate further the impact of CLL-directed treatments.This is a retrospective international multicenter study of the ERIC and the Campus CLL. It represents a continuation of a previous study conducted during the first wave of the pandemic . InvestiNinety-one institutions participated in the study providing information for a total of 1009 cases. After excluding cases with uncertain diagnosis  and those without a qRT-PCR positive test for SARS-CoV-2, we resulted in 941 patients to be studied, 190 of whom were included in the first study. The full list of participating countries is reported in Supplemental Table TP53 gene mutation status assessed by Sanger sequencing or next-generation sequencing (at last assessment); CLL treatment status; presence, number, and type of comorbidities, date of COVID-19, COVID-19 symptoms, COVID-19 management, treatment, complications, and outcome.Data extracted from the medical records included: baseline demographics; date of CLL diagnosis; IGHV gene somatic hypermutation status; cytogenetic status for chromosomes 11q, 13q 17p, and 12 determined by fluorescence in situ hybridization (at last assessment); OS was defined as the time from suspected COVID-19 to death or last follow-up date.In keeping with international practice, patients were deemed to have COVID-19 if a qRT-PCR assay test from a throat or nose swab was positive for SARS-CoV-2.Severe COVID-19 was defined as hospitalization and need of oxygen or admission into an intensive care unit; nonsevere/mild COVID-19 was defined as confinement at home or hospitalization without need of oxygen.Patients diagnosed with COVID-19 from the start of the pandemic until June 30, 2020, were considered to belong to the 1st wave of SARS-CoV-2, while patients diagnosed from July 1st until the completion of data collection were designated to the 2nd wave. The cut-off was decided based on the disposition of cases in our cohort , COVID-19 disease severity (where appropriate), CLL treatment status (at the time of COVID-19 and in the last 12 months), type of CLL treatment (at the time of COVID-19 and in the last 12 months), and measures taken for the management of COVID-19 . Also, we compared CFR between the two waves. To this end, to reduce the impact of confounding factors, we firstly tested for differences of the above-mentioned risk factors between the two waves. The statistical analyses were carried out in either all patients or subsets of the dataset . In the analyses for CFR, patients who were still under medical care were excluded. The significance level was set to 5%. In post-hoc comparisons, the correction of Bonferroni was used.Median (IQR) is used to describe numeric variables while frequencies and percentages are used for categorical. Both univariate and multivariate analyses were carried out, having COVID-19 disease severity, CFR, or OS as outcomes. As potential risk factors, we examined clinico-biological characteristics  were used for univariate analyses, while logistic regression was used for the multivariate. When necessary, we performed bias reduction techniques to the logistic model estimates by adjusting Firth’s logistic regression. Furthermore, for the comparison of CFR between the two waves, age was used as a confounder, since statistically significant older patients were identified in the first wave. For the comparisons of the numeric risk factors between the two waves, independent samples t-tests (Student’s or Welch’s) were conducted. The homogeneity of variance between the two groups was examined through Levene’s test. For the categorical risk factors, χ2 test or Fisher’s exact test were used.For COVID-19 disease severity and CFR, For OS, the log-rank test was used for the univariate analyses, and Cox regression was conducted for the multivariate.p value.For the multivariate analyses, we performed a two-level variable selection approach. At first, we obtained the statistically significant risk factors through univariate analyses and used them as risk factors for a multivariate model. We further explored the multivariate model by performing backward elimination using All statistical analyses were conducted using R 4.0.4. We used brglm package  for FirtWe included 941 patients with a median age of 69 years (IQR 61–77) at the time of SARS-CoV-2 infection. Most patients were diagnosed with CLL , while 38 and 16 patients were diagnosed with SLL and MBL, respectively. The majority  were male with a median number of comorbidities of 2. Baseline patient characteristics are shown in Table Regarding CLL history, 394 patients (41.9%) had never received treatment for CLL, while 547 (58.1%) had been treated with at least one line of treatment. Amongst treated patients, 320 (34%) were on treatment at the time of SARS-CoV-2 infection. The most common treatment category at the time of COVID-19 was BTKi monotherapy (56.3%), followed by venetoclax monotherapy (10.7%) and chemoimmunotherapy (9.1%). Detailed information about CLL-directed therapy is given in Table The majority of patients  were symptomatic at the time of documented SARS-COV-2 infection. Fever was the most common COVID-19 related symptom  patients were confined at home, while 695/941 (74.7%) patients were admitted to the hospital. Of the hospitalized patients, 177 (25.5%) were admitted to the ICU, 440 (63.3%) needed oxygen supplementation outside the ICU, and 78 (11.2%) were hospitalized without need of oxygen , CLL-directed treatment was stopped during COVID-19 infection , hypogammaglobulinemia , and coronary artery disease  remained statistically significant  were identified as risk factors of severity in the univariate analyses. In the multivariate model, age  and hypogammaglobulinemia  remained statistically significant predictors in the multivariate analysis    , cardiac failure  and treatment in the last 12 months  remained statistically significant in multivariate analysis , cardiac failure  and treatment status  appeared as statistically significant risk factors of OS in the multivariate analysis.Univariate and multivariate analyses of risk factors of OS are outlined in Table Comparison of OS between treated and untreated patients is depicted graphically in Fig. n = 169) with patients receiving venetoclax at the same time (n = 31) and patients who had received chemoimmunotherapy in the last 12 months (n = 85). Then, in a second comparison, we kept the first two categories and replaced the third one with patients who had received any anti-CD20-based therapy  in the last 12 months (n = 128). No statistically significant difference between the groups was noted in either analysis. Nonetheless, treated patients (in any treatment category) had a worse OS when compared to the untreated ones (p < 0.001) , while both groups had a worse outcome compared with untreated patients (p < 0.001)  Table .Table 6OWe here present the largest cohort published to date of patients with CLL and COVID-19. In this homogeneous disease-specific series, age, history of cardiac failure, and CLL treatment were the main risk factors for a dismal outcome.In our study, the finding that older patients with CLL often have severe disease, higher CFRs, and lower survival time is in line with data derived from the general population, where COVID-19-related fatality and time to death positively correlated with age , 23, 24.In our original cohort including patients from the first wave of SARS-CoV-2 , higher In this updated cohort, patients treated for CLL had a worse OS than untreated patients, while in both the report by Mato et al  and our previous report , no difference was found between treated and untreated patients, probably due to the lower number of cases , 15. Of Untreated patients had a better outcome also when compared separately with patients in different treatment categories . Patients who continued BTKi treatment during COVID-19 infection did not appear to fare worse than those who stopped the drug with a potential benefit albeit not statistically significant, suggesting that BTKi continuation definitely is not harming patients and may potentially benefit them by preventing respiratory failure and death or by keeping CLL under control . This isPatients treated with anti-CD20 antibodies, alone or in combination, had worse OS than untreated patients. This finding suggests that such treatment likely renders patients with CLL more susceptible to succumb to COVID-19 infection. That said, the small number of patients treated with the combination of anti-CD20 antibodies with novel agents in the present cohort hinders firm conclusions from being drawn regarding its precise impact on COVID-19 outcome.The CFR in the entire cohort was 27.4% and increased to 38.4% among patients with severe COVID-19. This indicates a remarkable consistency among different studies in patients with CLL and COVID-19 as well as in the different waves of the pandemic, confirming the unabated aggressiveness of the disease with time –16. In tIn the updated cohort, CLL-directed treatment was associated with a worse outcome. However, it had no impact on COVID-19 severity in either the orginal or the updated cohort. We suspect that investigators were more likely to be informed about patients on CLL-specific treatment with mild COVID-19 symptoms than untreated patients, since the former were followed up more closely and probably sought immediate guidance: thus, we may have missed more asymptomatic untreated patients.Dexamethasone and tocilizumab remain the only treatments that showed a mortality benefit in patients with COVID-19 , 30. TheWe acknowledge certain limitations in our study. As already mentioned, hospitalized and/or symptomatic patients were more likely to be captured in our cohort, while we probably missed some asymptomatic or mildly symptomatic patients who avoided undergoing testing and/or contacting their physician, especially during the first wave, when COVID-19 testing was not the rule. To avoid the caveats of a retrospective study and the lack of a control group in overestimating the mortality, we restricted the analysis to patients with severe disease and avoided broad generalizations about prevalence, morbidity, and mortality. Taking into account the multicenter, international design of the study, some heterogeneity in our patient population is to be expected as we included patients from different countries, diagnosed with COVID-19 during different waves of the pandemic and managed differently. Along this line, mirroring the course of the pandemic, some countries like Italy and Spain were overrepresented in the first wave, while the second wave featured more patients from other countries . Finally, we recognize that differences in the quality of health care and weather could influence our results. Indeed, only a small number of cases were diagnosed during the summer and none of the patients in our cohort was vaccinated for SARS-CoV-2. In addition, the lack of any difference in CFRs between the two waves illustrates that CLL and treatment-related immunosuppression may overshadow other risk factors of outcome.Taken together, our findings suggest that in patients with CLL and COVID-19, older age confers a worse prognosis, with increased mortality. Untreated patients had a better chance of survival than those on treatment or recently treated.Supplemental information


On May 13, 2020, Chicago established a free community-based testing (CBT) initiative for SARS-CoV-2, the virus that causes COVID-19, using reverse transcription–polymerase chain reaction (RT-PCR). The initiative focused on demographic groups and geographic areas that were underrepresented in testing by clinical providers and had experienced high COVID-19 incidence, including Hispanic persons and those who have been economically marginalized. To assess the CBT initiative, the Chicago Department of Public Health (CDPH) compared demographic characteristics, economic marginalization, and test positivity between persons tested at CBT sites and persons tested in all other testing settings in Chicago. During May 13–November 14, a total of 253,904 SARS-CoV-2 RT-PCR tests were conducted at CBT sites. Compared with those tested in all other testing settings in Chicago, persons tested at CBT sites were more likely to live in areas that are economically marginalized  and to be Hispanic . The cumulative percentage of positive test results at the CBT sites was higher than that at all other testing settings . These results demonstrate the ability of public health departments to establish community-based testing initiatives that reach communities with less access to testing in other settings and that experience disproportionately higher incidences of COVID-19.Because of limited access to SARS-CoV-2 diagnostic testing in the early phase of widespread transmission in Chicago, CBT sites began operations on May 13, 2020. The City of Chicago’s CBT initiative, with direction from CDPH and the Racial Equity Rapid Response Team,After overall COVID-19 incidence declined and transmission became increasingly localized, the number of fixed CBT sites were reduced from six to four on June 23, 2020, and a mobile testing strategy was begun. Mobile sites were deployed to zip codes with the highest 7-day average percentage of positive test results. Most mobile sites remained in place for 1–2 days, and many were redeployed more than once to the same location during the study period, if that location continued to have a high percentage of positive test results. CDPH and CORE promoted sites with messages in English and Spanish and partnered with community-based and faith-based organizations to identify and advertise CBT sites. Beginning September 23, 2020, persons seeking testing were asked to show a health insurance card or state identification at registration to allow Chicago to seek reimbursement from health insurance or the Health Resources and Services Administration (HRSA); showing either document was optional.Demographic characteristics, economic marginalization, and percentage of positive test results were compared between persons tested at CBT sites and persons tested at any other setting in Chicago. Characteristics of Chicago residents tested at all other settings were extracted from the Illinois National Electronic Disease Surveillance System.During May 13–November 14, approximately 1.6 million COVID-19 tests were conducted in Chicago, including 253,904 (16%) at CBT sites and 1,346,994 (84%) in all other testing settings. Overall, 11.1% of all SARS-CoV-2 test results at CBT sites were positive, with higher percentages of positive tests at CBT sites than in all other testing settings  . DiffereCompared with persons tested in all other settings, those tested at CBT sites were more likely to be aged <40 years  . Race anDuring May 13–November 14, 2020, approximately 1.6 million COVID-19 RT-PCR tests were conducted in Chicago, including approximately 250,000 (16%) through the city’s CBT initiative. The CBT initiative effectively reached communities disproportionately affected by COVID-19, including Black and Hispanic communities and persons living in zip codes with high levels of economic marginalization . However, in a separate, unpublished CDPH study, missing race and ethnicity data were imputed using probabilistic methods based on individual persons’ last name and U.S. Census tract of residence. This imputation did not materially change the general distribution of race and ethnicity in the sample.This study demonstrates the capacity of public health agencies to establish community-based testing sites that reach communities disproportionately affected by COVID-19 and that have less access to testing in other settings. Collaboration between public health entities and community-based organizations is integral to promoting equitable access to affordable COVID-19 testing (Chicago established a free community-based testing (CBT) initiative for COVID-19, focusing on groups underrepresented in testing and who experienced high levels of COVID-19.During May 13–November 14, 2020, a total of 253,904 tests were conducted at CBT sites. Compared with persons in other testing settings, those tested at CBT sites were more likely to be Hispanic and to live in areas that are economically marginalized. The proportion of positive test results was larger at CBT sites.CBT initiatives led by public health departments can reach communities with less access to testing in other settings and disproportionately higher COVID-19 rates.


Background: The Coronavirus Disease 2019 (COVID-19) pandemic has caused numerous unexpected challenges for many families, and these long-lasting demands likely contribute to higher stress for parents. The aim of this study was to describe changes in parent stress longitudinally from before (retrospective) to two timepoints during COVID-19. Stressors that influenced parenting and strategies to manage parenting difficulties at each timepoint during COVID-19 are also described.Methods: Parents  in the US with >1 child aged 5–18 years completed an online survey in May 2020  and in September 2020 . Surveys included the 10-item Perceived Stress Scale (PSS) and questions on parenting-specific stress, stressors that influenced parenting, and strategies to manage parenting difficulties during COVID-19. Retrospective report of pre-COVID-19 stress was assessed at T1; current stress was assessed at T1 and T2. Repeated measures analysis of variance examined changes in stress over time.Results: Parent's stress increased from before COVID-19 to T1 , and decreased by T2 (19.2 ± 6.0), but remained elevated above pre-COVID-19 values (p < 0.01). Most parents (71.1%) reported an increase parenting-specific stress from before COVID-19 to T1, which continued to increase for 55% of parents at T2. Common stressors that impacted parenting during COVID-19 were changes in children's routines, worry about COVID-19, and online schooling demands. Common strategies parents used to manage parenting difficulties included doing family activities together, keeping in touch with family/friends virtually, and keeping children on daily routines.Conclusions: Parent stress increased substantially during COVID-19 and has not returned to pre-COVID-19 levels, suggesting the need for enhanced mental health resources and supports. Public health interventions should address parenting-specific stressors and effective strategies for managing parenting difficulties to mitigate their deleterious impact. The Coronavirus Disease 2019 (COVID-19) has swept the globe causing new and unexpected challenges, including severe financial losses, concerns around contracting COVID-19, and mandatory stay-at-home orders disrupting families' daily routines. These challenges have contributed to a heightened awareness among clinicians  and publPrevious infection outbreaks have resulted in profound psychosocial consequences , 12, andEffective stress management is essential to mitigate the deleterious impact of COVID-19. To manage pandemic-related stress, professional organizations such as the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend strategies such as setting a routine, taking time to unwind, connecting with others, and staying informed while limiting the amount of news that causes distress , 5. HoweThe aim of this paper is to examine changes in parents' perceived stress, from before to throughout COVID-19. Data were collected at two key timepoints during COVID-19 (May and September 2020). Additionally, at each timepoint, pandemic-related stressors that parents report as having impacted their parenting during COVID-19, and strategies they found effective at managing stress and parenting difficulties are described.A longitudinal, observational study utilizing an online survey was conducted at two separate timepoints in May (T1) and September 2020 (T2). At T1, parents were asked about their general and parenting-specific stress before COVID-19 (retrospective report), as well as currently during COVID-19. Parents were also asked about stressors that currently influenced their parenting and strategies they found effective at managing parenting difficulties during COVID-19. The same survey was administered at T2 where parents only reported on their current perceptions of these constructs.n = 58 parents were not eligible). To ensure that all completed responses were valid , the study used Completely Automated Public Turing test to tell Computers and Humans Apart (CAPTCHA) features and the research staff continuously monitored all survey responses. Upon survey completion, each respondent was contacted to provide compensation and ensure the responses were from humans. A total of n = 603 parents completed the first survey, and n = 19 were excluded due to invalid survey responses from bots (resulting in n = 584). Of these, n = 433 also completed the second survey at T2 (74% retention). These analyses include the N = 433 who completed both surveys. Parents who did not complete the second survey were more likely to be Hispanic/Latino (p < 0.01) and non-White (p = 0.03). There were no other differences for demographic or perceived general stress scores between parents who did vs. did not complete the second survey. The study procedures and passive consent process were approved by the Institutional Review Board at [redacted] University.The first online survey administered at T1 occurred during the peak of government-mandated stay-at-home orders between April 30, 2020 and May 23, 2020, approximately 4 months after the start of the COVID-19 in the US  and a feNever (0) to Very Often (4) and summed. Total scores ranged 0 to 40, with higher scores indicating greater stress. Total scores were categorized to describe parents who experienced low stress (0–13), moderate stress (14–26), and high stress (27–40) based on previously established cutoff values  and since May 2020 (at T2).The 10-item Perceived Stress Scale (PSS) was used to measure general stress. At T1, parents completed the PSS when reflecting back on their feelings before COVID-19, and again considering their current feelings during COVID-19. At T2, parents completed the PSS only once when considering their current situation at that time. This reliable and valid scale is the most widely used psychological instrument to evaluate perceived stress , 21. Itef values –25. Parenot difficult at all, somewhat difficult, very difficult, or extremely difficult.Parenting difficulty was assessed at each timepoint using a single-item question asking, “How difficult have the past few weeks been for you to continue parenting in the same as you did prior to COVID-19?” Response options included At each timepoint, parents were provided with a list of nine possible stressors and asked to select which impacted their parenting during COVID-19. Stressors included a lack of money, lack of food, lack of time due to increased work tasks, lack of time due to increased household tasks, change in daily routines and structure, parents' worry/anxiety around COVID-19, child's worry/anxiety around COVID-19, child's online schooling at home demands, and child's desire to be with friends. Parents selected all that applied. There was a response option for “other,” where parents were prompted to provide a short answer response. This list of stressors was developed by experts on the research team, given the lack of existing measures assessing COVID-19-specific stressors for parents at the time this study was conducted.At each timepoint, parents were provided with a list of nine possible strategies and asked to select which of these they found effective at managing parenting difficulties during COVID-19. Strategies included using extended family for resources, using community resources, keeping in touch with family/friends virtually, keeping their child on a daily routine, doing family activities together, finding ways to effectively manage anxiety, controlling the information they seek on COVID-19, focusing on the big priorities and letting the small tasks go, and taking time for myself. Parents selected all that applied and could also choose “other” to provide an alternative short answer response. This list of strategies was developed by experts on the research team, given the purpose of examining COVID-19-specific strategies, and the lack of COVID-19-specific measures at study onset.At T1 only, parents answered demographic questions including parent age, sex, race, ethnicity, education, marital status, family income, insurance status, and the total number of children and adults in the home. Additional COVID-19-related questions were asked at both timepoints, including family diagnoses, working from home, job layoffs/furloughs, income changes, and unemployment benefits.p < 0.05.Means, standard deviations, and percentages were calculated for demographic and COVID-19-related variables as appropriate. A repeated measures analysis of variance model examined patterns of parents' perceived stress over time, from before to across COVID-19. Pairwise comparisons examined differences during COVID-19 at T1 and T2, relative to before COVID-19 (reference timepoint). Values are presented as mean [95% confidence interval (CI)]. A chi square test of independence was used to examine if the distribution of parents with high, moderate, or low stress differed at each timepoint. Furthermore, percentages were calculated for response options pertaining to factors causing parenting difficulties and strategies to manage parenting difficulties at T1 and T2. Thematic analyses were applied to the “other” responses provided, and categories were created. Two researchers (ELA and DS) created categories and independently rated each parent response into one or more categories. Ratings were compared between researchers, and discrepancies were discussed and resolved using a third person (MKB) when needed. Data were analyzed using SAS statistical software, version 9.4 , and significance was set at Parents were mostly female, White, and married or living with a domestic partner; almost half earned a family income of < $50,000/year . COVID-1p < 0.01). Specifically, scores increased from before COVID-19  to T1 , and decreased by T2 , but remained significantly higher than pre-COVID-19 values (p < 0.01). The average decrease from T1 to T2  remained significant, even after adjusting for pre-COVID-19 values (p < 0.01). Few parents reported high stress before COVID-19 (3.5%), while 22.4% reported high stress at T1 and 12.2% at T2 . These responses were categorized . Of these, the most common was engaging in hobbies/exercise . Other responses included making family changes , adapting their mental and spiritual outlook , and cutting back .continued to increase for just over half of all parents by September 2020. The majority of parents reported that it was difficult to continue parenting in the same way as they did prior to COVID-19, while almost half reported that parenting had become harder over the course of the pandemic. The most common factors that influenced parenting were changes in children's daily structure and routines, worry and anxiety around COVID-19, and demands related to children's online schooling at home. To manage these difficulties, most parents found that doing family activities together, keeping in touch with family/friends virtually, and keeping children on a daily routine were effective. These findings highlight the need to address the stress that families are experiencing and provide adequate resources to manage this stress during COVID-19.This study showed an increase in general stress from before COVID-19 (retrospective report) to May 2020; although stress decreased by September 2020, it remained relatively greater than retrospectively reported pre-COVID-19 values. Moreover, parenting-specific stress also increased from before COVID-19 to May 2020 and Around the height of government closures and stay-at-home orders (May 2020), one-in-five parents reported high stress, while three-in-four parents reported increased parenting-specific stress. Multiple other studies have found that high stress was a common initial reaction to this pandemic –30 and tThe most commonly reported factor that influenced parenting during COVID-19 was a change in children's daily structure and routines. Most children thrive under predictable routines, which makes them feel safe and secure, contribute to healthy habits, and lead to less problematic behaviors . StructuRoutines may include designated time for children's online schooling, which was another common factor that parents felt greatly impacted their parenting. During COVID-19, many parents have had to take on the additional role of teaching their children from home, amongst other work and household tasks. At a time when almost all schools were closed, two-thirds of parents report that children's online schooling impacted their parenting, which decreased slightly 4 months later, when some schools resumed in-person or hybrid instruction. Many parents expressed that limited time due to work and household demands were common stressors, which increased in prevalence over time. Parents are understandably overwhelmed by the many responsibilities and roles they have had to take on; for example, one parent mentioned that, “juggling all responsibilities at the same time instead of getting dedicated time for different responsibilities,” impacted her parenting. In addition to parents having to cope with these changes, children are also coping with the lack of sociability from friends and structure that school and extracurricular activities provide. In fact, just over half of parents at both timepoints reported that children's desire to be with friends have impacted their parenting. Doing family activities together at home and connecting with family and friends virtually are ways to help with coping, bonding, and providing sociability in an environment that limits social interactions , 31.In May 2020, another common stressor was managing parents' worry and anxiety around COVID-19. These findings are similar to another study that found reading/hearing about the severity and contagiousness of COVID-19 was the most commonly experienced stressor among a sample of US adults . With anFor some parents, prolonged periods of high stress may result in substantial mental health impacts including greater depression, anxiety, and reduced quality of life . These mStudy limitations include use of a self-reported questionnaire with retrospective parent reported regarding before-COVID-19. Obtaining baseline measures of parents' perceived stress was not feasible given the sudden onset of this pandemic. To minimize recall bias, the first survey was administered only a few months after the start of COVID-19, yet responses for pre-COVID-19 values were likely influenced by parents' current stress, the media, and other unmeasured biases. This study also used items developed by the research team to assess factors that influenced parenting and effective strategies to manage parenting difficulties. At the time of study onset, measures on COVID-19 specific stressors and parenting strategies did not exist, yet many are now available for future research . ResponsThis study provides timely data regarding the significant increase in parents' stress over the initial course of COVID-19, as stay-at-home orders were eased, and some children returned to school. Information from this study can be used to advocate for policies that support parents and families, including access to appropriate mental health resources to mitigate the negative impacts for those in need. Given the potential for an impending mental health crisis, adequate access to quality behavioral health services, including remote telehealth options, and workplace policies to accommodate families during this time are necessary for managing the chronic stress experienced by many parents. Specific parenting stressors and strategies parents found effective at managing parenting difficulties can be used to inform stress management initiatives and targeted prevention messaging specific to parenting challenges during the COVID-19 pandemic.The raw data supporting the conclusions of this article will be made available by the authors, upon request, without undue reservation.The studies involving human participants were reviewed and approved by Virginia Commonwealth University Institutional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.EA and MB conceptualized the research questions and obtained grant funding. EA completed data collection and quantitative data analyses. DS and EA completed qualitative data coding and analyses. EA drafted the initial version of the manuscript. All authors designed the study, interpreted the data, critically reviewed the manuscript, and approved the final version as submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies  expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses  resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination. The combination of inability to readily kill immunocompetent hosts, but rather use the respiratory system as an efficient vectoring pathway to infect others has made the resultant disease (COVID-19) one of the most prolific in human history, infecting hundreds of millions, and killing >2.7 million worldwide (Macaca mulatta) has emerged as a reasonable higher-order species from which advanced COVID-19 product evaluation studies can be performed. Experimental COVID-19 in the Rhesus is induced with a relatively large dose (1e+6 TCID50) of SARS-CoV-2 delivered mucosally  that results in development of a clinically mild to moderate upper respiratory infection, with resolution 14-21 days after experimental infection. This disease model has been used extensively in pathogenesis, vaccine, and therapeutic evaluations  in uninfected macaques using a surrogate ACE2/RBD binding ELISA and pseudovirus neutralization assay. Both measures provided prevailing neutralization and RBD binding levels over time, which allowed for optimal timing of planned infectious challenge. In another cohort of macaques, groups of animals were administered three dosage levels of C144-LS/C135-LS and then challenged mucosally with SARS-CoV-2 three days later. In a corollary study, the animals dosed in the kinetics study were delay-challenged with SARS-CoV-2 75 days after receiving antibody administration.via competitive RBD/ACE2 binding ELISA, persisted beyond initial administration, with levels detectable day 75 post administration remaining to 10% of initial values in these long-term animals. . Viral loads were significantly lower in the 20 and 6 mg/kg groups than the control in all three PCR analyses in the pharyngeal swab , chemokines , CD40L, PDGF-AA, PD-L1 and Granzyme B. Multiple differences were seen in the other dosage groups for IFN-α, IL-15, CD40L, PD-L1, CXCL13, and IP-10  than the lowest (2 mg/kg) dosage group , the receptor for SARS- CoV-2, offer a model of uniformly severe disease but data from this model requires cautious interpretation since it is likely that hACE2 is expressed in a variety of irrelevant cell types in this model. Siberian hamsters are likewise a model of moderate to severe disease and offer potential for testing novel therapeutics. Both of these models can be used for initial testing of therapeutic agents, but many of those agents will require testing in nonhuman primate models to assess translational potential before use in the clinic. The rhesus macaque model of COVID-19 recapitulates the mild to moderate disease shown in a large fraction of SARS-CoV-2 infected humans, but does not recapitulate the most extreme forms of the disease, including death. However, infection of macaques produces robust and quantifiable viral replication in respiratory and gastrointestinal tracts, as well as elevated pro-inflammatory cytokines, and acute virus- induced lesions in lungs , which cIn this study we used a combination of two mAbs (C135-LS and C144-LS) with well-defined neutralization profiles that target defined epitopes in the viral spike protein . Both weAn aspect of the COVID-19 pandemic of critical importance is the emergence of viral variants, some of which appear to be bona fide escape mutations from either natural or vaccine induced humoral immunity . ImportaTaken together, our data strongly suggest that prophylactic use of combinations of mAbs, such as those we tested, administered systemically can provide durable and potent protection from mucosal infection with SARS-CoV-2. Indeed, this particular mAb cocktail is in clinical trial to determine real-world efficacy . This particular mAb cocktail has not been tested head-to-head with others in trials or in current use so their efficacy cannot be directly compared. However, given the emergence of highly transmissible variants that also escape neutralization by some mAbs, it stands to reason that having as many mAbs available for prophylactic or therapeutic use would be of great benefit. This approach could enable protection from disease for individuals that either cannot receive the available vaccines for any reason, or that work in high-risk environments and require enhanced protection from the virus.The Tulane University Institutional Animal Care and Use Committee approved all procedures used during this study. The Tulane National Primate Research Center (TNPRC) is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC no. 000594). The U.S. National Institutes of Health (NIH) Office of Laboratory Animal Welfare number for TNPRC is A3071-01. Tulane University Institutional Biosafety Committee approved all procedures for work in, and removal of samples from, Biosafety Level 3 laboratories.via sequencing. VeroE6 cells were used for live virus titration of biological samples and were maintained in DMEM  with 10% FBS.Virus used for animal inoculation was strain SARS-CoV-2; 2019-nCoV/USA-WA1/2020 (BEI# NR-52281) prepared on subconfluent VeroE6 cells (ATCC# CRL-1586) and confirmed Macaca mulatta), between 3 and 11 years old, were utilized for this study. All Rhesus macaques (RMs) were bred in captivity at TNPRC. Three RMs were infused with 20 mg/kg of an equal (10 mg/kg each) combination of C-135-LS and C-144-LS monoclonal antibodies raised against SARS-CoV-2 spike protein and monitored for RBD binding as well as neutralizing activity routinely for 75 days to determine pharmacokinetics.A total of 19 Rhesus macaques  installation of viral inoculum . The three RMs used for pharmacokinetic studies were exposed similarly 75 days after initial infusion to assess responses to delayed challenge. Animal information, including time to challenge and mAb dosage, can be found in Sixteen of the RMs were infused with 20, 6, or 2 mg/kg mAb cocktail three days before challenge. They were then exposed The animals were monitored twice daily for the duration of the study, with collections of mucosal swabs  as well as fluids  were taken pre-exposure as well as post-exposure days 1, 3, and at necropsy. Blood was collected pre-exposure, as well as 1, 2, 3, 5 and at necropsy. Physical examinations were performed daily after exposure, and necropsy occurred between 7 and 9 days post-exposure. During physical examination, rectal temperature and weight of each animal was performed. No animals met humane euthanasia endpoints during this study. During necropsy, tissues were collected in media, fresh frozen, or in fixative for later analysis.Viral load in tissues, swabs and BAL cells and supernatant was quantified using RT-qPCR targeting the nucleocapsid (genomic and subgenomic) or envelope gene (subgenomic) of SARS- CoV-2. RNA was isolated from non-tissue samples using a Zymo Quick RNA Viral Kit  or Zymo Quick RNA Viral Kit  for BAL cells, per manufacturer’s instructions. RNA was eluted in RNAse free water. During isolation, the swab was placed into the spin column in order to elute the entire contents of the swab in each extraction. BAL supernatant was extracted using 100 μL, and serum was extracted using 500uL. Viral RNA from tissues was extracted using a RNeasy Mini Kit  after homogenization in Trizol and phase separation with chloroform.in vitro transcribed RNA of each sequence diluted from 108 down to 10 copies. Positive controls consisted of SARS-CoV-2 infected VeroE6 cell lysate. Viral copies per swab were calculated by multiplying mean copies per well by amount in the total swab extract, while viral copies in tissue were calculated per microgram of RNA extracted from each tissue.Isolated RNA was analyzed in a QuantStudio 6  using TaqPath master mix  and appropriate primers/probes Table S2Median Tissue Culture Infectious Dose (TCID50) was used to quantify replication-competent virus in swabs and BAL supernatant. VeroE6 ells were plated in 48-well tissue culture treated plates to be subconfluent at time of assay. Cells were washed with serum free DMEM and virus from 50uL of sample was allowed to adsorb onto the cells for 1 hour at 37°C and 5% CO2. After adsorption, cells were overlayed with DMEM containing 2% FBS and 1% Anti/Anti . Plates were incubated for 7-10 days before being observed for cytopathic effect (CPE). Any CPE observed relative to control wells was considered positive and used to calculate TCID50 by the Reed and Muench method.via immunofluorescence.Animals were humanely euthanized following terminal blood collection. The necropsy was performed routinely with collection of organs and tissues of interest in media, fresh frozen, and in fixative. The left and right lungs were imaged and then weighed individually. A postmortem bronchoalveolar lavage (BAL) was performed on the left lower lung lobe. Endoscopic bronchial brushes were used to sample the left and right mainstem bronchi. One section from each of the major left and right lung lobes  sample fresh, and the remaining lung tissue was infused with fixative using a 50 mL syringe, and saved in fixative. Fixed tissues were processed routinely, embedded in paraffin and cut in 5 µm sections. Sections were stained routinely with hematoxylin and eosin or left unstained for later analysis Histopathologic lesions identified in tissues were scored semiquantitatively by the same pathologist that performed the necropsies. Lesions were scored based on severity as the lesions being absent (-), minimal (+), mild (++), moderate (+++), or severe (++++). For statistical analysis of histopathologic lesions, the four treatment cohorts were grouped together, and the two control cohorts were grouped together and non-parametric pairwise comparisons were performed.Immunofluorescence staining was performed on 5um sections of Formalin-fixed, paraffin- embedded lung were incubated for 1 hour with the primary antibodies  diluted in NGS at a concentration of 1:1000). Secondary antibodies tagged with Alexa Fluor fluorochromes and diluted 1:1000 in NGS were incubated for 40 minutes. DAPI  was used to label the nuclei of each section. Slides were imaged with Zeiss Axio Scan Z.1 slide scanner.via the Surrogate Virus Neutralization Test (GenScript# L00847) using the included kit protocol modified per the following: Serum samples were diluted from 1:10 to 1:21, 870 in order to determine an IC50 for RBD/ACE2 binding. Pseudovirus neutralization testing of matched serum was performed using a SARS-CoV-2.D614G spike-pseudotyped virus in 293/ACE2 cells, with neutralization assessed via reduction in luciferase activity (The ability of antibodies in serum to disrupt the binding of the receptor binding domain (RBD) of SARS-CoV-2 spike protein to Angiotensin Converting Enzyme (ACE2) was assessed activity .via bead-based determination of cytokine and chemokine concentrations was performed using a Life Technologies Cytokine 37-plex Non- Human Primate Panel for Luminex platform  according to manufacturer instructions in BSL-2+ conditions. Samples were plated in duplicate utilizing included diluents and standards after clarification by centrifugation. Plates were read on a Bio-Plex 200 System . Heat maps of log2 fold change from raw fluorescence values were generated using the heatmap package in RStudio and normalized to baseline. Hierarchical clustering was unsupervised. Red was used for fold-change increase and blue was used for fold-change decrease.Characterization of the immune response Analysis of blood chemistries was performed using a Sysmex XT-2000i analyzer for EDTA collected plasma, or an Olympus AU400 chemistry analyzer for serum.via Spearman correlation. For histopathological scoring, comparisons were made via Mann- Whitney t-test. A two-way ANOVA with Tukey multiple comparisons test was utilized for cytokine/chemokine comparisons.Data analysis was performed using Graphpad Prism 9.0.2 software . Comparison of PCR viral load between groups was performed using a Kruskal-Walis ANOVA using the area under the curve of each individual combined into the dosage groups as compared to the control group. For comparison of TCID50 and day-by-day comparison of PCR viral load between groups, a Mann-Whitney t-test was used to compare between each dosage group and controls. Relationships between serum neutralization and viral loads were assessed The original contributions presented in the study are included in the article/The animal study was reviewed and approved by TULANE IACUC.All authors contributed to the production of this manuscript with individual efforts consistent with deservingly sharing authorship.This work was supported, in part, by National Institute of Allergy and Infectious Disease Contract HHSN272201700033I (to CR) and also supported, in part, by Grant OD011104 from the Office of Research Infrastructure Programs, Office of the Director, NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.