What are the incidence of and outcomes associated with neurologic manifestations in patients with COVID-19?In this cohort study of 3744 patients in 2 large consortia, neurological manifestations were found in approximately 80% of patients hospitalized with COVID-19; the most common self-reported symptoms included headache (37%) and anosmia or ageusia (26%), whereas the most common neurological signs and/or syndromes were acute encephalopathy (49%), coma (17%), and stroke (6%). Presence of clinically captured neurologic signs and/or syndromes was associated with increased risk of in-hospital death.These findings suggest that neurological manifestations are prevalent among patients hospitalized with COVID-19 and are associated with higher in-hospital mortality. This cohort study investigates the neurological phenotypes, incidence, and outcomes among adults hospitalized with COVID-19. The COVID-19 pandemic continues to affect millions of people globally, with increasing reports of neurological manifestations but limited data on their incidence and associations with outcome.To determine the neurological phenotypes, incidence, and outcomes among adults hospitalized with COVID-19.This cohort study included patients with clinically diagnosed or laboratory-confirmed COVID-19 at 28 centers, representing 13 countries and 4 continents. The study was performed by the Global Consortium Study of Neurologic Dysfunction in COVID-19 (GCS-NeuroCOVID) from March 1 to September 30, 2020, and the European Academy of Neurology (EAN) Neuro-COVID Registry (ENERGY) from March to October 2020. Three cohorts were included: (1) the GCS-NeuroCOVID all COVID-19 cohort (n = 3055), which included consecutive hospitalized patients with COVID-19 with and without neurological manifestations; (2) the GCS-NeuroCOVID COVID-19 neurological cohort (n = 475), which comprised consecutive patients hospitalized with COVID-19 who had confirmed neurological manifestations; and (3) the ENERGY cohort (n = 214), which included patients with COVID-19 who received formal neurological consultation.Clinically diagnosed or laboratory-confirmed COVID-19.Neurological phenotypes were classified as self-reported symptoms or neurological signs and/or syndromes assessed by clinical evaluation. Composite incidence was reported for groups with at least 1 neurological manifestation. The main outcome measure was in-hospital mortality.Of the 3055 patients in the all COVID-19 cohort, 1742 (57%) were men, and the mean age was 59.9 years . Of the 475 patients in the COVID-19 neurological cohort, 262 (55%) were men, and the mean age was 62.6 years . Of the 214 patients in the ENERGY cohort, 133 (62%) were men, and the mean age was 67 years . A total of 3083 of 3743 patients (82%) across cohorts had any neurological manifestation . The most common self-reported symptoms included headache (1385 of 3732 patients [37%]) and anosmia or ageusia (977 of 3700 patients [26%]). The most prevalent neurological signs and/or syndromes were acute encephalopathy (1845 of 3740 patients [49%]), coma (649 of 3737 patients [17%]), and stroke (222 of 3737 patients [6%]), while meningitis and/or encephalitis were rare (19 of 3741 patients [0.5%]). Presence of clinically captured neurologic signs and/or syndromes was associated with increased risk of in-hospital death  after adjusting for study site, age, sex, race, and ethnicity. Presence of preexisting neurological disorders  was associated with increased risk of developing neurological signs and/or syndromes with COVID-19.In this multicohort study, neurological manifestations were prevalent among patients hospitalized with COVID-19 and were associated with higher in-hospital mortality. Preexisting neurological disorders were associated with increased risk of developing neurological signs and/or syndromes in COVID-19. More reports describe concomitant COVID-19 neurological manifestations,28 The primary aims of this study were to (1) determine the incidence of COVID-19 neurological manifestations among global cohorts of hospitalized patients and (2) investigate the association of neurological manifestations with in-hospital mortality. A secondary exploratory aim was to identify potential risk factors associated with the development of neurological symptoms and signs or syndromes.To address this critical knowledge gap, 2 global consortia collaborated to establish worldwide incidence, type, and outcomes of neurological manifestations among patients hospitalized with COVID-19.28 and (2) the European Academy of Neurology (EAN) Neuro-COVID Registry (ENERGY), a prospective registry established to evaluate the incidence of COVID-19 neurological manifestations and their outcomes at 6 and 12 months.24 The central coordinating center of the GCS-NeuroCOVID established ethics approval for a multicenter study from the University of Pittsburgh, confirmed local ethics approval with a waiver of informed consent because of the observational nature of the study, and executed data use agreements with each participating site. All centers that participated in the ENERGY Consortium received ethics committee approval, and all patients in the ENERGY cohort provided written or verbal informed consent, depending on requirements at each site. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The study population was derived from 2 large consortia: (1) the Global Consortium Study of Neurologic Dysfunction in COVID-19 (GCS-NeuroCOVID), a large multicenter cohort study set up to systematically establish the incidence, severity, and outcomes of neurological manifestations among patients with COVID-19,25The GCS-NeuroCOVID and ENERGY consortia have complementary global geographic reach and established a formal partnership to harmonize common data elements in their respective case report forms (eTable 1 in the Three separate cohorts were included in this study: (1) the GCS-NeuroCOVID all COVID-19 cohort (3055 patients at 6 sites), which included consecutive patients hospitalized with COVID-19 with and without neurological manifestations; (2) the GCS-NeuroCOVID COVID-19 neurological cohort (475 patients at 9 sites), which included consecutive patients hospitalized with COVID-19 and neurological manifestations; and (3) the ENERGY registry (214 patients at 13 sites), which included patients with COVID-19 and neurological manifestations.28 The setting included secondary or tertiary centers caring for patients hospitalized with COVID-19. Inclusion criteria were patients aged 18 or older admitted to an acute care hospital with clinically diagnosed or laboratory-confirmed COVID-19. Exclusion criteria were patients with severe preexisting neurological dysfunction, such as baseline coma or vegetative state, that precluded determination of new neurological dysfunction. Race and ethnicity were recorded by local investigators based on patient self-identification during routine clinical care, history, and clinical examination. The GCS-NeuroCOVID Consortium included 2 separate cohorts. The all COVID-19 cohort included consecutive eligible patients over a 3-month study period regardless of neurological manifestations. The COVID-19 neurological cohort included consecutive eligible patients who were identified as having neurological manifestations over a 3-month study period. Both cohorts used the same standardized case report form.The design, rationale, and data elements of GCS-NeuroCOVID study have been previously published.28As part of the pragmatic design to promote feasibility, data elements were divided into core and supplemental sections. Core elements were a robust minimal data set available regardless of care settings and resource availability, posed minimal data collection burden on frontline clinicians, and were mandatory from all sites. Supplemental data elements captured additional clinical features, and reporting was encouraged but optional. By design, all data elements were available through history and clinical observation and were not dependent on diagnostic testing availability, which may exhibit center-specific variabilities due to service disruptions during a pandemic.30 The setting included primary, secondary, and tertiary care centers providing care to patients with COVID-19. Study inclusion criteria were as follows: aged 18 years or older, clinically diagnosed COVID-19, case ascertainment through neurological consultation, and patient informed consent. Neurologist members of the EAN or its affiliated national societies registered consecutive patients fulfilling the inclusion criteria. Race/ethnicity were not recorded, as most registered patients were White individuals; however, patients from populations with other ethnic origins were confirmed by the local investigator. The ENERGY study included both hospitalized and nonhospitalized patients; however, in harmonization with the GCS-NeuroCOVID study, only hospitalized patient data were reported for this study. ENERGY data elements captured general symptoms and signs and prominent comorbidities of COVID-19 as well as neurological symptoms, signs, or diagnoses.The procedures of the European registry are detailed in a study protocol administered to participating study sites.Neurological manifestations were differentiated into (1) self-reported symptoms  and (2) neurological signs or diagnoses captured by clinical evaluation . Overall incidence of neurological manifestations was reported in 2 composite groups: the all neurological group included any patient with at least 1 self-reported neurological symptom and/or clinically captured neurological sign or syndrome. To minimize ascertainment bias resulting from self-reported symptoms, a separate signs and syndromes group was defined to include patients with at least 1 clinically captured neurological sign or syndrome.Descriptive statistics were provided for demographic and clinical characteristics of each cohort, including incidence of composite and individual neurological manifestations. We reported the total sample size available for each core and supplemental data element. No imputation was performed for missing data. Due to the emergent nature of COVID-19 pandemic, there was no formal a priori sample size and power calculations. Because the ENERGY and GCS-NeuroCOVID COVID-19 neurological cohorts did not include the general hospitalized COVID-19 population, subsequent analyses of associations used only the GCS-NeuroCOVID all COVID-19 cohort to examine the association of neurological signs and/or syndromes with outcome at hospital discharge.We first evaluated the associations between demographic characteristics and neurological manifestations with the risk of in-hospital death using logistic regression and reported odds ratios (ORs) with 95% CIs from unadjusted and adjusted models. Models adjusted for known baseline factors associated with COVID-19 mortality, ie, study center, age, age squared , sex, race, and ethnicity. Presence of multicollinearity was assessed using the variance inflation factor. Because 1 of the 6 centers in the GCS-NeuroCOVID all COVID-19 cohort did not record body mass index , we only adjusted for BMI in secondary analyses. Because only 8 patients (0.3%) were missing 1 or more of the adjustment covariates, we performed a complete case analysis.P < .05. All tests were 2-tailed. All analyses were performed using JMP Pro version 14.0 (SAS Institute).We then explored the associations between demographic characteristics and preexisting neurological conditions with the risk of developing clinically captured neurological signs and/or syndromes using logistic regression. We report ORs and 95% CIs from unadjusted models and models that adjusted for baseline characteristics . The null hypothesis was rejected with Characteristics of all 3 cohorts are summarized in The cohorts consisted of predominantly White individuals ; men; patients with overweight, ie, BMI greater than 25 ; and older patients. All 3 cohorts had a high incidence of underlying medical conditions, such as diabetes, hypertension, and preexisting neurologic disorders. The proportion of patients receiving mechanical ventilation ranged from 44 of 214 patients (21%) in the ENERGY cohort to 186 of 475 patients (39%) in the COVID-19 neurological cohort. In-hospital mortality ranged from 30 of 209 patients (14%) in the ENERGY cohort to 113 of 460 patients (25%) in the COVID-19 neurological cohort.We separately report the incidence of neurological manifestations for each cohort, as their different entry requirements may affect observed calculated estimates. Only the GCS-NeuroCOVID all COVID-19 cohort approximately reflected the general hospitalized COVID-19 population based on entry criteria. All core data elements for neurological symptoms or clinically captured signs and/or syndromes are reported with less than 5% missing data from the GCS-NeuroCOVID study cohorts and less than 10% missing data from ENERGY registry . Only a Across all 3 cohorts, headache was the most common self-reported symptom , with 11Acute encephalopathy was the most common clinically captured neurological sign and/or syndrome in all cohorts (1845 of 3740 patients [49%]), with 1541 of 3053 patients (50%) in the all COVID-19 cohort, 254 of 475 patients (53%) in the COVID-19 neurological cohort, and 50 of 212 patients (24%) in the ENERGY cohort. The next most common neurological signs and/or syndromes were coma and stroke. The incidence of coma was 509 of 3049 patients (17%) in the all COVID-19 cohort, while coma was reported in 22 of 214 patients (10%) in the ENERGY cohort and 118 of 475 (25%) in the COVID-19 neurological cohort, resulting in a pooled estimate of 17% (649 of 3737 patients) across all groups. A total of 94 of 3054 patients (3%) had a stroke. In contrast, the 2 neurologically focused cohorts both reported a higher incidence of stroke, with 88 of 475 patients (19%) in the COVID-19 neurological cohort and 40 of 208 patients (19%) in the ENERGY cohort. The least common neurological signs or syndromes were meningitis and/or encephalitis  and myelopathy , an observation consistent across all 3 cohorts  across cohorts had any neurological manifestation. In the all COVID-19 cohort, 2439 of 3054 patients (80%) had any neurological manifestation, either self-reported and/or clinically captured . Excluding self-reported symptoms, incidence of any clinically captured neurological sign or syndrome  was 53% (1628 of 3055 patients) in the all COVID-19 cohort and between 81% and 89% in the other 2 neurologically focused cohorts .In the all COVID-19 cohort, 1098 of 1312 women (84%) had any neurological manifestation, and 1341 of 1742 men (77%) had any neurological manifestation . A totalThe presence of any neurological symptom or clinically captured sign or syndrome was associated with a higher risk of in-hospital death  . When liSelf-reported neurological symptoms  were associated with a reduced risk of in-hospital death, while clinically captured neurologic signs or syndromes were associated with an increased risk of death . After aUsing data from the all COVID-19 cohort, we found that multiple baseline characteristics, including age, sex, race, ethnicity, and preexisting neurological disorders, were associated with an increased risk of developing clinically captured neurological sign or syndrome with COVID-19 . In a moTo our knowledge, this is the first and largest study of neurological manifestations in patients hospitalized with COVID-19 across different cohorts and geographic regions. Neurological manifestations ranged from self-reported symptoms to more severe neurological signs or syndromes captured by clinical evaluation. Overall, 80% of these patients exhibited at least 1 new neurological symptom, sign, or syndrome, and 55% had at least 1 neurological sign or syndrome captured on clinical evaluation. We observed that acute encephalopathy was the most common neurological sign or syndrome with an incidence of 50% across the 3 separate cohorts, while meningitis and/or encephalitis and myelopathy were the least common, with incidences of 0.1% and 0.2%, respectively. Furthermore, the presence of neurological signs or syndromes with COVID-19 significantly increased the risk of dying during acute hospitalization after adjusting for variations by study site and baseline characteristics. Taken together, these observations highlight the importance of neurological manifestations in COVID-19 and their potential impact on disease outcome.33 In our large multicenter all COVID-19 patient cohort, we observed a 50% incidence of acute encephalopathy. Meanwhile, our 2 cohorts with neurological manifestation as entry criteria observed incidences of encephalopathy between 24% and 53%, likely reflecting differences in case ascertainment and severity of illness in the underlying populations. Acute encephalopathy carried a 5-fold higher risk of in-hospital death after adjusting for known risk factors, including age. This was consistent with another multicenter study31 showing acute encephalopathy in COVID-19 was more prevalent in older patients with medical comorbidities, with higher need for critical care and worse 30-day mortality.In addition to reporting global incidence of COVID-19 neurological manifestations, we reported the incidence of specific common neurological symptoms, signs, and syndromes across 3 different cohort types. This reporting was important, as it highlighted variations in rates based on region and cohort composition. Aggregating all data and presenting only pooled data could have resulted in inaccurate estimates and an overstatement of actual signs and syndromes. Thus, the presentation of the data as distinct cohorts allowed for precise reporting of parameters, which was needed to definitively build the science around these conditions within the setting of COVID-19 infection and to compare outcomes between patients with COVID-19 with and without neurological manifestations. Existing data from prior cohorts report incidence of acute encephalopathy ranging from 8% to 84%.35 Our all COVID-19 cohort observed stroke incidence at 3% in the hospitalized COVID-19 population, while our neurologically focused COVID-19 neurological cohort and ENERGY cohort reported a higher stroke incidence (19%), suggesting that clinical neurological syndromes, such as stroke, may be overrepresented in populations captured by neurological entry criteria. Coma among patients with COVID-19 has been reported,36 but its incidence is not known. Our study found coma incidence ranges from 10% to 25%. The higher incidence of acute encephalopathy and coma in GCS-NeuroCOVID cohorts compared with the ENERGY registry likely reflected differences in population characteristics, such as the proportion of patients receiving mechanical ventilation.The incidence of other common neurological syndromes, such as stroke and coma, varied among cohorts in our study. Existing studies with variable designs, sampling methods, and case definitions have reported wide ranges for stroke incidence, from 1% to 46%.37 and between 25% and 68% for anosmia or ageusia39 The observation that self-reported neurological symptoms, such as headaches, were associated with lower risk of death requires consideration. A separate retrospective study38 also found that headache was associated with lower mortality in 576 patients hospitalized with COVID-19. The observed association between self-reported neurological symptoms and lower mortality may reflect ascertainment bias, where such data may be insufficiently captured in patients with higher disease severity, particularly in the hospitalized COVID-19 population.Measured incidence of self-reported neurological symptoms from our collective cohorts was within the range from existing literature: between 13% and 60% for headache41 In addition, we found that presence of neurological signs or syndromes with COVID-19 carried a 5-fold higher risk for in-hospital death after adjusting for differences between study sites and baseline characteristics. It is important to note that we did not record the time or duration of the neurological observation and its association to in-hospital mortality. Particularly among those with advanced illness, this is an important consideration and should be included in future research. Despite this limitation, our findings were consistent with the growing evidence from smaller cohort studies45 and supported the conclusion that neurological manifestations with COVID-19 are an important risk factor for mortality. As such, a formal neurological consultation may be warranted when neurological signs or symptoms are suspected among individuals who test positive for COVID-19.Consistent with existing reports, advanced age, male sex, White race, and higher BMI were associated with higher mortality.46 Additionally, less common neurological manifestations were recorded as supplemental data elements. Although the denominators in these initial estimates were small, as not all sites recorded this information, our preliminary findings highlighted possible presence of these phenotypes among hospitalized cohorts. To our knowledge, this was the first report to detail these syndromes, and it warrants additional investigation in future research on neurological manifestations in COVID-19. As such, these important questions will be addressed in future tiers of the GCS-NeuroCOVID consortium studies. Meanwhile, this observation provides a first general direction for identifying patients with COVID-19 who may be at higher risk of developing neurological signs or syndromes.Our exploratory analysis found that in addition to baseline characteristics and underlying medical conditions, having a preexisting neurological disorder was independently associated with increased risk of developing neurological signs or syndromes with COVID-19. In our pragmatic study design, we did not categorize preexisting neurological disorders, nor did we classify potential etiologies of COVID-19 neurological signs or syndromes, which may have included exacerbation of existing neurological pathology, development of de novo neurological syndromes, or neurological complication of systemic illness.This study has limitations, including factors inherent in global cohort study design and data collection in the midst of a pandemic and the fact that this was not a formal population-based study. Both the GCS-NeuroCOVID and ENERGY consortia used a pragmatic design to facilitate rapid study launch and data collection by frontline clinicians, as traditional research resources were limited during pandemic surges. Both prospective and retrospective data capture were permitted, and this may have led to ascertainment bias. There were no a priori sample size and power calculations, and study sample size was based on available data from registered sites. Use of a global, multicenter approach may have resulted in heterogeneity of reporting due to clinical settings and interobserver variabilities. To limit infection exposure risk related to data collection, interrater reliability was not performed; however, all sites used standardized data definitions and assessment guides to minimize variability. Because of the variations in care delivery and research resource availability across global sites, our data set captures different cohort types. Some sites were able to provide a control cohort and denominator for calculating incidence of neurological manifestations by submitting data on all patients with COVID-19 patients, including those without neurological manifestations . Other sites reported data solely among patients with COVID-19 and neurological manifestations . While these differences limit the possibility of merging data across cohorts, inclusion of these different cohorts allowed for comprehensive reporting across different populations. Furthermore, our study only reports outcomes at hospital discharge and did not report detailed causes of death. Follow-up studies are underway to determine the associations of neurological manifestations with longer-term outcomes and to capture delayed onset neurological manifestations beyond acute hospitalization.This multicenter cohort study found that neurological manifestations in COVID-19 were highly prevalent and associated with premature mortality. Using a global network with standardized protocols and common data elements is critical to facilitate further studies to understand COVID-19 neurological manifestations, including disease progression, associations with long-term outcomes, pathobiological mechanisms, and societal impact.


Experimental dental resin composites incorporating copper-doped mesoporous bioactive glass nanospheres (Cu-MBGN) were designed to impart antibacterial and remineralizing properties. The study evaluated the influence of Cu-MBGN on the mechanical properties and photopolymerization of resin composites. Cu-MBGN were synthesized using a microemulsion-assisted sol–gel method. Increasing amounts of Cu-MBGN  were added to the organic polymer matrix with inert glass micro- and nanofillers while maintaining a constant resin/filler ratio. Six tests were performed: X-ray diffraction, scanning electron microscopy, flexural strength (FS), flexural modulus (FM), Vickers microhardness (MH), and degree of conversion (DC). FS and MH of Cu-MBGN composites with silica fillers showed no deterioration with aging, with statistically similar results at 1 and 28 days. FM was not influenced by the addition of Cu-MBGN but was reduced for all tested materials after 28 days. The specimens with 1 and 5% Cu-MBGN had the highest FS, FM, MH, and DC values at 28 days, while controls with 45S5 bioactive glass had the lowest FM, FS, and MH. DC was high for all materials (83.7–93.0%). Cu-MBGN composites with silica have a potential for clinical implementation due to high DC and good mechanical properties with adequate resistance to aging. Conventional restorative materials have several drawbacks, with one of the greatest being the occurrence of caries surrounding them, the so-called secondary caries . Seconda2) have been shown to enhance apatite formation on the surface of both ACP and BG composites [The issue of secondary caries has enticed our research group to develop remineralizing bioactive materials with the addition of amorphous calcium phosphate (ACP) ,4,5,6 anmposites ,4,5. Simmposites ,10. NoneIn commercial composites, the silane coupling layer is a bifunctional molecule that simultaneously creates a covalent Si–O–Si bond to the filler particles and copolymerizes with the methacrylate groups in the resin phase. The synergy between the filler and the resin increases the mechanical strength of the composite material by arresting crack propagation . On the To counteract the abovementioned issue and avoid silanization of bioactive fillers, mesoporous bioactive glass nanospheres seem to be a suitable alternative. Mesoporous particles have a higher surface area than dense spherical ones due to a porous structure. Theoretically, low-viscosity resin monomers could penetrate pores and remain mechanically interlocked in their position after polymerization . TherefoMesoporous bioactive glass is currently in the spotlight of regenerative medicine. In addition to dense bioactive glass particles possessing the ability to release calcium, phosphorus, and silicon ions , mesoporStaphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis [Zheng et al. developed copper-doped mesoporous bioactive nanospheres (Cu-MBGN) by a sol–gel method using a Cu/L–ascorbic acid complex as the precursor of Cu . This medermidis ,27,28 asdermidis ,24.If added to conventional resin monomers and silanized inert micro- and nanofillers, the end product could be a multifunctional dental composite with highly desirable properties, namely antibacterial, ion-releasing/remineralizing, and improved mechanical strength. To our knowledge, no similar composite materials have been synthesized and investigated so far.Bimodal approach: A material containing only Cu-MBGN fillers and inert silanized microfillers was compared to control materials. For the inert control material, a composite containing inert silica nanofillers and inert microfillers was used. The bioactive control consisted of conventional bioactive glass 45S5 and inert microfillers.Trimodal approach: Similar to commercial materials, three types of fillers were used, namely inert silanized microfillers, inert silanized silica nanofillers, and various amounts of unsilanized Cu-MBGN fillers. Inert and bioactive control materials had identical composition as in the bimodal approach but with the total filler amount adjusted to 70 wt %.This study aimed to examine the influence of the addition of Cu-MBGN with proven antibacterial effect on selected mechanical and curing properties of experimental resin composites. Two strategies were tested in this study:The first null hypothesis of the bimodal strategy was that the addition of Cu-MBGN would not influence the flexural strength (FS), flexural modulus (FM), Vickers microhardness (MH), and DC in comparison to inert silica fillers or commercial bioactive fillers. The second null hypothesis of the trimodal strategy was that the combination of silica and increasing amounts of Cu-MBGN would not improve the tested properties  over Cu-MBGN alone.2–10.2CaO–4.4CuO (in mol %) as reported in the literature [Cu-MBGN were synthesized using a microemulsion-assisted sol–gel method as reported in our previous work ; the detterature .1,2 radiation.XRD patterns of Cu-MBGN and 45S5 BG  powders were recorded at room temperature with a Panalytical Aeris powder diffractometer  using CuKαThe commercial 45S5 BG and Cu-MBGN powders were sputter-coated with 5 nm Au/Pd layer (90% Au/10% Pd), evaporated with argon plasma at 6 kV using the Precision Etching Coating System .FE-SEM images of prepared samples were taken with a scanning electron microscope JSM7000F  linked to the energy-dispersive X-ray analyzer EDS/INCA 350 .Bisphenol A–glycidyl methacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) were purchased from Merck, Darmstadt, Germany. An identical resin matrix containing 60:40 weight ratio of BisGMA: TEGDMA with a photoinitiator system  and ethyl-4-dimethylamino benzoate ) was used for all materials. Resin was heated to 60 °C prior to admixture of fillers.Four types of fillers were used in this study, as shown in The materials were mixed in the absence of blue light using an asymmetrical centrifugal mixer  at gradually increasing speed up to 2700 rpm.Group testing the bimodal approach with 65 wt % total filler load andGroup testing the trimodal approach with 70 wt % total filler load used for investigating 1, 5, and 10 wt % Cu-MBGN composites with silica fillers.Eight experimental resin composites were prepared, divided into two groups:Each group contained both inert control  and bioactive control  in adequate amounts corresponding to the total filler load .3 was filled with the composite paste, covered with polyester strips on both sides to prevent oxygen-inhibited polymerization layer formation, and pressed with a weight to extrude excess material. The specimens were light-cured three times on each side with overlapping exposures, i.e., six times in total. The light guide of the curing unit was always in direct contact with the polyester strip. Composite specimens were photopolymerized for a total of 120 s (6 × 20 s) with a light-curing unit . The upper side that was polymerized first was marked, and the specimens were stored in saline solution in the dark in an incubator at 37 ± 1 °C for 1 or 28 days. Ten specimens were prepared per material and time point.FS and FM were measured according to ISO/DIN 4049:1998 using the three-point bending test . Custom-After the designated time, the specimens were removed from the saline solution, dried, and tested immediately with the side that was irradiated first facing the jig. The customized universal testing machine Ultratester  was used at a 1 mm/min crosshead speed until specimen failure.Vickers microhardness (MH) was measured using Vickers hardness testing machine CSV-10 . The specimens prepared for the three-point bending test and aged for 1 or 28 days in saline solution at 37 °C were polished to remove the surface resin-rich layer with 4000 grit silicon carbide paper and 0.05 µm aluminum oxide slurry. The measurements were made with a 100 g load and 15 s dwell time at the specimen surface. Six specimens per material were subjected to five measurements per depth. The data from different measuring points on the same specimen were pooled and treated as a statistical unit.−1. For each spectrum, 300 scans were recorded.The measurements were made using FT-Raman spectroscopy . The excitation source was a NdYaG laser (1064 nm), kept at 400 mW laser power and resolution of 8 cmn = 5) to determine the mechanical properties. Spectra of the uncured composites (n = 5) were acquired correspondingly. Kinetics add-on for MATLAB  was used for spectral analysis.DC was measured on the top and bottom surfaces of the specimens that were previously stored in saline solution for 1 day .Mean values of FS, FM, MH, and DC were compared among the experimental materials using one-way ANOVA with Tukey’s post-hoc adjustment for multiple comparisons. Pairwise comparisons  were compared using a two-tailed The results shown in The results are depicted in The MH  was geneThe results are shown in Incorporation of antimicrobial and remineralizing components into a dental resin composite could be an effective strategy for preventing secondary caries. In this study, experimental dental composites with Cu-MBGN were developed to mitigate this issue. Trimodal approach with the addition of Cu-MBGN combined with silica nanofillers and inert glass microfillers seemed to benefit the durability of their FS and MH, especially after 28 days of exposure to saline solution. Conversely, FS, FM, and MH of the corresponding control materials containing 45S5 bioactive glass with irregular microsized particles were the lowest at the same time point. Both null hypotheses were rejected.The design of a bioactive dental restorative material is challenging. Demanding oral conditions of repeated masticatory load and constant changes in pH, temperature, chemical erosion, and enzymatic degradation require a mechanically resistant and stable material. On the other hand, bioactive materials interact with living tissue and release certain substances. Thus, material deterioration over time, at least to some extent, is expected. The balance between these opposites is delicate, and the materials should always be tested after intermediate or long-term exposure to an aqueous environment similar to saliva. To minimize unwanted material degradation, stable inert fillers with reinforcing properties were combined with bioactive fillers. Owing to the large reactive surface of Cu-MBGN, bioactivity is expected even when introduced in smaller quantities, allowing five times higher share of inert fillers. In this study, we opted to compare the sole effect of Cu-MBGN particles on the mechanical and curing properties of composite bimodal mixtures to inert and commercial bioactive control before examining the combined effect of Cu-MBGN and silica nanofillers in a trimodal approach.Bimodal 10-CuBG without silica fillers showed lower FS than the corresponding inert (10-Si) and bioactive control (10-BG). The strength of the resin composites is mainly controlled by the amount of fillers, their size, and the quality of their bond to resin . The mesOur SEM analysis showed a significant difference in the shape and size of two bioactive glasses used in the present study. Irregular shape of 45S5 BG is typical for a melt-quench method, while microemulsion-assisted sol–gel method developed by Zheng et al.  typicallUnlike FS, the FM and MH were enhanced by introducing Cu-MBGN, manifested by the higher values for 10-CuBG than 10-Si and 10-BG. The increase in FM is directly correlated to the filler loading but independent of the interfacial adhesion . GeneralHowever, higher filler volume cannot explain the low FM of 10-BG specimens. Commercial BG fillers previously showed a dose-dependent decrease in FM and FS in composites with identical resin matrix and fillers and similar filler amounts as in the present study . High hyIn conclusion, the incorporation of Cu-MBGN in bimodal composite mixtures led to improvement of polymerization, stiffness, and hardness but degradation of strength.To control the strength reduction caused by the Cu-MBGN addition, further refinement and adaptation of the composite structure was necessary. The majority of contemporary commercial nanofilled composite materials consist of microfillers with a minor share of nanofillers. Smaller particles occupy the space between larger particles and increase the material’s packing density . This apThe trimodal filler mixture was apparently more successful than the bimodal approach. Cu-BG composites with silica  kept high FS values after 28 days and were statistically similar to values after 1 day. The reinforcing effect of Cu-MBGN particles could be ascribed to a combination of resin interlocking in the porosities of Cu-MBGN and additional reinforcing effect of silica, which prevented composite deterioration in water after 28 days. On the other hand, a drop of FS in the inert control 15-Si could be related to silane water degradation, leading to hydrolytic degradation and resin plasticization .Similar to the bimodal approach, in CuBG composites with silica, the gradual replacement of silica with Cu-MBGN also provided a higher volume and higher FM and MH, thus retaining the favorable stiffness and hardness. The results are in agreement with other studies ,31,32.All the tested materials achieved high DC values except for 10-CuBG-Si. The high surface area of mesoporous nanoparticles limits their addition to resin. A higher amount of resin is necessary to wet each particle, leading to a steep increase in the composite’s viscosity with higher Cu-MBGN inclusion. Uniform dispersion of nanoparticles becomes increasingly difficult with higher amount. If unsuccessful, particles group together due to weak van der Waals attractive forces ,31. Mate3/g with a 50% volume fraction [A key to uniform dispersion of nanoparticles in general, and especially mesoporous particles, in resin composites is the mixing process. Numerous techniques have been developed to overcome this obstacle ,31. A hifraction . AlthougTrimodal filler mixture, in particular materials with 1 and 5% of Cu-MBGN, demonstrated high polymerization, strength, modulus, and hardness.The bimodal combination of Cu-MBGN and microfillers elevated the stiffness and hardness of the resin composites while decreasing strength. However, trimodal CuBG composites with silica and microfillers demonstrated durable strength after 28 days and increased elastic modulus and hardness, with very high polymerization efficiency. Due to these characteristics, the trimodal filler mixture appears to be a promising path for future investigations of antibacterial properties and ion release. Within the limitations of this study, we expect that the incorporation of Cu-MBGN as an addition to inert silica and glass microfillers in dental resin composite could result in an effective and resistant remineralizing restorative material.


Older adults (≥65 years of age) bear a significant burden of severe disease and mortality associated with influenza, despite relatively high annual vaccination coverage and substantial pre-existing immunity to influenza. To test the hypothesis that host factors, including age and sex, play a role in determining the effect of repeated vaccination and levels of pre-existing humoral immunity to influenza, we evaluated pre- and post-vaccination strain-specific hemagglutination inhibition (HAI) titers in adults over 75 years of age who received a high-dose influenza vaccine in at least four out of six influenza seasons. Pre-vaccination titers, rather than host factors and repeated vaccination were significantly associated with post-vaccination HAI titer outcomes, and displayed an age-by-sex interaction. Pre-vaccination titers to H1N1 remained constant with age. Titers to H3N2 and influenza B viruses decreased substantially with age in males, whereas titers in females remained constant with age. Our findings highlight the importance of pre-existing immunity in this highly vaccinated older adult population and suggest that older males are particularly vulnerable to reduced pre-existing humoral immunity to influenza. In the United States (U.S.), there are an estimated 4 million incident cases per year in older adults, accounting for 90% of deaths associated with influenza5. The U.S. Centers for Disease Control and Prevention (CDC) recommends annual influenza vaccination for prevention of influenza infection and complications in people 6 months and older6. The high-dose inactivated influenza vaccine (HD-IIV) is available to older adults and has demonstrated superior efficacy over the standard-dose vaccine in older age groups7. Seasonal influenza vaccination coverage is relatively high in older adults, with >60% of older Americans being vaccinated annually, compared to <40% vaccination coverage in the 18–49 age group8.Seasonal influenza is an important public health burden in older adults (people ≥ 65 years of age), particularly the oldest and frail subset11, is believed to be the primary reason for the reduced effectiveness of influenza vaccines observed in older adults14. Repeated annual vaccination may also have a negative effect on vaccine-induced humoral immune responses as well as vaccine effectiveness (VE). For example, a recent observational test-negative study using ten years of vaccination history found that in older adults, VE decreases with increasing numbers of previous vaccinations but that vaccination continues to offer some level of protection15. Another study over eight seasons in the general adult population found that VE to H3N2, but not influenza B virus, is reduced among individuals with frequent vaccination history compared to those without prior vaccination16. Age-specific effects have also been observed in this context, with a reduction in influenza vaccine immunogenicity observed with repeat vaccination in teenagers, but not adults17. In addition, a meta-analysis found heterogeneous effects of repeated vaccination overall and that when negative effects are observed, they are most pronounced for H3N218. In contrast, a recent systematic review and meta-analysis concluded that the available evidence did not support a reduction in VE with consecutive repeat vaccination, but that certainty in the evidence was low19. Case–control studies in both Australia and Spain found beneficial effects of repeated annual vaccination on VE in older adults21, and an observational population-based study in Sweden found no differences in VE between those who had been vaccinated in the current season only and those who had been vaccinated in both the current and previous seasons22. Based on the conflicting evidence, multi-season clinical studies to address the effects of aging and repeated vaccination have been recommended18.Age-related immunosenescence, defined by a decline in cellular and humoral immune function combined with a chronic low-grade inflammatory phenotype (CLIP), or inflammaging23. Broad pre-existing immunity is thought to have negative consequences, as pre-existing antibodies can suppress the response to novel influenza virus strains by reducing the amount of available antigen or epitope masking25. A theoretical benefit of HD-IIV is that pre-existing antibodies cannot sequester the increased amount of antigen delivered, and, thus, more antigen is available to activate memory B cells and elicit a protective response28. To our knowledge, however, the impact of pre-existing immunity in the context of HD-IIV has not been adequately characterized.Mechanistically, pre-existing immunity generated to various influenza virus exposures over time can have an important impact on the outcome of vaccination. According to the immune imprinting theory, the memory response established by an individual’s first influenza exposure has a lifelong effect on subsequent immune responses to infection or vaccination29. The immunological differences between males and females in immune responses are largely attributed to sex hormones and sex chromosomes32. Inflammaging also contributes significantly to age- and sex-related differences in immune responses and vulnerability to infections, as discussed in two recent reviews in the context of SARS-CoV-2 infection and ongoing pandemic33. The relationship between frailty and influenza vaccine responses is debated in the literature, with one study reporting frailty having a negative effect34, others reporting no effect39, and others still reporting a positive effect40. Finally, in older adults, obesity, as measured by BMI, is significantly associated with decreased hemagglutination inhibition (HAI) titers and percentage of switched memory B cells41. Whether host related factors, including sex and age, explain variation in pre-existing immunity following repeated vaccination has not been reported. We hypothesize that the variation across studies in estimates of the effects of pre-existing immunity and repeated annual vaccination is partly caused by failure to adequately account for heterogeneity and interactions among host factors that likely differ across studies. To address this knowledge gap, we used a longitudinal cohort of older adults over 75 years of age who had received high-dose, trivalent inactivated influenza vaccine (HD-IIV3) in at least four out of six influenza seasons to estimate the impact of repeated vaccination on the antibody response to HD-IIV3 and its dependence on the intersection of age, sex, frailty, BMI, and pre-existing immunity.In addition to age, other host factors including sex, frailty, and body mass index (BMI) can impact vaccine responses in older adults. Females have been found to mount greater antibody responses to HD-IIV than males42. Trends in the change of frailty status since baseline were calculated as the difference between the first and last years of participation and differed by sex. A greater proportion of males improved in frailty status, whereas more females either did not change or progressed in frailty status. A greater proportion of males also experienced changes in BMI over the course of the study, while BMI did not change for females.Over the six influenza seasons from 2014–2015 to 2019–2020, 90 individuals participated in at least four study seasons and 433 doses of HD-IIV3 were administered. The strains included in each vaccine and the study protocol are described in Fig. 45) were high. This was particularly true for influenza B, where 98% of the participants were seroprotected prior to immunization. Post-vaccination, >94% of participants achieved seroprotection for H1N1 and H3N2, while 100% of participants were seroprotected against influenza B. Because of the lack of variability in post-vaccination seroprotection, this outcome was omitted from further analysis. Fold-rise in titers and rates of seroconversion, as defined by ≥4-fold titer increase46, were relatively low but were highest for H3N2. There were no sex differences in any post-vaccination outcomes. Together, these data indicate that pre-existing and post-vaccination strain-specific HAI titers remained high among older adults who were repeatedly vaccinated with HD-IIV3.Pre- and post-vaccination strain-specific HAI titer outcomes to the three HD-IIV3 vaccine strains are summarized and disaggregated by sex in Table We then assessed the relationships between pre-defined host factors  and post-vaccination strain-specific HAI titer outcomes. When controlling for pre-vaccination titers and influenza season, neither age, frailty, nor BMI individually had statistically significant associations with post-vaccine titers  and post-vaccination HAI titers outcomes was investigated to evaluate potential negative effects of repeated annual vaccination on the humoral immune response to future vaccines. Vaccination status prior to study enrollment could not be verified, but leveraging the longitudinal design of our study, we sought to quantify whether post-vaccination outcomes declined with additional annual vaccination, and whether this effect differed by sex. There was a non-significant negative trend between the number of years participated and the fold-rise in titers Fig. . These tp < 0.0001 for testing the null hypotheses that the slope equals zero for each vaccine strain), such that greater pre-vaccination titers were associated with a smaller fold-rise. Analyses were repeated using post-vaccination titers  Fig. , H3N2 s, and infNext, we assessed the relationships between age, frailty, BMI, and pre-vaccination titers Fig. . Neitherp = 0.152; females: 0.35 units per decade, p = 0.267), nor was the sex difference in slope (p = 0.676) , they tended to decrease with age in males , leading to a significant sex difference in age slopes   but decreased by 0.78 units per decade in males (p = 0.023) .To further interrogate the sex-specific effects of aging, expanded models controlling for frailty and BMI, in addition to influenza season, were constructed. Coefficients from the base  and expanded models are shown in Table 76) Fig. . For H3N10) Fig. . For inf23) Fig. . Like H3p = 0.037), and the decrease in males was driven primarily by people in the 75–80 age category  . Conversely, increasing titers to influenza B with age in females were driven by individuals in the 75–80 age category , whereas for males there was a sharp decline in HAI titers that occurred in participants 90–95 years of age  . Taken together, these data illustrate sex-specific effects of aging on pre-vaccination antibody titers to H3N2 and influenza B, but not H1N1.Both the base and expanded models were then amended to include cubic splines to obtain more granular estimates of the effects of age on HAI titers for males and females. Coefficients for base and expanded non-linear models are shown in Table 12) Fig. . Thus, t01) Fig. . Here, t48, analyses were repeated controlling for sex rather than allowing age effects to vary by sex. For H3N2 and influenza B, where the effect of aging was found to be significantly different in males as compared to females, the estimates derived by controlling for sex  of the CDC has recommended annual influenza vaccination for anyone aged 6 months and older since 201053.Because older adults are disproportionally burdened by severe disease and mortality from seasonal influenza, significant effort has been devoted to improving annual vaccination coverage for this vulnerable population. Older adults, particularly those who are over 75 years of age, can thus have decades of repeated annual influenza vaccination. Cumulatively, repeated annual vaccination can lead to high pre-vaccination titers, which we observed in this study and previously reported in younger adult healthcare workers, where mandatory vaccination policies result in exceptionally high rates of immunization57. Influenza vaccines are unique in this respect due to the recommendation for yearly immunization and exceptional antigenic diversity, which alter the dynamics of waning immunity. The constant pre-vaccination HAI titers with age in females seen in our study suggest that females benefit from a booster effect from each successive annual vaccination that appears to prevent antibody waning. This influenza-specific effect has been reported elsewhere, where samples collected from individuals over a 20-year period revealed longitudinal increases in neutralizing titers to influenza58. However, our data suggest that this effect is absent in males for H3N2 and influenza B. The reasons for this sex difference are unknown, but may be attributable to the compounding effects of females developing stronger responses to influenza infection and vaccination throughout adulthood59, leading to a more robust repertoire of memory B cells that recognize conserved epitopes on drifted virus strains. It is speculated that in older adults, consistently inferior responses among males may manifest as a lack of memory B cells that can be boosted by drifted viruses to counteract waning of antibody over time, thus resulting in decreasing pre-vaccination titers with age.For many vaccines, antibody titers wane over time60 and the influenza B/Victoria lineage61. In addition, the global co-circulation of B/Yamagata and B/Victoria lineages leads to increased exposure to divergent antigens62. Accordingly, over the six influenza vaccine seasons included in our study, and in the past decade, vaccine antigens were significantly more variable for H3N2 and influenza B than for H1N163. It is thus possible that repeated exposure to the same H1N1 strain sufficiently boosted male steady-state immunity to mask sex differences in the immune response. Conversely, for H3N2 and influenza B, sequential exposure to drifted viruses required robust and broad responses to allow for boosting of steady-state immunity, which may have only been present in females. Another possible explanation is immunological imprinting in youth, as it has a lifelong impact on subsequent immune responses to influenza infection and vaccination64. Individuals in our cohort, born from 1916–1941, may have been exposed to H1N1 in their youth, while the 1918 pandemic virus continued to circulate, but were likely exposed to H3N2 and influenza B later in life65. It is, therefore, possible that strong immune imprinting to H1N1 virus strains masked the sex differences otherwise observed for H3N2 and influenza B.Notably the sex difference was absent for responses to H1N1 vaccine antigens. A possible explanation for this lies in the differing evolutionary rates of the three vaccine viruses. H1N1 viruses experience slower evolution than H3N2 viruses66. Relying solely on serological samples also prohibited mechanistic investigation at the cellular level. In-depth studies of cellular and transcriptional mechanisms underlying the sex differences observed in this cohort are on-going. Third, the lack of racial diversity in our cohort must be noted, as it prohibited us from investigating race as a host factor of interest, which should be considered in future studies. Although the study lacked racial diversity, the cohort was diverse in terms of age at vaccination, allowing us to study effects in the ‘oldest’ old subset. In addition, we were unable to ascertain vaccination history for the participants prior to enrollment in the study. Previous research suggests that influenza vaccination coverage is similar among older men and women67, such that it is unlikely that gender differences in vaccination history confounded the results observed. Finally, a major strength of this study is the intersectional approach to analysis, which allowed for interrogation of effects both between and within groups , leading to a richer and more nuanced interpretation68.Our study had several strengths and limitations. First, this was an observational study that was not specifically designed to interrogate sex differences in the immune response to influenza vaccination. To overcome small yearly sample sizes, six influenza seasons were pooled together, and statistical methods were used to control for annual variation in vaccine virus strains and repeated measurements on participants. The resulting multi-season nature of this work improves generalizability to future influenza seasons. Secondly, the humoral immune response to vaccination was strain-specific HAI titers, which are the standard in the field, but lack the functional quality of microneutralization assaysIn conclusion, we demonstrate that in highly vaccinated older adults, pre-vaccination HAI titers, rather than age, sex, BMI, frailty, or repeated vaccination, predict post-vaccination parameters of humoral immunity. These pre-vaccination titers change with age in a sex-specific manner, such that older males are particularly vulnerable to lower levels of pre-existing humoral immunity. These findings provide a basis for future studies to investigate the predictive value of host factors and vaccination history in protection from influenza, which could ultimately be a valuable tool in a clinical setting. Further research should focus on elucidating the mechanisms underlying this sex difference, as well as novel vaccination strategies to harness the breadth of pre-existing immunity in older adults to provide better protection against influenza for this vulnerable population.42. After a pre-vaccination blood draw, participants received HD-IIV3 . A second blood sample was collected between 21 and 28 days after vaccine administration  were excluded. Study participants came to the Clinical Research Unit at Johns Hopkins Institute of Clinical and Translational Research on the Johns Hopkins Bayview Medical Center campus, or study visits were conducted at participants’ home as needed. A detailed medical history was obtained, vital signs were measured and frailty was assessed as per the Fried Frailty PhenotypeWritten, informed consent was obtained from all participants. The study protocol was approved by the Johns Hopkins School of Medicine Institutional Review Board. The study is registered on clinicaltrials.gov (NCT02200276).69. Briefly, serum was incubated with type III neuraminidase to eliminate non-specific inhibitors and then with turkey red blood cells to adsorb non-specific agglutinins. Two-fold serial dilutions of sera, beginning at a 1:10 dilution, were then performed in duplicate, and sera were incubated with influenza virus (4 hemagglutination units/25 µl). Turkey red blood cells were then added, and the titer defined as the highest dilution in which hemagglutination of turkey red blood cells was inhibited.Validated HAI assays were performed by Sanofi Pasteur and used to quantify antibody titers against the three influenza virus strains  included in each season’s vaccine42. Participants with three or more out of these five characteristics were defined as frail, those with one or two as prefrail, and those with none as non-frail. BMI was calculated based on measured height and weight and used as a continuous variable. Influenza season  was used as categorical variable so as not to imply a linear relationship from year-to-year. The influenza season was included as a dummy variable in analyses to account for possible confounding due to variation in the vaccine composition over time. In sensitivity analyses, viral strain was used as an alternative approach to control for confounding due to antigenic drift. Number of years of study participation was defined as the number of vaccines administered to an individual as part of the study at the time that the outcome was measured each year. Number of years of study participation was used as a time-varying continuous predictor ranging from 1 to 6 .Sex was used as a dichotomous variable based on self-report. Age was calculated based on the date of vaccination and used as a continuous variable. The frailty assessment was based on the presence or absence of five measurable characteristics: slowed motor performance , poor endurance and energy (by self-report of exhaustion), weakness (by grip strength), shrinking , and low physical activity (by self-report)2 scale to achieve an approximately normal distribution. The fold-rise in titer was calculated as post-vaccination titers divided by pre-vaccination titers, and log10 transformed to achieve a normal distribution. Seroconversion was defined as achieving a fold-rise ≥4 and used as a binary outcome. Seroprotection was defined as a titer ≥40 and used as a binary outcome.Geometric mean titers were calculated both pre- and post-vaccination. For regression analysis, titers were transformed to a log70. Following standard risk factor analysis procedure, the contributions of host factors of interest were first assessed individually. Based on the a priori hypotheses of this analysis, fixed effects of the base models for post-vaccination outcomes adjusted for influenza season, pre-vaccination titers and included interaction terms to allow the effect of the host factor to differ for males and females. Fixed effects of the base models for pre-vaccination titers adjusted for influenza season and included interaction terms to allow effects to differ by sex. Where significant sex differences were found, further analysis controlled for additional covariates, and used cubic B-splines to investigate non-linear relationships71. The relative goodness-of-fit of various models were compared using Akaike’s Information Criterion. For graphs, predictions were capped at 95 years of age due to low sample size and large uncertainty in estimates above 95 years. Coefficients were considered statistically significant if 95% confidence intervals did not span the null value of zero . Analysis was performed in Stata 15 (StataCorp).To account for repeated measures on participants, multi-level mixed-effects models with random intercepts on the individual were used. Although there was missing data in our study, we do not anticipate substantial departure from the missing at random assumption. The mixed-effects method was selected because it is considered to be robust in addressing “non-informative” missing dataFurther information on research design is available in the Supplementary InformationReporting Summary


The aim of this study was to assess differences in cervical cancer screening and treatment outcomes by HIV status in a routine programmatic setting with a high generalized HIV prevalence. Women living with HIV (WLHIV) are at heightened risk of developing cervical cancer and the World Health Organization recommends all WLHIV who are sexually active be screened, regardless of age. In 2018, Namibia’s Ministry of Health and Social Services introduced a screen-and-treat approach using visual inspection with acetic acid (VIA) and ablative treatment with cryotherapy or thermocoagulation with a focus on screening HIV-positive women due to Namibia’s 11.5% prevalence of HIV in women aged 15–49. Using program data from October 2018 to March 2020 from seven of the country’s 14 regions, we calculated descriptive statistics and chi-square tests to test the statistical significance of differences in VIA-positivity, ineligibility for ablative treatment, treatment completion, and same day treatment completion by HIV status. Between October 2018 and March 2020, the program conducted 14,786 cervical cancer screenings. Among 8,150 women who received their first VIA screening, more WLHIV screened VIA-positive (17%) than HIV-negative women (15%). This difference was statistically significant (p = 0.02). Among 2,272 women who screened VIA-positive at any screening, 1,159 (82%) completed ablative treatment. This suggests ablative treatment is feasible and acceptable in resource-limited settings. WLHIV were also more likely to complete treatment than HIV-negative women (p<0.01). Differences in health seeking behavior of sub-populations as well as resource availability between service delivery points should be considered for further investigation. Going forward in order to strengthen program implementation and expand screening access and uptake further investigation is needed to determine cancer incidence by HIV status, age, and time since last screening to assess cases that are averted as well as potential rates of overtreatment. Cervical cancer is the most common cause of cancer-related deaths among women in low- and middle-Human Development Index (HDI) countries . Its incCervical cancer is highly preventable through vaccination against the types of human papilloma virus infection (HPV) most likely to cause cervical cancer, screening of all women, and treatment for precancerous lesions. Women living with HIV (WLHIV) are at heightened risk of developing cervical cancer, with data showing up to a six-fold increase in incidence compared to HIV-negative women . StudiesThe World Health Organization’s (WHO) cervical cancer elimination strategy has set ambitious targets for all countries to achieve by 2030: 90% of all girls vaccinated for HPV before age 15, 70% of all adult women screened by age 35 and again by age 45, and 90% of women identified with precancerous lesions treated .Beyond this general guidance, data were scant from randomized trials or programs in areas with a high burden of HIV that could further guide policy makers in refining their cervical cancer prevention approach. The 2021 WHO guidelines incorporate newer data into the recommendations but continue to cite low-certainty evidence for age to start screening for cervical cancer among WLHIV . There iNamibia has a generalized HIV epidemic with an estimated prevalence of 11.5% among those aged 15–49 years: 14.8% among women and 8.0% among men . In combThe Namibian Ministry of Health and Social Services (MoHSS) adopted cervical cancer prevention guidelines in March 2018 . AlthougThis study describes results from the first 18 months of a cervical cancer screening program which provided VIA as part of routine HIV services in seven of Namibia’s 14 regions . These data are part of ongoing program monitoring and improvement as Namibia moves toward the WHO cervical cancer elimination goals. The analysis provides valuable insights into cervical cancer screening outcomes and treatment completion by HIV serostatus and age.The Namibia Cervical Cancer Screening and Treatment Programme launched in October 2018 with the training and certification of health care providers in VIA and cryotherapy and the establishment of those services in public health facilities in the Khomas region. The program was subsequently expanded to public health facilities in six additional regions, based on the volume of HIV-positive women active in care and treatment within the MoHSS cervical cancer screening target age range. The program also offered ablative treatment with cryotherapy and, beginning in September 2019, treatment with thermocoagulation.The MoHSS eligibility criteria for VIA included all women age 25–50 who were ever sexually active if HIV-negative or age 20–50 if HIV-positive. As part of routine service delivery, standard MoHSS written consent was documented for screening and treatment; no supplemental consent or contact with clients was sought for this retrospective analysis. Women who have had a hysterectomy with the cervix removed were ineligible for VIA screening. For HIV-positive women, VIA was recommended every three years or, if a woman screened VIA-positive and received treatment, every year until she screened VIA-negative for three consecutive years. For HIV-negative women, VIA was recommended every five years or, if she screened VIA-positive and received treatment, every year until she screened VIA-negative for three consecutive years. Women of unknown HIV status followed the HIV-positive screening guidelines.Women screening VIA-positive were eligible for cryotherapy if the lesions occupied less than 75% of the transformation zone, the entire lesion was visible, the lesion did not extend into the cervical os and the cryotip could completely cover the lesion. Ineligibility criteria included visible abnormal blood vessels, presence of polyps or scarring that prevented full contact between the cryotip and cervix, pregnancy, and severe cervicitis. Eligibility criteria for thermocoagulation were similar to cryotherapy except that the thermocoagulation tip does not need to cover the lesion completely. If screening results indicated treatment, the client could elect same-day treatment or delayed treatment, as needed. If the lesions did not meet the above criteria, the clients were referred for large loop excision of the transformation zone (LLETZ) which required a return visit based on the availability of a trained provider at centrally located sites. Clients with suspected cancer were not considered VIA-positive and were referred for treatment.De-identified data were abstracted from paper-based health facility cervical cancer screening registers into Excel. These registers are the primary data collection and reporting tool used by MoHSS for cervical cancer screening. Data quality checks are performed monthly to identify outlying data and changing trends, verify data, and rectify register data if needed. Variables of interest were age, HIV status, screening type, VIA outcome, and treatment outcome. Screening type included first screening, re-screening, and one-year post-treatment follow-up screening. Data on cytology-based screening and treatment outcomes were not captured in the register. Treatment outcomes for suspected cancer cases were not captured at the referring health facility and the de-identified data used for this study prevented stable record matching of biopsy results at the referral facility.We calculated descriptive statistics using Stata, version 15, and used chi-square tests to test the statistical significance of differences by HIV status and VIA-positivity, ineligibility for ablative treatment, treatment completion, and same day treatment completion using an alpha of 0.05. The study obtained all required approvals from the Office of the Executive Director of the Namibian MoHSS and received a non-research determination from the University of Washington Institutional Review Board. The study was reviewed in accordance with the U.S. Centers for Disease Control and Prevention human research protection procedures by the Office of the Associate Director for Science and was determined to be a non-research, public health program activity. The information included in this cross-sectional analysis was routinely collected, programmatic data. Consent was not obtained since the data used for analysis were anonymized.Between October 2018 and March 2020, a total of 121 healthcare workers  were trained in VIA and ablative treatment. Ten doctors were also trained to perform LLETZ and cervical biopsies starting in February 2020. Four nurse mentors also provided supportive supervision including review of cervicograms, as well as monitoring the WHO program performance benchmark indicators as part of routine quality assurance. During its first 18 months, the Namibian screen-and-treat program conducted a total of 14,786 screenings, including 8,150 first screenings, and provided 1,804 instances of treatment with ablative treatment or LLETZ at 50 public health facilities in the above stated regions.Among 8,150 women receiving their first VIA screening, 4,805 (59%) were HIV-positive, 3,202 (39%) were HIV-negative, and 143 (2%) had unknown HIV status see . Among WOf the 1,308 VIA-positive women receiving their first screening, 437 (33%) were ineligible for cryotherapy or thermocoagulation treatment. Ineligibility for ablative treatment was more common among WLHIV (35%) compared to HIV-negative women (31%) at first screening although this difference was not statistically significant (p = 0.36). WLHIV were more likely to present with suspected cancer (16%) compared to HIV-negative women (11%) at first screening.Among 2,272 women who screened VIA-positive, including first screening, follow up, and rescreening, 1,159 (82%) completed ablative treatment. Treatment completion was higher among WLHIV (86%) compared to HIV-negative women 77%, see . This diComplications following ablative treatment occurred in 6 of 733 (0.8%) WLHIV compared to 2 of 426 (0.5%) HIV-negative women see .Namibia’s cervical cancer screening and treatment program provided 14,786 VIA screenings in the selected regions over the first 18 months. We found significantly higher rates of VIA-positivity, treatment completion, and ineligibility for ablative treatment among WLHIV when compared with HIV-negative women.The significantly higher rates of VIA positivity among WLHIV in our study 17% vs 15%) align with other published literature, although the difference is not as large as seen in some African programs. Results ranged from 40% vs 20% in Zambia, 14% vs 5% in South Africa, 12% vs 3% in Tanzania and 8% vs 2% in Cote d’Ivoire –16. The % vs 15% Our findings support the integration of cervical cancer screening programs into HIV services and align with the Sigfrid et al. systematic review that found integration was feasible and acceptable to WLHIV . HoweverNamibia’s treatment completion rates suggest that ablative treatment is feasible and acceptable in resource-limited settings. Logistical challenges for providing cryotherapy, such as availability of the compressed gas refill tanks, may have negatively impacted treatment completion rates and time to treatment when only cryotherapy was available. Programs in Zambia and Malawi found similar overall treatment completion rates when using cryotherapy and thermal ablation, respectively ,27. HoweThe post-ablative treatment complication rate in our routine service settings was low. There is limited published data on adverse events following ablative treatment, however the published literature suggests complications are rare .Ineligibility for ablative treatment serves as a proxy for size and severity of the lesion. Among all VIA-positive women receiving their first screening, 437 (33%) were ineligible for ablative treatment, higher than the 21% ineligibility rate found in Nigeria . In our The Namibia policy to start cervical cancer screening at age 20 for WLHIV seems appropriate. Among 20-24-year-old women, more HIV-positive women were VIA-positive than HIV-negative women . However, it is unclear how many of these lesions would have self-cleared with time, especially among younger women, and how many would have progressed to cervical cancer. Of the 65 cases of suspected cancer at first screenings, 4 cases were among WLHIV aged 20–24. More information on the pathology of these cases and ongoing monitoring of cancer incidence by age, HIV status, and last screening date is needed to guide policymakers in refining screening policies, especially the age at which to begin screening and the impact of VIA on reducing cancer incidence. Further research on knowledge, attitudes, and practices around HPV and cervical cancer screening and treatment in Namibia could strengthen awareness campaigns and counseling strategies.This study used aggregate, observational program data. Data quality checks performed indicate that the data are reliable, and efforts were made to cross-check data with clinical records. However, it is not possible to discern the number of unique women screened or treated; rather, the number of screenings and treatments conducted is reported. Similarly, it is not possible to confirm whether the same women who screened positive were also those who were treated in the reporting period or to track one-year post-treatment follow-up visits.The Namibia Cervical Cancer Screening and Treatment Programme successfully provided, as part of routine HIV service delivery, 14,786 screenings in the first 18 months of implementation. The program continues to expand, supporting Namibia’s progress towards the WHO cervical cancer elimination targets. These results support the use of VIA programs in high HIV burden settings until widespread HPV testing to triage clients is feasible. Further research into the implementation considerations for incorporating HPV tests into screening and launching an HPV vaccination program might also support policymakers. Further investigation is needed into cancer incidence by HIV status, age, and time since screening to estimate potential rates of overtreatment and cancer cases averted in young women as well as ongoing analyses such as return rates for one-year post-treatment follow-ups and VIA-positivity following treatment.S1 File(DOCX)Click here for additional data file.


MyGeneRank, to conduct a prospective observational cohort study (NCT03277365) involving the automated generation, communication, and electronic capture of response to a polygenic risk score (PRS) for coronary artery disease (CAD). Adults with a smartphone and an existing 23andMe genetic profiling self-referred to the study. We evaluated self-reported actions taken in response to personal CAD PRS information, with special interest in the initiation of lipid-lowering therapy. 19%  of participants provided complete responses for baseline and follow-up use of lipid-lowering therapy. 20% (n = 19/95) of high CAD PRS vs 7.9% (n = 8/101) of low CAD PRS participants initiated lipid-lowering therapy at follow-up . Both the initiation of statin and non-statin lipid-lowering therapy was associated with degree of CAD PRS: 15.2% (n = 14/92) vs 6.0% (n = 6/100) for statins  and 6.8% (n = 8/118) vs 1.6% (n = 2/123) for non-statins  in high vs low CAD PRS, respectively. High CAD PRS was also associated with earlier initiation of lipid lowering therapy . Overall, degree of CAD PRS was associated with use of any lipid-lowering therapy at follow-up: 42.4% (n = 56/132) vs 28.5% (n = 37/130) . We find that digital communication of personal CAD PRS information is associated with increased and earlier lipid-lowering initiation in individuals of high CAD PRS. Loss to follow-up is the primary limitation of this study. Alternative communication routes, and long-term studies with EHR-based outcomes are needed to understand the generalizability and durability of this finding.We developed a smartphone application,  Early studies exploring the behavioral impact of genetic risk found no significant influence on behavior, though they have generally been performed in a context where specific actionability was limited both due to the broad nature of direct-to-consumer genetic risk panels and the limited risk stratification conveyed by early polygenic risk estimates7. Contemporary, indication-specific and prospective studies on PRS utility are limited.The utility and effectiveness of polygenic risk scores (PRS) in health-decision making has been the recent subject of intense interest and debate11. While there remains some debate regarding the precise utility of CAD PRSs in risk stratification15, high polygenic risk has been independently and repeatedly associated with enhanced benefit from lipid-lowering therapy21. This allows a CAD PRS to act as a “risk-enhancer” in any CAD preventive health-decision making scenario, beyond its contribution to risk stratification23  are of special interest given that CAD is a highly heritable condition and the leading cause of preventable death in the developed world7, a few focused clinical studies investigating patient actions in response to CAD PRS have been pursued26. These pilot studies were relatively small (n = 100–200) and investigated the clinical profiles of individuals receiving a clinical risk score vs a clinical risk score and PRS (rather than across PRS risk tiers). These studies were also recruited and conducted in clinical settings with direct clinical staff interaction in the communication of risk. Encouragingly, an improvement in clinical profiles was observed for all individuals receiving a PRS, regardless of their degree of genetic risk. A more recent Finnish study, Kardiokompassi, also enhanced with electronic risk visualization, has reported similar preliminary findings supporting a role in risk reduction broadly27.Beyond multi-disease direct-to-consumer genetic studiesMyGeneRank, of CAD PRS communication, where participants join through self-referral from the community and provide informed consent electronically. Study participant response to CAD PRS information is self-initiated and self-guided with the support of interactive risk visualizers. Study participant response is gauged through electronic questionnaire. The objective of the study was to determine whether communication of a CAD PRS was associated with initiation of lipid-lowering therapy.Here we describe a prospective, app-based study, 23. The overall study population is largely comprised of clinically low-risk (<7.5% 10-year ASCVD risk based on PCE) individuals of European ancestry, with 73% of study participants and 63% of study respondents belonging to the clinical low-risk category at enrollment. Relative to the baseline study participant population, study respondents were more likely to be male , of elevated age (47% vs 30% age >50 years old), non-smoker (2.6% vs 6.4% smokers), and currently on lipid-lowering medication (33% vs 23%). These differences enriched study respondents for individuals of intermediate clinical risk (20% of study respondents vs 13% of study participants) equally across CAD PRS tiers (Table Study participant and study respondent (those responding to follow-up) characteristics at baseline are presented in Table Of the 3800 study participants, 1053 28%) provided their immediate reaction to learning their CAD PRS by expressing their degree of (dis)agreement with 12 statements. The statements where reactions differed significantly by CAD PRS category are presented in Table 8% providSimilarly, study participants were surveyed for their intended actions after receiving their CAD PRS. Specifically, participants were asked (1) “Now that you know your coronary artery disease genetic risk score, do you intend to make any changes to your use of statins?”, and (2) “Now that you know your coronary artery disease genetic risk score, do you intend to meet with a physician to discuss these results?” Study participants reported intentions to meet with their physician (2-fold difference in high vs low CAD PRS) and begin the use of statins (4-fold difference in high vs low CAD PRS) in strong association with their degree of genetic risk Table .Table 3SAt 6-months follow-up or later, 253 study respondents proactively answered this survey within the study app while 459 study respondents answered the email survey. Follow-up times were 1-year on average for app-based respondents and 1.5 years on average for email-based respondents. We gauged the initiation and/or intensification of lipid-lowering in two ways: (1) by comparing responses relating to lipid-lowering therapy use at baseline and follow-up, and (2) by asking about attribution of changes in lipid-lowering to receipt of the PRS at follow-up.p-value = 0.018). Similarly, study respondents of high CAD PRS and not taking non-statin lipid-lowering therapy at baseline, initiated non-statin lipid-lowering therapy at ~3-fold the rate of low CAD PRS individuals . Discontinuation rates were low and not associated with degree of genetic risk  “Are you currently taking or have you previously taken a statin?” and (2) “Are you currently taking or have you previously taken any medication, other than a statin, used to treat high cholesterol?” The comparison of baseline vs follow-up responses is presented in Table sk Table .Table 4In = 11 of 19) report speaking with their physician about their results. For low CAD PRS lipid lowering initiators, 44% (n = 4 of 9) report speaking with their physician about their results.We also asked directly about lipid-lowering actions attributed to receipt of the CAD PRS through the following three questions: (1) “Did you meet with a physician to discuss your coronary artery disease risk score?,” (2) “After receiving your coronary artery disease genetic risk score, did you make any changes to your use of statins?,” and (3) “After receiving your coronary artery disease genetic risk score, did you make any changes to your use medications, other than statins, used to treat high cholesterol?” In response to this question, a clear difference in response to the PRS by level of engagement was apparent Table . Proacti24, our results conflict with prior reports in that we observe a weak association of lipid-lowering with degree of genetic risk at baseline (Table n = 101) vs 9.9% (n = 284) vs 20% (n = 95) of study respondents initiate any lipid-lowering therapy in low, average, and high CAD PRS individuals respectively . The average age of study respondents who initiate lipid-lowering therapy also differs significantly . Overall lipid-lowering therapy initiation occurred at >2-fold the rate and >10 years earlier in study respondents receiving a high vs low CAD PRS.Finally, we compare the rate of statin, non-statin, and combined lipid-lowering therapies at baseline and follow-up across study respondents Table . While wne Table . This asne Table . When coHere we report the results of a real-world, fully-digitized, and participant-centric approach to communication of CAD genetic risk via a PRS combined with clinical risk evaluation. We find that communication of a CAD PRS is associated with improved and earlier alignment of risk reducing interventions with degree of genetic risk. The rate of lipid-lowering therapy initiation 20%) in high CAD PRS individuals is remarkable given the overall low clinical risk profile of study participants, at least according to the Pooled Cohort Equations (PCE). We also find that CAD PRS information is perceived to be understandable, actionable, and does not induce health anxiety  reporting initiation of lipid lowering therapy. The 25-year-old male scored an extreme 98th percentile CAD PRS and reported a non-HDL cholesterol of 189 mg/dL. In contrast, the 35-year-old male and female scored at the 82nd and 85th CAD PRS percentile, respectively, but both also report moderately high total cholesterol levels, a family history of heart attack for the male, and a diagnosis of diabetes for the female. Thus, of the three youngest lipid lowering initiators; one reports a definitive class I indication for the initiation of lipid lowering therapy (diabetes), one reports a likely class I indication for initiation of lipid lowering therapy (significant hyperlipidemia) along with an extreme CAD PRS, and the third reports a risk enhancer but does not meet guidelines for the initiation of lipid lowering therapy overall. Therefore, even in those lipid lowering initiators with the lowest PCE risk, communication of the CAD PRS did not induce major deviations from guideline recommendations. In most cases, communication of the CAD PRS served to reinforce guideline recommendations.Singular risk factors, like hypercholesterolemia or diabetes status can provide a basis for early lipid lowering initiation without achieving the typical PCE risk threshold of 7.5%. Similarly, it has been recently suggested that, an extreme CAD PRS could also provide a singular basis for early initiation of lipid lowering therapyThere are a series of important limitations which restrict the generalizability and significance of our findings. First, the study population consists of a convenience sampling of “early adopters,” predominantly of European ancestry, who have already generated their genetic profiles from 23andMe, and who may be more engaged in preventive health in comparison to the general population. For example, the enrichment of non-smoker study respondents supports the notion that the worried well and early adopters drive some of the statistical signal we observe. Further avenues for the delivery of CAD PRS information must be explored to generalize the impact to different communities.Second, our study highlights some of the limitations of pure electronic communication of risk and provides some early indications that the association between lipid-lowering therapy and CAD PRS can be further amplified and generalized by deeper engagement with participants and/or with treating physicians as well as genetic counselors. For example, differences in attribution of response from app-based (proactive—more engaged) vs email-based (reactive—less engaged) responses suggest the influence of CAD PRS communication may diminish overtime if not reinforced. This engagement effect appears to be more important than the refinement of risk communication, as a stronger association of preventive behavior with degree of CAD PRS is observed in app-based follow-up Table , even th29, and that the observed drop-out rate is equal across study groups and the characteristics of study participants and study respondents remain balanced across study arms at follow-up. Moreover, there is no correlation between the CAD PRS and baseline ASCVD risk factor characteristics , and no correlation between the CAD PRS and clinical risk are induced by differences in participant decisions to respond to the follow-up survey , which can lead to biases in trial results. However, we also note that this follow-up rate is substantially higher than similar digital studies, like the Apple Health study, where the follow-up rate is <1%8.7%, whiFourth, our primary outcome is self-reported usage of statins and other lipid-lowering treatments. Approximate PCE scores were reported to the majority of study participants using qualitative risk factor responses. Whether adequate lipid lowering is achieved, or whether self-reported initiation leads to durable adherence is unknown. Our findings should be replicated in additional studies including objective and/or EHR-based outcomes.30, as well as access to different capabilities may have influenced the consistency of our results. Importantly, much of the study follow-up overlaps with the ongoing SARS-CoV-2 pandemic, which has reportedly decreased the use of primary care interventions overall, including cholesterol testing31. We expect these issues have diminished our ability to detect the alignment we observe between the CAD PRS and risk reducing actions.On the other hand, several issues also bias us against observing a signal in this study, especially the changing landscape overtime as this study was conducted. Individual level changes in the scoreOverall, we find that communication of a high CAD PRS was associated with increased and earlier initiation of lipid-lowering. Further objective, long-term follow-up studies are required to confirm this finding and to dissect the role of CAD PRS communication in adherence to and intensification of lipid-lowering therapy in individuals taking sub-optimal statin dose or other lipid-lowering medications. Longer term objective studies are required to determine whether communication of a CAD PRS can extend its influence from initiation to intensification, adherence, and persistence of lipid-lowering therapy, as well as improved hard outcomes. Finally, we suggest that PRS utility studies should be executed in indication-specific contexts to accurately gauge their influence on early prevention.The study protocol “MyGeneRank” (NCT03277365) was approved by the Scripps IRB Protocol #: IRB-16-6835. Informed consent was collected electronically in the study app. Individuals were consented for publication at enrollment. This research conforms with the principles of the Helsinki Declaration.32. The first version of the study app calculated and returned a 57-SNP CAD PRS based upon the latest CAD GWAS meta-analysis at the time33. An update was launched in December 2019 expanding the application to Android, adding Spanish language, adding a genetic counseling option, and improving the overall interface. The CAD PRS was also updated to calculate and return a 163-SNP CAD PRS based upon the prior score plus the latest efforts in identifying potential causal alleles34 and weights from the latest CAD GWAS meta-analysis35. The derivation and calculation of these PRSs are described in Supplementary Note 30. Screenshots of the app interface at launch are presented in Fig. The MyGeneRank study was launched in August of 2017 as solely an iOS application available in the USA. The initial development and basic design is described previouslyAdults (≥18 years old) with access to a smartphone and existing genetic data from 23andMe were eligible to participate in the MyGeneRank study. Participants download the study application and are presented with an eligibility screen and series of screens summarizing the consent, a complete consent form, HIPAA authorization, and participant bill of rights. PDF copies of these documents are available to study participants within the app, and participants may withdraw from the study at any time. Study participants are asked to link their 23andMe genetic data via API, optionally provide access to their mobile health data via Apple Health and Google Fit, and answer three survey questionnaires. These questionnaires include: (1) a 28-question initial demographic and CAD health and history survey answered prior to the return of results, (2) a 14-question psychosocial survey delivered immediately after the return of results aimed at gauging study participant intentions and feelings in response to their CAD PRS, and (3) a follow-up CAD health survey with questions overlapping with the initial health and history survey. The follow-up survey is made available to study participants in the study app at 6-months post-results but could be answered at any time after 6-months post-results. Follow-up responses were also obtained via email-based electronic survey disseminated via an email-based outreach campaign from November 2020 to January 2021. Three questions were added to the baseline and follow-up survey, and four questions to the psychosocial survey with the December 2019 update.Participants self-referred to the study organically, primarily through word of mouth and social media announcements. A total of 3,800 participants, after duplicate removal, connected their existing 23andMe genetic data with the MyGeneRank system. Approximately, 80% of study participants joined the study prior to the December 2019 update. Of the individuals enrolling prior to the score update, 2.5% responded to the psychosocial survey and 67% responded to the follow-up CAD health survey after receiving their updated score. 253 (6.7%) of participants responded proactively to the app-based follow-up survey. 459 (12%) of participants responded to an email-based follow-up survey after re-contact. Participants were able to skip survey questions, leading to differences in the number of study respondents answering each individual question. Herein, we refer to “study participants” as any enrolled individual. “study respondent” refers to those enrolled individuals whose response includes the final follow-up survey. The study remains open to enrollment with indefinite follow-up, this manuscript is an interim analysis following our time-limited email-based campaign to collect outcomes from current participants. The overall study flow and number of participants responding at each stage is summarized in Fig. 32. PCE risk is calculated based upon self-reported PCE risk factors. Quantitative measures are used when provided by study participants. Out-of-range values are adjusted to the top or bottom of the range of the PCE calculator as necessary. If a quantitative value is not provided, it is assigned based on survey response to qualitative risk factor questions  an interface integrating the CAD PRS with the 10-year ASCVD Pooled Cohort Equations (PCE) derived risk, and 2) a percentile rank view where the percentile rank is calculated relative to simulated individuals of similar genetic ancestry  available to the participant at the time of their response was used. For the statin and non-statin lipid-lowering initiation subgroup analysis, the subgroup is defined as those individuals indicating non-use of lipid-lowering therapy in the baseline survey.U test for high vs low CAD PRS individuals. Likert scores were coded as 1: strongly disagree, 2: somewhat disagree, 3: neutral, 4: somewhat agree, 5: strongly agree. Standard errors are provided in tables providing Likert score outcome comparisons.Binary choice survey response comparisons are made with a standard one-tailed two-proportion z-test for high vs low CAD PRS individuals. Standard errors for these proportions are a direct function of the provided proportion and sample size. Accordingly, sample sizes per exposure group are provided in each table reporting binary choice survey outcomes. Likert score responses and age comparisons were made using the Mann–Whitney 36.Individuals with missing data were discarded on a survey item-by-item basis to maximize sample size for each comparison. Effect modifying variables are internally controlled via “natural randomization” (by CAD PRS status) and the lack of correlation between PCE risk and the CAD PRS, which results in balanced clinical risk factors across comparison groups (see Table Further information on research design is available in the Supplemental DocumentSupplemental Tables S1-S4.IRB ProtocolReporting SummaryNPJ DigiMed Checklist