The 2020–2025 Dietary Guidelines for AmericansBRFSS is an annual, state-based, random-digit–dialed telephone survey of health-related behaviors representative of noninstitutionalized adults aged ≥18 years in the United States and participating territories.Previously developed scoring algorithms were used to estimate the percentage of each state’s population who met fruit and vegetable intake recommendations. Development of the methodology  and lowest among males (10.1%). Meeting vegetable intake recommendations was highest among adults aged ≥51 years (12.5%) and lowest among adults with low income (6.8%).States can use this information to tailor efforts to populations at high risk  and to implement enhanced interventions, policies, and programs that help persons increase fruit and vegetable consumption to support immune function and prevent chronic diseases.


Enterococcus species are the third most common organisms causing central line-associated bloodstream infections (CLABSIs). The management of enterococcal CLABSI, including the need for and timing of catheter removal, is not well defined. We therefore conducted this study to determine the optimal management of enterococcal CLABSI in cancer patients.Enterococcus bacteremia between September 2011 to December 2018. After excluding patients without an indwelling central venous catheter (CVC), polymicrobial bacteremia or with CVC placement less than 48 h from bacteremia onset we classified the remaining 397 patients into 3 groups: Group 1 (G1) consisted of patients with CLABSI with mucosal barrier injury (MBI), Group 2 (G2) included patients with either catheter-related bloodstream infection (CRBSI) as defined in 2009 Clinical Practice Guidelines for the Diagnosis and Management of Intravascular Catheter-Related Infection by the Infectious Diseases Society of America (IDSA) or CLABSI without MBI, and Group 3 (G3) consisted of patients who did not meet the CDC criteria for CLABSI. The impact of early (< 3 days after bacteremia onset) and late (3–7 days) CVC removal was compared. The composite primary outcome included absence of microbiologic recurrence, 90-day infection-related mortality, and 90-day infection-related complications.We reviewed data for 542 patients diagnosed with p = 0.003). In non-CLABSI cases (G3), CVC retention  was significantly associated with a higher success rates compared to early CVC removal (< 3 days) .Among patients in G2, CVC removal within 3 days of bacteremia onset was associated with a trend towards a better overall outcome than those whose CVCs were removed later between days 3 to 7 (success rate 88% vs 63%). However, those who had CVCs retained beyond 7 days had a similar successful outcome than those who had CVC removal < 3 days (92% vs. 88%). In G1, catheter retention  was associated with a better success rates than catheter removal between 3 and 7 days  could be associated with better outcomes in patients with Enterococcal CLABSI whose CVC withdrawal is clinically indicated. In contrast, it seemed that catheter retention was associated to higher success outcome rates. Therefore, future studies are needed to clearly assess this aspect. Enterococcal central line-associated bloodstream infections (CLABSIs) are increasingly common and can be associated with poor outcomes, especially in the oncologic patient population. Optimum catheter management in such infections is yet to be fully defined.Enterococcus species are the third most common cause of central line–associated bloodstream infections (CLABSIs), the optimal management of these infections remains unclear  chronic comorbid conditions [E. faecium and hospitalization in a critical-care setting. The poor outcomes of VRE BSIs were reported in a retrospective review of 7128 adult and pediatric patients who had received their first HSCT. Multivariable models showed that VRE-BSI was associated with higher non-relapse mortality and lower overall survival [E. faecium (90%). While the emergence of E. faecium as a pathogen with poorer outcomes than E. faecalis has been well reported in the literature [nditions , 17. In survival . In our terature , in our Our study was limited by its retrospective nature, particularly in that the indications for CVC management were not consistently documented. The decision to remove the CVC was also based on the decision of the treating physician, with no clear pattern or time frame and often without a documented rationale and irrespective of clinical status at point of removal. Another limitation is that given the retrospective design of the study, patients were not followed on a defined prospective clinical protocol. Therefore, not all patients had follow-up daily blood cultures, although it is a standard practice of care to repeat blood cultures, and some patients had missing data for some variable. Hence, these patients were excluded in the final analysis. This reduced the number of cases and may have impacted the statistical significance of our data. We also depended on phenotypic susceptibility pattern to identify recurrent isolates, use of more accurate methods was not possible given the retrospective nature of the study .In cases of Enterococcal CLABSI where CVC removal is clinically indicated, early removal < 3 days could be associated with better outcomes than CVC removal between 3 and 7 days. In contrast, CVC retention  showed best success rates among the groups. Further prospective data are needed to determine the best approach.


Pregnancies occur during HIV-1 vaccine clinical trials, despite requirements for women of reproductive potential to use effective contraception. Deployment of an effective HIV-1 vaccine regimen will likely target adolescents and young adults and therefore safety for pregnant and breastfeeding women will need to be addressed.We performed a retrospective, cross-protocol analysis to identify and compare pregnancy outcomes reported in 53 Phase 1 and Phase 2a HIV-1 vaccine clinical trials conducted by the HIV Vaccine Trials Network (HVTN).Two thousand six hundred seventy-three women of reproductive potential were identified and 193 pregnancies were reported. 39 of 53 (74%) studies had at least one pregnancy reported with an overall pregnancy rate of 3.15 per 100 woman-years (w-yr). While active contraception use was required during study participation, 13 of the 53 studies also contained a long-term follow up period during which pregnancy was no longer discouraged. The pregnancy rate during main study participation was 3.09 per 100 w-yr, while pregnancies occurred at a slightly greater rate in the long-term follow up period (3.22 per 100 w-yr). Adverse pregnancy outcomes were reported at similar rates between vaccinees and placebo recipients when vaccine vectors, adjuvant used, or geographic region were examined.Although there is considerable heterogeneity amongst the different vaccine trials, there appears to be no obvious indication of increased risk of adverse pregnancy or birth outcomes in these early phase HIV-1 vaccine studies. More complete data on pregnancy outcomes should be collected in early phase HIV-1 vaccine clinical trials to better inform subsequent efficacy trials.The online version contains supplementary material available at 10.1186/s12879-021-06431-x. Some significant progress has been made this decade to develop a safe and effective vaccine that protects against HIV infection. UNAIDS estimates there were 1,600,000 new adult HIV infections in 2018, the majority in those aged 15–24 years, with young women in Sub-Saharan Africa bearing the brunt of the epidemic [For females of reproductive potential, sex that could lead to HIV infection may also result in pregnancy. Conversely, the perinatal period is a time of heightened risk for new HIV infection –5. AdditSince its formation in 2000, the HIV Vaccine Trials Network (HVTN) has opened more than 80 vaccine trials with over 20,000 volunteers enrolled . Study pThis retrospective, cross-protocol analysis included all pregnancies reported during Phase 1 and Phase 2a HVTN vaccine clinical trials, including during long-term follow-up (LTFU) periods. Studies that did not involve active vaccination with an HIV vaccine were excluded, and only those studies for which participants and study staff were unblinded to treatment assignment prior to December 31, 2018 were included. Efficacy (Phase 2b or Phase 3) studies, some of which only enrolled participants born male, were excluded from this analysis. Of note, though efficacy studies are not included in this analysis, the HVTN 503 efficacy study data have previously been published .Study participants were healthy volunteers without HIV-1 infection ages 18 through 50 years who were at low risk for acquiring HIV as per standard criteria . The indPregnancy report and outcome data gathered from study Case Report Forms (CRFs) were tabulated from 53 phase 1 and 2a HIV vaccine trials conducted by the HVTN between 2002 and 2018. These trials were conducted in multiple countries, though the majority of participants were from the United States. Pregnancies were reported from time of enrolment through final study visit. The vaccination phase varied by study from a single injection up to 36 months.Additionally, 13 studies also contained a long-term follow up (LTFU) period after the vaccination phase was completed for continued safety monitoring during the next 28 to 42 months during which pregnancies were also reported. To normalize across studies and different study phases, pregnancy rates per 100 woman-years (w-yr) of study participation were calculated.Female participants of reproductive potential were identified according to the individual study criteria. This included being assigned female sex at birth, and not having reached menopause (no menses for 1 year) or having undergone a hysterectomy, bilateral oophorectomy or tubal ligation. All reported pregnancies were verified by clinical research site staff using urine or serum testing.The date of the participant’s last menstrual period (LMP) was obtained from CRF data. In the case where only the month and year were reported, a date of the 15th of the known month was used as an estimate. Additionally, if an LMP was not reported, it was estimated using the outcome date and reported gestational age at outcome. Similarly, if an outcome was reported without an outcome date, the outcome date was estimated from the LMP and the reported gestational age. All pregnancies had either an LMP or outcome date reported.Sites were encouraged to attempt to obtain outcome information from pregnant participants as soon as possible, even after the participants’ study participation had concluded. As most studies were blinded until after the final scheduled visit by all participants had occurred, outcomes reported during the active phase of studies were generally collected in a blinded fashion. Pregnancy outcome data, where known, were categorized as follows: therapeutic/elective abortion (as reported on the CRF), spontaneous abortion (< 20 weeks gestation), spontaneous fetal demise and/or stillbirth (≥20 weeks), premature live birth (< 37 weeks), or full-term live birth (≥37 weeks). Outcomes of spontaneous abortion, spontaneous fetal demise and/or stillbirth, and premature live birth were considered to be adverse outcomes. Adverse events of moderate or greater severity, including congenital anomalies, occurring within a window of 7 days prior to the pregnancy outcome and 56 days following the outcome were also compiled and tabulated from CRF data.Treatment assignment information (receipt of study product or placebo) was classified according to type of product administered , viral vector type and adjuvant. Participants were considered in receipt of product if they received at least one study product administration. Placebo recipients were grouped for analysis. Participants were also separated according to whether vaccinations were discontinued due to the identification of the incident pregnancy or whether the full course of vaccinations was completed prior to the identification.Participants born female who were not considered of reproductive potential according to the protocol in which they participated were excluded. Pregnancies reported via standardized CRFs were compiled and tabulated. The total number of participants reporting being assigned female sex at birth, of reproductive potential and having received at least one dose of an experimental HIV vaccine or placebo served as the denominator. Descriptive statistics were used to summarize the participant characteristics, pregnancy rates, and outcome data. The associations between pregnancy outcomes and study factors, including treatment assignment, product type, adjuvant, injection status, and participant geographic region, were evaluated using Fisher’s exact test .We reviewed data collected during 53 completed HVTN HIV-1 vaccine Phase 1 and 2a clinical trials conducted from March 27, 2001 and unblinded by December 31, 2018. Table The two earliest studies (HIVNET 026 and HVTN 203) required that site investigators reviewed “adequate birth control methods” with the participants and described specific types in study-specific ancillary documents. The 51 protocols that followed used a standard protocol template that specified a list of effective barrier, hormonal, and surgical contraceptive methods that were to be reviewed and discussed with all participants who were of reproductive potential prior to enrolment. In addition, 3 of these required that participants in South Africa agree to use two effective methods of contraception. Contraception use was required throughout the study in 47/53 studies (89%), even after the active vaccination phase was completed. Contraception use was required only during the active phase of vaccination for the remaining six studies. All studies required that sites perform pregnancy tests on the day of vaccine receipt and that negative results were obtained prior to study product administration. During LTFU, no contraception was required as pregnancy was no longer discouraged, but pregnancies and outcomes were still reported. While contraception use was required in all trials, those data are not presented in this analysis; the most recent contraception use and/or cessation of contraceptive usage was not collected on pregnancy-related CRFs.The studies included in this analysis enrolled 2673 females identified as being of reproductive potential in 15 countries , although 34/193 (18%) of pregnancies occurred prior to completion of the planned vaccine regimen and therefore led to early termination of vaccination. The pregnancy rate during main study participation was 3.09 per 100 woman-years. As pregnancy was no longer discouraged after the main study phase was completed, pregnancies occurred at a slightly greater rate in the LTFU phase  occurred at higher frequency in vaccinees vs placebo recipients. Amongst the 193 pregnancies reported following enrollment in HVTN studies through December 31, 2018, there were 22 maternal or fetal AEs of moderate or greater severity reported, all deemed unrelated to study product  had unknown outcomes and were not included in the analysis of outcomes (13 amongst active vaccine recipients). Of the 175 pregnancies with known outcome, 32 resulted in an elective/therapeutic abortion (18%). Of the 143 pregnancies not terminated, 105 had full-term live births (73%) and adverse pregnancy outcomes were reported in 38 (27%). The adverse outcomes included 26 spontaneous abortion (< 20 weeks), 9 premature births (5.1% of known outcomes), and 3 spontaneous fetal demise and/or stillbirth (≥ 20 weeks), corresponding to a rate of 25.6 stillbirths per 1000 outcomes.P = 0.90). This lack of signal held true for viral vectored vaccines . Additionally, when participants were divided into groups based on whether their vaccination series was stopped due to the identification of the pregnancy, no difference was seen in rates of adverse pregnancy outcomes in those whose vaccinations were stopped versus those who completed the vaccinations prior to pregnancy onset, . We also did not observe a difference in rates of adverse pregnancy outcomes between geographical regions . Further comparisons between outcome sub-categories, including adverse outcomes vs therapeutic/elective abortions, adverse outcomes vs live births, and therapeutic/elective abortions vs live births, revealed no statistically significant differences in any of the comparison groups  Fig. . This senes Fig. B as wellnes Fig. C . A similar finding was described in the groundbreaking RV 144 trail in Thailand . Not sur0 w-yr. AThe Global Alignment of Immunization safety Assessment in pregnancy (GAIA) consortium  is one oOur analyses provide a descriptive overview of the safety of prior HIV vaccines in pregnancy and provide reassuring data for estimating pregnancy safety in the large HVTN efficacy trials which are currently ongoing. Taken together, our data do not support an increased frequency of adverse pregnancy outcomes or pregnancy-related AEs in participants who have previously received experimental HIV-1 vaccines.However, our study has limitations. Due to the diversity of the study populations across all included protocols, it is difficult to provide a comparator population for relative frequencies of pregnancies or adverse events to what is seen outside of the clinical trial context. The vaccine study products used were quite diverse and the number of pregnancies in recipients of any specific product was quite low. All participants who were capable of becoming pregnant were counselled to avoid pregnancy, contraception methods were reviewed throughout the trial periods, and negative pregnancy tests were confirmed prior to study product administration on the day of all vaccinations. Most pregnancies occurred after product administration had been completed, with many occurring over a year following last vaccination. Therefore, these data do not definitively establish that any of the products studied in HVTN trials are safe to administer during pregnancy.However, these data do provide an estimated safety event rate which can inform sample size calculations for any future HIV-1 vaccine studies which specifically recruit pregnant women or participants who are considering pregnancy in the near future. As has been argued in the context of COVID-19 –31, whenWe did not identify any apparent indications of increased risk of adverse pregnancy or birth outcomes in the early phase HIV-1 vaccine studies we analyzed. As there was considerable heterogeneity amongst the different vaccine trials, we suggest that more complete data on pregnancy outcomes should be collected in early phase HIV-1 vaccine clinical trials to better inform eventual efficacy trials.Additional file 1: Fig. S1. Pregnancy outcomes in HVTN vaccine studies that occurred at any time following enrollment.Additional file 2: Table S1. Details of Study Products Received.Additional file 3: Table S2. Comparison of Pregnancy Outcomes.Additional file 4: Table S3. Pregnancy Detail Table.


Coronavirus disease 2019 (COVID-19) resulted in a worldwide pandemic that at the time of this writing has caused over 400,000 deaths within the United States. During the pandemic surge in New York City, NY, a number of military Medical Corps (MC) and Nurse Corps (NC) providers were mobilized in direct support of critical care capabilities through expansion intensive care units. In the course of the deployment, high rates of neurological-related manifestations associated with COVID-19 infection were directly observed by our military provider teams which will be described and supporting literature highlighted.This is organic information absorbed in real time during the early stages of the pandemic in New York City.The neurological manifestations of COVID-19 varied in presentation and severity. Cerebral vascular injuries documented included strokes, iatrogenic intraparenchymal hemorrhage, hypoxia-related changes and sequelae, as well as acquired diseases secondary to delayed treatment of other primary neurologic disease states. Hypercoagulable and inflammatory markers  were commonly elevated, and anticoagulation became a key factor in disease treatment and to help mitigate the downstream neurologic sequelae associated with this disease.Here we present these initial findings to lay the groundwork for more robust clinical studies moving forward.  Triservice military medical teams were rapidly mobilized to provide aid and services during the coronavirus disease 2019 (COVID-19) pandemic surge in New York City, NY, of 2020. These military medical assets consisted of physicians, nurses, and associated healthcare support staff who were assigned to platforms including the Navy Medicine Support Team (NMST), the Army Urban Augmentation Medical Task Forces (UAMTFs), and Air Force Reservists which were integrated into various New York City (NYC) civilian hospitals. At the time of drafting of this manuscript, the city of New York had documented over 207,693 confirmed cases and 17,127 COVID-19-associated fatalities [In April 2020, nearly 1600 U.S. Navy Reservists were mobilized in support of COVID-19 alone . SpecifiGiven the early experiences of our team, a number of neurological and neurosurgical diseases, most predominantly stroke and hemorrhage, presented during the management of COVID-19 ICUs. Within a large public city hospital in NYC, identification of these neurological manifestations and establishment of treatment protocols was critical in reducing short- and long-term morbidity associated with neurologic injury associated with COVID-19 infection. These manifestations will be outlined in the following sections with suggested treatment considerations with supporting literature reported. Cerebrovascular accidents and strokesContinuing evidence introduces the pro-thrombotic and inciting microvascular injury and nature that COVID-19 infection may manifest. Mao et al. documented some of the earliest studied neurologic presentations and observations of hospitalized COVID-19 patients treated in Wuhan, China. Across 214 patients, 45.5% had a neurologic presentation with COVID-19 infection which included acute cerebrovascular events, impaired consciousness, and/or neuromuscular injuries. Severity of disease was defined primarily by respiratory symptomology based on community-acquired pneumonia standards. Of those patients suffering from severe COVID-19 infection sequelae, 5.7% of patients had an acute cerebrovascular disease as confirmed via chart review and/or supported by imaging analysis. Other associated symptoms consisted of loss of taste or ageusia in 5.6% and loss of smell or anosmia in 5.1% of cases, respectively, including both severe and less acuity cases having confirmed COVID-19 infection [These observed data have been reproduced in various studies within the United States. For example, Oxley et al. presented case series level data explicitly showing stroke in patients less than the age of 50. These neurologic injuries were determined to be large vessel strokes in patients ranging in age from 33-49 years old. A 33-year-old patient illustrated in this publication specifically delayed seeking care within the mechanical intervention window secondary to concerns of COVID-19 risk if she were to present to a medical center for evaluation and/or treatment. Neurological treatment of cases reported in this series included supportive care measures, basic and advanced medical therapies, certain endovascular procedures, and in one case surgical care via need for a hemicraniectomy . In another large series reported by Zhou et al., 191 cases from both Jinyintan Hospital and Wuhan Pulmonary Hospital were evaluated for causes of mortality with a direct correlation to inflammatory status identified. Multivariable regression analysis showed increased odds of in-hospital death associated with older age , higher Sequential Organ Failure Assessment (SOFA) score , and d-dimer measurements greater than 1 µg/mL  on admission. Nearly 48% of cases had a medical comorbidity [In support of the aforementioned studies, the following stroke case was treated within our ICU. A 39-year-old male with no significant past medical history presented to the emergency room suffering from hemiplegia following a night of excessive alcohol intake Anecdotally, two-thirds to three-fourths (66-75%) of ICU patients with COVID-19 infection are on therapeutic doses of anti-coagulation via either oral, transdermal, and/or intravenous routes. Patients on therapeutic anticoagulation possess existing concern for transformation of spontaneous intracerebral hemorrhages (IPHs). In fact, this scenario represents the second most common subtype of stroke seen, and fatality in these cases can reach over 60% within one year . SpontanIn recent reports, a role for extracorporeal membrane oxygenation therapy for certain severe COVID-19-infected patients may exist, however, a known complication of this therapy includes IPH . FinallyCOVID-19-associated hypoxiaFrom an incidence standpoint, the most common neurological presentation associated with COVID-19 infection is brain hypoxia secondary to the pulmonary restraints and respiratory distress this disease commonly manifests. For example, a 65-year-old male with a medical history significant for hypertension, chronic obstructive pulmonary disorder, and asthma presented six days prior to hospitalization with symptoms of fatigue, body aches, anorexia, fevers, cough, and progressive dyspnea or shortness of breath. Over the next four days his symptoms progressed, which necessitated his presentation to his primary care provider with worsening of his baseline wheezing and increased exertional dyspnea and shortness of breath. He was noted to have a positive COVID-19 PCR test and was subsequently started on azithromycin and prednisone. On the day of his hospital admission, he was found by his wife with cyanotic hands and frothing as well as sputum coming from his nasal and oral airways. Emergency medical service (EMS) was initiated, and upon EMS arrival the patient’s initial oxygen saturation was noted to be in the 30s. The patient was transferred to the emergency room where additional laboratory and radiologic findings showing a white blood cell count of 23,000, a C-reactive protein level of 116, a d-dimer level of 512, and a chest x-ray showing bilateral infiltrates. Care was rapidly escalated to inpatient ICU admission and initiation of intravenous antibiotic therapy of vancomycin, piperacillin/tazobactam, and metronidazole for concerns of superimposed pneumonia on top of COVID-19 infection was implemented.On hospital day #1, the patient was acutely found to be bradycardic to the 50s and hypoxic to an oxygen saturation of the 40s. Noted to be in asystole, advanced cardiac life support (ACLS) was initiated with return of spontaneous circulation (ROSC) in 10 minutes. The patient experienced high plateau pressures requiring switch from volume control to pressure control ventilatory management. As was apparent in a number of individuals requiring high positive pressures, the patient unfortunately suffered a pneumothorax necessitating chest tube placement. He required increasing vasopressor support, neuromuscular paralytics, steroids, and was placed in a prone position for persistent hypoxia and an arterial oxygen partial pressure/fractional inspired oxygen (PaO2/FiO2) <100. The steroids resulted in a transient diabetic ketoacidosis and GI-related bleed treated with pantoprazole IV drip. His accompanying myoclonic jerks were treated with lorazepam. After his three-day ICU course of care, he had persistent hypoxia with oxygen saturation in the 70s despite maximized ventilatory settings. Unfortunately, the patient’s neurologic status deteriorated with a CT scan of the head showing patchy hypodensities along all watershed areas concerning for diffuse hypoxia, and the patient eventually expired due to his hemodynamic instability coupled with neurologic injury.Hospital-acquired COVID-19 transmissionConcern for hospital-acquired COVID-19 infection has been seen and validated . This inDelayed care scenariosSignificant concern in both the lay and public press surrounds the concern for patients delaying necessary care out of fear of acquiring COVID-19 infection. As reported within the Oxley et al. series, one particular patient with a large vessel stroke missed her stroke intervention window due to such concerns . In a reOur medical care team witnessed such cases during our deployment. Specifically, a 47-year-old female presented to our ICU as a transferred from the ER with “the worst headache of her life”. She delayed going to a hospital for six days since her headache onset despite having intermittent hemiplegia and confusion due to concerns of COVID-19 exposure risk. She was noted to have a Hunt and Hess Grade 3, Fischer grade 1 subarachnoid hemorrhage with hydrocephalus illustrated by the prominence of the temporal horns bilaterally on imaging. CT angiography and subsequent formal angiography illustrated a ruptured posterior communicating artery aneurysm that was coiled via endovascular intervention. This is appreciated in Figure Lessons learned and next steps…As highlighted through the supporting literature and case-based examples, the mobilization of our military medical teams into areas of COVID-19 pandemic outbreaks has presented interesting medical treatment challenges and opportunities to reflect and apply lessons learned to future pandemic responses. Naturally, solutions to the proposed challenges of COVID-19 will develop as more is understood regarding the virus and its disease process. However, certain neurological manifestations associated with COVID-19 infection are important to identify as appropriate treatment will aid in lessening neurological-associated morbidity and mortality. Neurologic sequelae related to hypoxia require rapid intervention and support in improving the underlying respiratory status and condition. The cellular and microvascular damage caused by the virus has obvious deleterious effects directly on the pulmonary system which directly contributes to the hypoxic state commonly seen in these patients. Prone positioning has been shown to improve survival as well as permit the posterior and inferior lung spaces to be less restricted and thus improve oxygen exchange for many patients suffering from COVID-19 infection. Additionally, certain therapeutic medications have beneficial impact on improving respiratory function. Recent data from the U.K. and supported through our ICU early experiences illustrated that steroids such as dexamethasone reduced deaths by one-third in ventilated patients  and by one-fifth in other patients receiving oxygen only  . With a Attention and care in neurologic examinations can provide early warning signs in the setting of intraparenchymal hematoma and stroke. Early management changes that could improve outcomes, e.g. stopping anticoagulation in patients with IPH or in providing mechanical thrombectomy and pharmacologic anticoagulation in patients suffering from thrombogenic strokes, are critical in addressing and reducing adverse neurologic short- and long-term morbidities. Patient, patient’s family, nurse, and provider education of early neurologic compromise signs or symptoms to look for in patients with COVID-19 infection should be discussed and understood. It’s important to recognize that data from the DEFUSE 3 trial has increased the potential window for mechanical intervention in stroke patients up to 16 hours . This wiAs COVID-19 disease burden slows, the ability to isolate positive COVID-19 infected patients from non-positive patients will become a significant safety and treatment factor for many medical centers throughout the globe as pandemic spread occurs. Not only is this true at the local hospital level but also within systems or geographical levels where patient transfers and movement are necessary. Disaster management algorithms should look to include not just units within the medical center level but also coordinated throughout hospital systems at a local, city, or even state level if at all possible as resources become strained or pinched in certain regions. For various hospital systems, this type of integration is openly challenged by the realities of competing hospital’s payor-mix considerations. However, with the growing onslaught of hospital consolidation in the market, this has the potential to allow for greater coordination of resources [The most important and immediate work can be done on patient education. Patients need to be educated and informed about seeking proper care for medical conditions despite any justified fears they may have from threat of COVID-19 infection. This should be direct education to the patient through standard media techniques as well as primary care provider to patient education. Such educational endeavors certainly have the potential to improve neurological outcomes in patients delaying emergency medical evaluations and treatment. Educating patients on the importance of identifying major medical threats to their health and the need to seek medical care is the most direct and impactful change that could dictate care at the local and community levels. Educating patients, systems of providers, and ancillary healthcare staff about how to best communicate with patients is essential to inform them of urgent treatment needs and where care can be rendered, relieve concerns that are heightened during trying times such as the current pandemic, and aid in understanding or empowering individuals to be active in their health and medical care needs. This situation obviously extends well beyond neurological issues and can impact oncologic care, pediatric vaccinations, preventative medicine, cardiac care, and essentially all other medical conditions which had been impacted by concerns that COVID-19 had throughout various health care markets and regions. Neurological manifestations and potential solutions are visually displayed in Figure The COVID-19 pandemic affects not only the respiratory status of those infected, but also can contribute to end-organ dysfunction and neurologic compromise. Knowledge of such neurologic manifestations must be assessed and addressed in the initial stage and throughout the care of COVID-19-infected patients in order to prevent and reduce adverse neurologic injury when encountered. Familiarity and integration of the latest stroke intervention criteria combined with the best possible neurologic examination are critical given the many manifestations that this virus has on the human body. Further investigation is warranted to improve the understanding of this disease at a pathophysiologic, infectious disease, radiographic, and clinical as well as basic science level. As the military medical systems are called into action, we must quickly incorporate fast-changing disease treatment paradigms while applying the scientific process to identify best practices and lessons learned to apply in further missions to combat the pandemic. Open communication and supportive educational endeavors on critical care training are essential to ensure that our military medical teams are effectively able to address this disease when called upon during current and future crises related to COVID-19.


We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two black-box methods that can interpret genome regulatory sequences for cell type-specific or condition specific patterns. We show that these methods identify relevant transcription factor motifs and spacings that are predictive of cell state-specific chromatin accessibility. Finally, we integrate these methods into framework that is readily accessible to non-experts and available for download as a binary or installed via PyPI or bioconda at  Within the genome are the instructions to build all the cell types that make up the human body. However, understanding these instructions and how and when these instructions go wrong in cancer or genetically inherited disease is an open problem. Deep neural networks provide powerful models to learn the relationship between DNA sequence and functional consequence across many different cell types, such as whether a particular stretch of DNA is accessible and genes in that region can be expressed or is inaccessible and therefore genes are inactive. Despite these advances, a major setback in deep learning is that it is challenging to understand what patterns of DNA sequence that a deep learning model has learned to associate with a particular genomic function, whether these patterns are significant, and how to determine whether these patterns are specific to a particular cell type or are general “housekeeping” patterns that function across many cell types. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two methods which allow us to evaluate the significance of particular patterns of DNA sequence features on models trained to predict function across multiple cell types, and apply this to problems of transcription factor binding and DNA accessibility across multiple cell types. PLOS Computational Biology Methods paper.This is a de novo DNA sequence motifs that influence local cell type-specific accessibility in the context of a massively parallel reporter assay [Neural networks have been increasingly helpful in understanding and predicting cellular function from genomic sequence in tasks such as transcription factor binding ,2, chromer assay . Here, wer assay . A cell Recovering interpretable biological hypothesizes from trained deep neural network models has been a challenge given the complexity of the models. Contemporary approaches for interpreting deep learning models of genomic sequence features utilize three main approaches :Interpretation of model and individual sequences for important nucleotides using model saliency and motif generation from input sequences –11 or saInterpretation of model convolutional filters as position weight matrices ,4,12,13.Model response to in silico “designed” sequences ,14–17.a priori that particular combinations of features (patterns) that could have important effects, such as transcription factor motifs. Interpretation methods make use of our a priori knowledge about the patterns of features such as DNA motifs and relationships between patterns such as spacing to investigate what features the model has learned are overall most important through evaluating model response to “designed” DNA sequences in silico. However, these approaches have yet to explore how to assign the significance of these effects and how to interpret whether these effects are differential between two conditions, such as cell types, when applied to multi-task classification models.Methods in class 1 such as DeepSHAP ,18 and EHere we present a method of assigning both effect scores and significance values to DNA sequence feature patterns of interest, such as transcription factor motifs or grammars such as particular transcription factor motifs and motif spacings, that differentiate between cell types or conditions from a multi-task neural network . Signifiy, over N training examples, we can estimate the overall expected probability of y to be:In contrast to methods that analyze a single input sequence or a model, we build upon the use of “designed sequences” to create a computational method to measure the learned differential effect of a transcription factor motif or other DNA sequence pattern on the output of a multi-task classification model . These pp, such as a transcription factor motif  as the signed-rank test statistic of the ratios:c1 and c2 based upon the average log2 ratio of predictions:We then compute the Differential Expected Pattern Effect between two classes Rm represents the rank of the absolute log ratio of the expected pattern effect of the pattern, p, differentially between two classes c1 and c2, on a sequence m.In addition to computing the magnitude of the DEPE between two classes, we compute the significance of this effect:Our goal is to apply the DEPE to discover cell type-specific transcription factor activity from chromatin accessibility, but to first validate that DEPE can correctly identify differential transcription factor activity, we apply the DeepAccess architecture to predict DNA binding from ChIP-seq data. In contrast to chromatin accessibility where multiple or specific combinations of transcription factors may be responsible for activity, ChIP-seq should produce sequences enriched by the binding of a single DNA binding protein. Therefore, if a DeepAccess model has successfully learned differential DNA sequence patterns between two ChIP-seq experiments, DEPE should identify the DNA binding motifs of those transcription factors.We evaluated the effectiveness of the DEPE by evaluating how well it could identify sequence motifs that were responsible for differential ChIP-seq binding. We trained a single DeepAccess model to predict the DNA sequences bound by six ChIP-seq experiments for the transcription factors ELF1, MEF2A, CTCF, USF1, TCF12, and NRF1 in mouse erythroleukemia (MEL) cells from ENCODE . The traWe then used EPE and Differential EPE to investigate chromatin accessibility. First, we hypothesized that we can use EPE to investigate preferential spacing of Oct4 (Pou5f1) and Sox2 motifs within accessible DNA in stem cells, as these two transcription factors are known to cooperatively bind. We tested the EPEs for multiple spacings of Oct4 and Sox2 motifs  and findWe have increased the utility of our method by providing an executable version of DeepAccess for the Linux operating system as well as a python package available via PyPI and bioconda . Using KThe DeepAccess system, including methods for estimating EPE and Differential EPE, is available as a PyPI package and on bioconda. We also provide a binary of the DeepAccess system which avoids typical installation problems with the evolution of Python packages by packaging all dependencies including the Python interpreter within our software. Even when using package managers such as pip and conda which are present with frameworks such as Selene , Kipoi , and pyyWhile previous work has explored the contributions of neural network architecture, training methodology, and model interpretation to learn the relationship between DNA sequence and differential DNA binding , these nWhile DeepAccess was developed specifically for identifying cell type-specific sequence features from chromatin accessibility, Differential Expected Pattern Effect can be used to discover condition-specific sequence features from many types of experimental genome-wide sequencing data. We expect that DeepAccess will provide a useful approach for discovering sequence features that drive differential cell fates, and that the system we provide will make this capability accessible to a wide audience.Reads were trimmed for adaptors and low-quality positions using Trimgalore (Cutadapt v0.6.2)  and aligWe used as input to DeepAccess 200bp regions centered at the GPS-called ChIP-seq binding event. DeepAccess then uses these regions to create a training data set of 87,114 100bp sequences which can be labeled as binding events for any combination of the 6 tested transcription factors, including regions where none of the 6 transcription factors are predicted to bind. We define a region as a ChIP binding event for a transcription factor if more that 50% of the 100bp region overlaps the 200bp region centered at a GPS binding event. The DeepAccess model was then trained for 5 epochs with default parameters.https://zenodo.org/record/4908895#.YL6YpR0pDfY.Methods for DeepAccess and differential nucleotide importance with DeepAccess were previously described in Hammelman et al., 2020. Briefly, we trained DeepAccess on ATAC-seq data from ten cell types: stem cell, fibroblast, hepatocyte, endoderm, beta pancreatic cell, alpha pancreatic cell, cardiomyocyte, skeletal muscle, dopaminergic midbrain neuron, and spinal motor neuron  using biFor ATAC-seq processing, reads were trimmed for adaptors and low-quality positions using Trimgalore (Cutadapt v0.6.2) . Reads wChIP-nexus binding data for Oct4, Sox2, and patch cap control  were dowFor all experiments we used as our synthetic distribution sequences 24 randomly selected genomic locations that were not overlapping ATAC-seq accessible regions in all ten mouse cell types. We provide these regions with the DeepAccess executable as default backgrounds for new experiments.DeepAccess executable was generated with pyinstaller (v4.1).S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigScatter plots of GIA (A-C) or EPE/DEPE (D-F) of effect sizes of 356 HOCOMOCOv11 mouse motifs on predictions of CTCF binding from a trained DeepAccess model on balanced class data (x-axis) compared to 2x, 10x, and 30x more CTCF binding sites: USF1 binding sites (y-axis) (A) GIA of CTCF binding (B) GIA of USF1 binding (C) GIA of CTCF vs USF1 differential binding (D) EPE of CTCF binding (E) EPE of USF1 binding (F) DEPE of CTCF vs USF1 binding.(TIF)Click here for additional data file.S3 Fig(A) EPE of Hoxc9 motif on EPE from DeepAccess model trained to predict motor neuron chromatin accessibility shows EPE of Hoxc9 motifs are low for positions in the beginning of the DNA sequence and high for positions at the middle and end of the DNA sequence. (B) Scatter plot of EPEs for 356 HOCOMOCOv11 mouse transcription factor motifs when insertion is at position 0 compared to position 50 indicate early insertion site in DNA sequence results in different EPE values for transcription factors, with some having lower EPEs and others having higher EPEs. (C) Scatter plot of EPEs for 356 HOCOMOCOv11 mouse transcription factor motifs when insertion is at position 70 compared to position 50 indicate early insertion site in DNA sequence results in similar EPE values for all transcription factors. (D) Heatmap of Pearson’s r of EPEs for DeepAccess motor neuron accessibility for insertions at all starting positions within the DNA sequence shows most insertion positions result in highly similar EPEs except for early insertion positions. (E) Heatmap of Pearson’s r of DEPEs for DeepAccess motor neuron vs fibroblast differential accessibility for insertions at all starting positions within the DNA sequence shows most insertion positions result in highly similar EPEs except for early insertion positions. (F) Row z-score normalized EPEs for DeepAccess motor neuron accessibility at each position show most motifs have higher EPEs when inserted at a position in the center of the DNA sequence. Some motifs (like Sp motifs) have higher EPEs at the beginning of the DNA sequence.(TIF)Click here for additional data file.S4 Fig(A) Estimates of EPE for 356 HOCOMOCOv11 transcription factor motifs on predicted motor neuron accessibility from a learned DeepAccess model. M sequences are selected randomly from regions closed in all 10 cell types, open in all 10 cell types, promoters which are closed in all 10 cell types, and promoters which are open in all 10 cell types. Shade indicates distribution size . Boxplot box indicates median and quartiles and whiskers extend to 1.5 times the inter-quartile range. Dots are outliers outside 1.5 times the inter-quartile range. (B) Estimates of significance of EPE for size and type of sequence used to estimate total sequence distribution. (C) Comparison of EPEs for 356 HOCOMOCOv11 transcription factor motifs for default (randomly selected 24 closed) compared to randomly selected closed (row 1), randomly selected open (row 2), promoter closed (row 3), and promoter open (row 4) shows while values of EPE dependent on selection of sequence distribution, rank remains relatively invariant.(TIF)Click here for additional data file.


Coffee is a widely consumed beverage that is naturally bitter and contains caffeine. Genome-wide association studies (GWAS) of coffee drinking have identified genetic variants involved in caffeine-related pathways but not in taste perception. The taste of coffee can be altered by addition of milk/sweetener, which has not been accounted for in GWAS. Using UK and US cohorts, we test the hypotheses that genetic variants related to taste are more strongly associated with consumption of black coffee than with consumption of coffee with milk or sweetener and that genetic variants related to caffeine pathways are not differentially associated with the type of coffee consumed independent of caffeine content. Contrary to our hypotheses, genetically inferred caffeine sensitivity was more strongly associated with coffee taste preferences than with genetically inferred bitter taste perception. These findings extended to tea and dark chocolate. Taste preferences and physiological caffeine effects intertwine in a way that is difficult to distinguish for individuals which may represent conditioned taste preferences. The consumption of these plant-based beverages has been associated with a lower risk of chronic diseases such as type 2 diabetes, cardiovascular diseases, and several types of cancer9. Although plausible underlying biological mechanisms have been identified, more research is needed to establish the causal role of these beverages in human health10. Thus, understanding determinants of beverage choice and consumption level is important to inform research and public health strategies.Coffee and tea are among the most widely consumed beverages in the world20. Furthermore, a variant near a gene encoding an olfactory receptor (OR5M8) has been associated with coffee intake. As coffee and tea have a bitter taste, it is also plausible that genetic variants related to bitter taste perception affect coffee and tea consumption. However, none of the loci identified in GWAS of coffee and tea intake overlap with TAS2R loci associated with taste perception of bitter compounds including propylthiouracil (PROP)/phenylthiocarbamide (PTC), caffeine, and quinine in GWAS  of coffee and tea drinking behavior have identified genetic variants involved in the metabolism and physiological effects of caffeine as determinants of the amount of these beverages consumed  that may yield genetic variants not reported previously by GWAS of total coffee and tea consumption.The current study uses genetic, dietary, and food preference (“liking”) data available from the UK Biobank (UKB) and two US cohorts, the Nurses’ Health Study (NHS) and Health Professionals Follow-up study (HPFS). We first test the hypothesis that published GWAS-confirmed variants related to taste are more strongly associated with black coffee consumption than with the consumption of total coffee or coffee with added sweetener or milk because their effects would not be masked by coffee taste manipulation. We focus on coffee but extend this hypothesis to tea since we expect similar but weaker associations of taste-related genetic variants with tea consumption, because tea is reportedly less bitter than coffee29. Participants provided informed written consent, completed touchscreen questionnaires on sociodemographic factors, lifestyle, and medical history followed by an interviewer-administered questionnaire, physical assessment, and biospecimen collection30. Subsets of the cohort have returned for follow-up assessments and have completed additional on-line questionnaires. The latter are the primary source of data for the current analysis. This study was covered by the generic ethical approval for UKB studies from the National Research Ethics Service Committee North West–Haydock , and all study procedures were performed in accordance with the World Medical Association Declaration of Helsinki ethical principles for medical research.In 2006–2010, the UKB recruited over 502,633 participants aged 37–73 years at 22 centers across England, Wales, and Scotland32. Detailed collection methods for coffee and tea intake and methods for estimating added milk and sweetener are provided in Supplementary Methods Unsweetened coffee consumers were defined as participants who never reported the addition of sweetener to coffee and never reported consumption of cappuccinos or lattes. No-milk coffee consumers were defined as participants who never reported the addition of milk to coffee and never reported consumption of cappuccinos or lattes. Black coffee consumers were those previously defined as both unsweetened and no-milk coffee consumers. Sweetened coffee consumers were defined as participants who always reported the addition of sweetener to coffee or who only consumed cappuccinos or lattes. Milk coffee consumers were defined as participants who always reported the addition of milk to coffee or who only consumed cappuccinos or lattes. Consumption data for coffee drinkers not defined as milk coffee consumers were set missing for genetic analysis but set to 0 for trait correlation analysis; the same approach was applied to other coffee consumption traits. Similar criteria were applied when defining different tea consumers while considering the relatively less detailed data collected for this beverage. Herein, tea-prepared-black refers to tea prepared without sweetener and milk to avoid confusion with tea type . The comprehensive list of coffee and tea traits account, in part, for cultural differences in how these beverages are prepared between UK and US cohorts.In 2009–2012, a subset of 122,292 participants who completed the baseline assessment center visit also completed at least two of five on-line 24 h dietary recalls33. Questionnaire items were randomized on a participant basis to reduce any bias that may occur due to tiredness. Participants were asked to rate how much he/she like each presented item on a scale from 1 (extremely dislike) to 9 (extremely like). Alternatively, they were given the option to select “Never tried” or “Prefer not to answer”. The current study focused on the following questionnaire items: liking for coffee with sugar, liking for coffee without sugar, liking for tea with sugar, and liking for tea without sugar. We also considered other bitter- and sweet-related items: liking for bitter foods, liking for dark chocolate, liking for Brussel sprouts, liking for cabbage, liking for bitter/ale and liking for sweet foods. In preliminary analysis, (1) liking for bitter vegetables was only weakly correlated with intake of bitter vegetables (r < 0.16) and liking for bitter foods (r < 0.10) and (2) liking for or intake of bitter vegetables were only weakly associated with coffee and tea traits (r < 0.15). Therefore, we did not pursue genetic analysis of bitter vegetable traits as results would unlikely be relevant for coffee and tea traits. After excluding 31 participants with liking score ranges of less than 4 across all 140 food items , up to 181,974 participants had data on food items of interest for the current analysis.In 2019, UKB participants with valid emails were invited to complete a food preferences questionnaire  analysis35.All UKB participants were genotyped using genome-wide arrays as detailed previouslyOf this UKB genetic sample, up to 126,599 individuals completed coffee- or tea-related liking scales and up to 86,006 participants had detailed coffee or tea consumption data based on 24-h recalls. Up to 61,955 participants had both liking and dietary intake data.36.Self-reported smoking status, physical activity, Townsend deprivation index, education, income, employment status as well as technician-measured body weight and height were collected during the UKB baseline assessment as described in detail previously37. In 1976, the NHS enrolled 121,700 U.S. female registered nurses aged 30–55 years38. Participants completed a mailed questionnaire on medical history and lifestyle characteristics every 2 years and a validated semi-quantitative food frequency questionnaire (FFQ) every 2–4 years39. All participants provided informed consent and study protocols were approved by the institutional review boards of Brigham and Women's Hospital and Harvard School of Public Health.In 1986, the HPFS enrolled 51,529 U.S. male health professionals aged 40–75 yearsWe considered diet data collected by the FFQ administered closest to and before the 2018 supplementary questionnaire described below. For NHS this was the 2010 FFQ and for HPFS the 2014 FFQ. For each FFQ item, participants were asked how often, on average, they had consumed a specified amount of each beverage or food over the past year. The participants could choose from nine frequency categories . Categorical measures of intake were converted to servings or cups/day by using the midpoint of each category; those reporting 6 or more servings or cups/day were assigned a daily intake of 6. The current analysis focused on coffee (regular or decaf) and tea  but also considered beer and dark chocolate as done for UKB. Total dietary energy intake and macronutrient composition of the diet were also derived from FFQs.40. Participants were asked “How do you usually drink your coffee or tea?” and for each beverage he/she could mark all response items as appropriate: “I do not drink this beverage”, “Black (nothing added)”, “Milk or cream”, “Non-dairy creamer/whitener”, “Sweetener ”, “Non-caloric sweetener ”. Participants were also asked “Do you avoid or drink less coffee because it tastes bitter?” (yes or no), “Do you avoid or drink less tea because it tastes bitter?” (yes or no). After one mailing, 5173 NHS (80% response) and 2940 HPFS (70%) returned the questionnaire.In 2018, more detailed questions regarding coffee and tea drinking behavior were included on a supplementary questionnaire mailed to NHS and HPFS participants previously selected for GWAS whom had not completed a supplementary questionnaire in 201041. To allow for maximum efficiency and power, we pooled HPFS and NHS samples genotyped on the same platforms and for each of the resulting datasets we imputed SNPs based on the 1000 Genomes (version 1.1 2016) cosmopolitan reference panel. Detailed methods and quality assurance pertaining to these genetic datasets have been reported elsewhere41. Any samples that had substantial genetic similarity to non-European reference samples were excluded from genetic analysis.Genetic data contributing to the current study were obtained from independent GWAS case–control studies nested within the cohorts, initially designed for outcomes of type 2 diabetes, coronary heart disease, gout, kidney stone, open-angle glaucoma, venous thromboembolism, prostate cancer (HPFS only), pancreatic cancer, colon cancer, mammographic density (NHS only), endometrial cancer (NHS only), ovarian cancer (NHS only) and breast cancer (NHS only). Studies were genotyped on Affymatrix, Illumina, Omni, OncoArray or HumanCoreExome platformsOf the participants with high-quality genetic data, 4295 NHS and 2447 HPFS participants had survey data on coffee or tea behaviors. Because these data were used in conjunction with FFQ data to derive a quantity of specific type of coffee consumed that aligned with that derived for UKB (see above for definitions), we excluded 286 HPFS and 175 NHS who did not complete an FFQ. We also excluded coffee data from 130 HPFS and 246 NHS who changed coffee drinking status (yes/no) between the FFQ and survey. The same approach was taken for tea data resulting in the exclusion of 497 HPFS and 836 NHS participants. In total, up to 2123 HPFS and 4064 NHS participants with genetic and coffee or tea data were included for the current analysis.Smoking status, physical activity, height, and body weight of participants were self-reported and obtained from questionnaires administrated to the entire NHS (2014) and HPFS (2016) cohorts preceding the 2018 supplementary questionnaire.25  using ANOVA. Significantly higher mean food liking scores were reported by participants who were younger, male, and had lower BMI compared to their respective counterparts (P < 0.0001). Because mean score differences were small, ranging from 0.19 (sex) to 0.03 (age), we only adjusted for these factors in our primary analyses as opposed to performing stratified analyses. The distributions of all coffee- and tea-related traits of interest were highly skewed and thus non-parametric tests were applied. Bivariate Spearman correlations were used to evaluate correlations among traits.All statistical analyses were performed using the SAS statistical package  unless indicated otherwise. Potential bias in use of the 9-point liking scale (unrelated to the content of the items) was evaluated by comparing mean scores of all 140 food items by age (at or above/below the median of 67 years), sex, smoking and BMI  avoiders due to bitter taste (yes vs no) using a logistic regression analysis. Given the highly correlated coffee and tea traits as well as the confirmatory and hypothesis testing nature of the current analysis, statistical significance was defined as P < 0.002 (0.05/23 SNPs) for both UKB and US (NHS/HPFS) cohorts; correcting only for the number of independent SNPs tested. Differences between beta-coefficients  in UKB were declared significantly different when their corresponding 95% confidence intervals did not overlap; an approach considered highly conservative48. Nevertheless, emphasis is placed on results consistent across UK and US cohorts.For UKB, multivariable-adjusted generalized linear modelling (GLM) was used to examine the association between each SNP (independent variable) and each continuous coffee/tea trait (dependent variable), adjusting for age, sex, smoking status, genotyping array and the top 20 PCs. Additional adjustments for BMI, physical activity, education level, Townsend index of socio-economic status, employment status, self-reported diabetes and heart disease and intake of total energy, alcohol, and other macronutrients (expressed as a proportion of energy) did not substantially change the results and thus we present results for the more basic model only. For NHS and HPFS, GLM was also used to examine the association between each SNP and each continuous coffee/tea trait, adjusting for age, smoking status, genotyping array and GWAS-specific case–control status. Results for NHS and HPFS were meta-analyzed with fixed effects using METAL49. Genome-wide significant (P < 5 × 10–8) SNP-trait associations were followed up in NHS and HPFS when possible and using statistical models as described above for candidate SNP-analysis.We performed GWAS of continuous coffee and tea traits in UKB . The rank-based inverse (blom) normal transformation was applied to each trait prior to GWAS and we excluded SNPs with MAF < 0.05 and INFO scores < 0.4. We performed genome-wide linear regressions using PLINK2 assuming an additive genetic model and adjusting for age, sex, smoking status and top 20 PCs. FUMA was used for displaying, pruning and annotating UKB summary-level resultsTable The consumption of coffee and tea prepared black was more common in the US cohorts than the UKB, because US participants were less likely to add milk to these beverages than UKB participants Table . CorrelaADORA2A), are previously reported GWAS-confirmed loci for indicated traits. Following, we present associations between our novel consumption/liking traits and i) GWAS-confirmed loci for bitter taste perception and ii) GWAS-confirmed loci for coffee/tea consumption behavior. We annotate statistically significant (P < 0.002) associations in Tables and note nominally significant (0.002 < P < 0.05) associations below. We then present results from GWAS of our novel consumption/liking traits in UKB applying the traditional GW-significance threshold (P < 5 × 10–8) along with replication in NHS/HPFS.Candidate “Taste” and “Behavior” loci selected for the current analysis are presented in Supplementary Table TAS2R19 (rs10772420 A) was significantly (P < 0.002) inversely associated with liking coffee with sugar (β = − 0.04) in UKB. Similarly, the variant was nominally (0.002 < P < 0.05) associated with less liking of tea with sugar (β = − 0.03) and greater liking of tea without sugar (β = 0.03). In UKB, the variant was nominally associated with lower coffee intake (β = − 0.02), regardless of the type of coffee. In the US cohorts, the variant was nominally significantly associated with higher intake of black (β = 0.12), unsweetened (β = 0.07), and no-milk (β = 0.10) coffee, but not substantially with total or other types of coffee. Although the TAS2R19 variant was significantly associated with less liking of dark chocolate (β = − 0.03) in UKB, it was not associated with intake of tea, beer, or dark chocolate in any of the cohorts.Associations between genetic variants related to taste perception and the liking and intake of different types of coffee, tea, and other bitter foods in UKB are shown in Table TAS2R38 variants were also inversely associated with liking dark chocolate , but not associated with liking coffee, tea, or bitter foods. However, the same variants were significantly associated with lower coffee intake (β = − 0.02), and higher tea intake (β = 0.03). The effect estimates were greatest for sweetened tea (β = 0.06) but not significantly different from other tea types. Results in the US cohorts were directionally consistent for coffee and tea intake but were not significant.PROP-taste sensitive TAS2R14 alleles were nominally associated with higher liking for coffee and intake of caffeinated coffee, regardless its preparation type. In the US cohorts this variant was nominally associated with higher intake of dark chocolate  but not with intake of coffee or tea.The caffeine-taste sensitive TMEM18, GCKR, POR, ADORA2A (rs2330783), CYP1A2 , AHR, CYP2A6, SEC16B, OR5M7P, ENSA, and MLXIPL were significantly associated with total coffee intake . Variants near AHR (rs4410790 C), CYP1A2 (rs2472297 C), and OR5M7P (rs597045 A) were more strongly associated with higher intakes of caffeinated than decaffeinated coffee intake. Variants near AHR (rs4410790 C), CYP1A2 (rs2472297 C), ABCG2 (rs1481012 A), ADORA2A (rs2330783 G), CYP2A6 (rs56113850 C), MC4R (rs66723169 A), SEC16B (rs574367 T), POR (rs17685 A) and TMEM18 (rs10865548 G) were exclusively or more strongly associated with liking coffee without sugar (P < 0.002) than liking coffee with sugar, but their association with higher coffee intake did not vary substantially by preparation type. Variants near ALDH2, EFCAB, FIBIN, NRCAM, PDSS2, and BDNF were not associated with coffee consumption in the current UKB sample, regardless of how coffee was usually prepared.Variants near ORA2A rs230783, CYAHR (rs4410790 C) and CYP1A2 (rs2472297 C) were also significantly associated with higher total tea intake (P < 0.002). This association was stronger for tea with milk than for tea-prepared black, and stronger for caffeinated than for decaffeinated tea. TMEM18 (rs10865548 G) was significantly associated with lower tea intake and ADORA2A (rs5751876 C) was significantly associated with higher tea intake regardless of how it was prepared. ABCG2 (rs1481012 A) was significantly associated with liking tea without (β = 0.06) but not with sugar, and was associated with higher tea intake regardless the type. MC4R (rs66723169 A) and SEC16B (rs574367 T) were significantly associated with less liking of tea with sugar (β = − 0.04), but neither was significantly associated with tea intake.Variants near ADORA2A (rs2330783 G), AHR (rs4410790 C), CYP1A2 (rs2472297 C), CYP2A6 (rs56113850 C), and POR (rs17685 A) associated with higher coffee intake in published GWAS were also significantly (P < 0.002) associated with greater liking of dark chocolate with effect estimates similar to those for liking coffee without sugar. The ADORA2A rs5751876 C variant previously associated with caffeine-induced anxiety and wake promotion was nominally inversely associated with all coffee-liking traits  and more strongly and significantly with less liking of dark chocolate . The same pattern of results was observed for the correlated ADORA2A rs5760444 C variant  previously linked to coffee intake in GWAS of Asians17. ADORA2A (rs2330783 G) was also significantly associated with higher dark chocolate intake and lower beer intake. No other behavior-loci were associated with dark chocolate intake in UKB. In post-hoc analysis we examined SNP associations with milk chocolate in UKB and observed statistically significant associations of ADORA2A (rs2330783 G), AHR, CYP1A2, and POR with liking milk chocolate, but not milk chocolate intake. The directions of associations with liking milk chocolate were opposite to those reported for liking dark chocolate. None of the loci was associated with liking of bitter foods.Variants near AHR (rs4410790 C) and CYP1A2 (rs2472297 T) were significantly associated with higher total coffee intake and effect sizes tended to be larger for black, unsweetened, or no-milk coffee than for coffee with added milk or sugar. CYP1A2 (rs2472297 T) was significantly associated with caffeinated but not decaffeinated coffee. A similar but smaller difference in association between caffeinated and decaffeinated coffee was observed for the AHR variant. The odds ratio (95% CI) of reporting ‘avoiding coffee because of its bitterness’ for each additional allele was 0.83  for CYP2A6 (rs56113850 C), 0.64  for ADORA2A (rs2330783 G), and 1.13  for ADORA2A (rs5751876 C).Few associations of the evaluated genetic variants with coffee intake met statistical significance (P < 0.002) in US cohorts (Supplementary Table novel genome-wide significant (P < 5 × 10–8) loci based on a GWAS leveraging the new and more refined coffee and tea phenotypes in UKB  was associated with lower unsweetened tea intake in the UKB (P = 2.3 × 10–8) and US cohorts (P = 0.02). TAS2R (rs2418224 G) was associated with greater liking of tea with sugar (P = 1.6 × 10–8) and coffee with sugar (P = 4.3 × 10–6) and less liking of tea without sugar (P = 0.0003) and coffee without sugar (P = 0.001) in the UKB. The same variant was nominally associated with liking sweet foods  but not bitter foods, dark chocolate or beer (results not shown). While not associated with consumption behavior in UKB, this TAS2R variant was associated with lower intakes of black , unsweetened , and no milk  coffee in the US cohorts.Only associations of new variants at 22q11.23  and quinine (rs10772420 A) perception; explaining about 2% and 6% of the phenotype variance, respectively25. The quinine-taste sensitive variant (rs10772420 A) has also been associated with caffeine perception but the effect is weaker and in the opposite direction to that for quinine25. We previously examined taste-related variants in relation to total coffee and tea intake in UKB. In that analysis, the quinine-taste sensitive TAS2R variant was associated with lower coffee intake and higher tea intake, whereas the caffeine-taste sensitive TAS2R variant was associated with higher coffee intake and lower tea intake21. We now extend that genetic research by using data on food liking and intake of different types of coffee and tea available for a subset of UKB participants. We show that the caffeine-taste sensitive variant (rs2708377 C) is associated with consumption of coffee regardless of how it is prepared but tends to be more strongly associated with caffeinated than with decaffeinated coffee. This result reiterates that caffeine-learned behavior , may explain the preference for this naturally bitter tasting chemical52. Coffee, tea, and chocolate do not contain quinine. We observed significant to nominal associations between several of our novel traits and quinine-taste variation (rs10772420) but in directions difficult to interpret. The opposing effects of this variant on quinine and caffeine perception25 or complex bitter-sweet taste interactions53 may underlie the unusual pattern of associations but merits further study. The well-studied PROP-sensitive TAS2R38 variants were associated with lower coffee and higher tea intake as we reported previously21. The direct association between these PROP-sensitive variants and tea consumption was especially strong for sweetened tea suggesting a bitterness-threshold at which tea becomes unpleasant tasting for individuals with these variants. The specificity of these associations with coffee/tea intake (vs. coffee/tea liking), which was not observed for rs2708377 and rs10772420, may be due to chance or perhaps a latent trait distinguishing intake from hedonic traits55.We examined genetic variants that have previously been associated with bitter taste perception in relation to coffee and tea related traits. In previous GWAS, variants in the TASR2 variant in the 12p13.2 region (rs2418224 G) that was associated with greater liking of tea with sugar. The nominally significant and opposite directions of effect on liking sweetened and non-sweetened versions of the beverages, the lack of association with other bitter taste traits, and the association with liking sweet foods, together suggest an effect of this SNP on general sweet-perception or preference. Our GWAS analysis in UKB also pointed to FTO, a well-established obesity locus. An FTO variant , previously associated with higher BMI, was associated with higher unsweetened coffee intake and liking and less liking of sweetened coffee and tea. In our previous GWAS of bitter and sweet beverages the same FTO variant was associated with higher coffee intake in the UKB but this result was not replicated in US cohorts19. However, the same variant was associated with lower SSB intake in both the UKB and US cohorts. A recent GWAS in UKB also observed associations between FTO variants and total sugar intake56. Taken together, our current study findings are likely attributed to sweet taste as opposed to coffee or bitter taste and the inability to replicate UKB FTO-coffee associations in US cohorts may be due to population differences in the social or food environment. Variants in MC4R, SEC16B and TMEM18 that associated with coffee consumption in previous GWAS are also GWAS-confirmed obesity loci. In the current study, the coffee-increasing variants  were inversely associated with liking sweetened coffee and tea. None of these loci were associated with SSB or sweet taste perception in GWAS56.Our GWAS identified an additional independent and novel intake did not vary substantially by preparation type. The stronger association with liking coffee without sugar was unexpected for loci with known roles in caffeine metabolism or physiological effects, but not in taste perception, such as AHR, CYP1A2, POR, CYP2A6 and ADORA2A. Again, these findings suggest that taste/smell and caffeine effects are not as distinct as expected. Individuals consuming more coffee because of a genetic predisposition to increased caffeine metabolism or tolerance may learn to associate coffee/caffeine bitter taste with the favorable physiological effects of caffeine. Our genetic findings align with results of a small clinical study by Masi et al.57. Individuals with a higher caffeine metabolism rate  gave lower bitterness ratings for espresso coffee samples and caffeine, but not quinine, solutions and added less sugar to coffee57.Most SNPs identified in previous GWAS of coffee consumption were replicated in the UKB subsample used in the current study. Replicated SNPs were generally more associated with liking coffee without sugar than coffee with sugar but their association with increased coffee AHR and CYP1A2 are the strongest and most robust signals in GWAS of coffee and caffeine intake20. Consistent with the role of the enzymes encoded by these genes in caffeine metabolism58, we observed stronger associations of these variants with caffeinated than with decaffeinated coffee in all cohorts included in the current study. We also observed that OR5M7P (rs597045 A) was more strongly associated with caffeinated than decaffeinated coffee in UKB. This variant is not associated with caffeine perception in GWAS25. OR5M7P is a pseudogene upstream of OR5M8, one of many genes encoding specific olfactory receptors which function in the perception of smell. Smell and taste are highly related but why this variant affects caffeine-and not taste-related traits is unclear but may be another case in-point of conditioned taste preferences.SNPs in ADORA2A, encoding the adenosine 2A receptor, a target for caffeine which mediates the psychostimulant effect of the drug58. As such, we did not expect SNPs in ADORA2A to be differentially associated with coffee and tea traits defined independent of caffeine content. ADORA2A (rs5751876 C) is thought to increase sensitivity to caffeine as it has been associated with greater caffeine-induced anxiety and alertness and lower caffeine intake in several candidate gene studies59. In the current study, this variant was associated with less liking of coffee (with or without sugar) and while not associated with coffee consumption it was associated with higher tea intake in the UKB. Taken together, these results suggest that individuals with ADORA2A (rs5751876 C) avoid heavy caffeine intake. Since coffee contains twice the amount of caffeine than tea, these individuals may prefer tea over coffee. In US cohorts, rs5751876 C carriers were more likely to avoid coffee because it tastes bitter; a finding that further illustrates how some individuals are unable to separate the physiological effects of caffeine from taste preferences. ADORA2A (rs2330783 G) was associated with liking coffee without sugar and higher coffee intake without differences in association by type of coffee. In US cohorts, rs2330783 G carriers were less likely to avoid coffee because of its bitter taste. Our GWAS of unsweetened tea in UKB identified variants near ADORA2A  not in LD with those described above that was also associated with unsweetened tea in the US cohorts. Unlike AHR and CYP1A2, none of the SNPs in ADORA2A differentially associated with caffeinated and decaffeinated coffee or tea. To our knowledge, adenosine receptors do not play a role in taste perception. Whether our results for ADORA2A rs2330783 and rs3788372 are mediated by taste or caffeine is unclear and warrants further investigation.We investigated three SNPs in ADORA2A, CYP1A2, CYP2A6, AHR and, to a weaker extent, POR variants associated with higher coffee intake (US cohorts) and liking (UKB) were also associated with increased dark chocolate intake (US cohorts) and liking (UKB). No association or associations in the opposite direction were observed with liking and intake of milk chocolate and other bitter foods, suggesting that caffeine  may be underlying the observed associations with dark chocolate. Dark chocolate contains more caffeine per weight than milk chocolate60 and while the amount is still less than the content in coffee and tea it may be detected by individuals sensitive to caffeine. Dark chocolate is also a unique source of theobromine, another methylxanthine with psychostimulant effects63.66. Specifically, the 24-h diet collections in UKB may not reflect usual intake and the FFQs used in the US cohorts may be prone to reporting errors. In addition, liking traits were only available for UKB and not for the US cohorts. The US cohorts included a non-representative group of elderly whose sense of taste and small may be reduced and beverage choices more likely affected by medical issues. Several genetic variants that were associated with coffee or caffeine intake in previous GWAS studies were not replicated in the current study. That probably reflects the smaller sample size in the sub-cohorts with more detailed information used in the current analysis. In addition, the association between ALDH2 variation and coffee consumption has only been reported in Japanese14 and given the low frequency of these variants in populations of European ancestry the lack of replication in the current study was expected. Finally, there are currently no published GWAS-SNPs for perception of other bitter compounds in coffee such as maillard reaction products, cafestol, chlorogenic acid derivatives or other uncharacterized coffee-bitter compounds69 thus limiting our candidate SNP-approach.Strengths of the current study include the use of novel and comprehensive coffee and tea traits in independent cohorts. Nevertheless, several limitations need to be considered. The liking and dietary intake measures used are subject to bias and measurement error as discussed in detail previously70. Whether a genetic instrument captures total coffee/caffeine intake, only certain types of coffee, or not only coffee but a broader characteristic, impacts the interpretation and translation of studies. For example, evidence for a causal relationship between black coffee and type 2 diabetes is very different than a causal relationship between coffee and type 2 diabetes. A cautionary approach to genetic instrumental variable studies is particularly relevant now that weaker non-genome-wide significant variants are included in such studies.Genetic markers of coffee and caffeine consumption are increasingly used as instrumental variables to seek causal insight to coffee/caffeine and healthIn summary, our genetic analysis suggests the psychostimulant effects of caffeine outweighs the bitterness of caffeine. A greater preference for caffeine based on genetic differences in the physiological effects of caffeine leads to a stronger preference for the taste/smell of coffee and dark chocolate. Similarly, greater sensitivity to the adverse physiological effects of caffeine was associated with avoiding the taste of coffee. Taste preferences and physiological caffeine effects thus seem to become entangled in a way that is difficult to distinguish for individuals. These potential examples of conditioned taste preferences or aversions merit further clinical investigation. This apparent disruption of an innate aversion to bitter taste and its genetic correlation with coffee preferences has important relevance to food and beverage development as well as genetic epidemiological studies of coffee.Supplementary Information 1.Supplementary Information 2.


Scientific Reportshttps://doi.org/10.1038/s41598-019-51137-5, published online 11 October 2019Correction to: The original version of this Article required clarification, as the limitations of the approach were not clearly stated, and the marginal association between Complete Kawasaki Disease and the first dose of 13-valent pneumococcal conjugate vaccine was not placed into adequate context.The text in the Abstract,“We did not detect a significant increased risk for overall KD among PCV13 recipients. However, a significant association between PCV13 and Complete KD was noted following receipt of the first dose of PCV13.”Now reads,“We did not detect a significant increased risk for overall KD among PCV13 recipients.”The text in the Discussion,“We did not detect a statistically significant increased risk for KD among PCV13 recipients. However, an approximate two fold increased risk of Complete KD within the 28 day risk interval following receipt of the first dose of PCV13 compared to control periods outside the risk window was noted. There is an urgent need to confirm this finding in future studies. Subsequent doses of PCV13 did not result in an increased risk of Complete KD. The results were robust in sensitivity analysis when we used monthly age-intervals or included vaccinated cases only in our models. Reassuringly, all KD cases with onset within the risk interval were treated (Intravenous Immunoglobulin (IVIG) and aspirin) and none had evidence of coronary artery sequelae in their final echocardiogram.”Now reads,“We did not detect a statistically significant increased risk for KD among PCV13 recipients, for any of the four doses. However, an approximate two fold increased risk of Complete KD within the 28 day risk interval following receipt of the first dose of PCV13 compared to control periods outside the risk window was noted. This could be a chance finding as no adjustment was made for multiple comparisons. Of the 8 associations tested, in all but one test we found no evidence for an association between PCV13 and KD risk. Subsequent doses of PCV13 did not result in an increased risk of Complete KD. Reassuringly, all KD cases with onset within the risk interval were treated (Intravenous Immunoglobulin (IVIG) and aspirin) and none had evidence of coronary artery sequelae in their final echocardiogram.”The text in the Conclusion,“In conclusion, using a self-controlled case series design, we did not detect a statistically significant association between PCV13 and overall KD. However, there was a statistically significant association between PCV13 and Complete KD following receipt of the first PCV13 dose in children < 2 years old. There was no evidence of any coronary artery sequelae following appropriate treatment for Complete KD following receipt of PCV13. Hence, clinical vigilance with early diagnosis and appropriate treatment of KD remains important. There is an urgent need for more studies in other settings to verify this signal in view of the potential impact on the risk-benefit of PCV13 immunization programmes globally.”Now reads,“In conclusion, using a self-controlled case series design, we did not detect a statistically significant association between PCV13 and overall KD. Although post hoc subgroup analyses of Complete KD (uncorrected for multiple comparisons) suggested a marginal association (for first PCV13 dose in children < 2 years old), this may well represent a type I error. There was no evidence of any coronary artery sequelae following appropriate treatment for Complete KD following receipt of PCV13. Hence, whilst our data overall suggests that the vaccine does not pose a risk, ongoing surveillance for any adverse events (including KD) related to vaccination is warranted. Early diagnosis and appropriate treatment of KD remains important.”This has now been corrected in the PDF and HTML versions of the Article.