The COVID-19 pandemic causes severe morbidity and mortality. This multi-country study aimed to explore risk factors that drive mortality in COVID-19 patients who received neither dexamethasone nor remdesivir. We analyzed a cohort of 568 survivors and 507 non-survivors from China, European regions, and North America. Elderly males ≥70 years accounted for only 25% of survivors, but this rate was significantly higher in non-survivors from China (55%), European regions (63%), and North America (47%). Compared with survivors, non-survivors had more incidences of comorbidities such as cerebrovascular disease and chronic obstructive pulmonary disease . Survival analyses revealed age, male gender, shortness of breath, cerebrovascular disease, and COPD as mortality-associated factors. Survival time from symptom onset was significantly shorter in elderly versus young patients (median: 29 versus 62 days), males versus females (median: 46 versus 59 days), and patients with versus without comorbidities (mean: 41 versus 61 days). Mortality risk was higher in elderly males with comorbidities than in young females without comorbidities . Elderly male survivors with comorbidities also had longer hospital stays than other survivors . Overall, the high mortality risk in elderly males with COVID-19-associated comorbidities supports early prevention and critical care for elderly populations. With a strong potential of sustained human-to-human transmission, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can quickly spread to vulnerable populations such as elderly individuals with aging-related immune disorders.Coronavirus Disease 2019 (COVID-19) is a global pandemic that is causing significant, widespread increases in morbidity and mortality, while effective antiviral treatments are still under development [+CD8+ T cells and cardiac troponin I were found to be important risk factors based on a small cohort of 21 non-survivors and 158 survivors [Early studies of COVID-19-associated mortality and risk factors have mostly involved small cohorts of non-survivors in Wuhan –7. For iTo reveal mortality-associated factors, our study collected a large cohort of survivors who recovered from COVID-19 and non-survivors who died of the disease before May 1, 2020. Due to the authorization of remdesivir on May 1 and dexamethasone on June 17 that may affect our results of mortality-related factors, our data collection initially retrieved clinical records of COVID-19 patients who received neither remdesivir nor dexamethasone. By using epidemiological and clinical records of COVID-19 patients from different countries, this multi-country study revealed a high-risk population of elderly patients with specific comorbidities. Given the rapid spread of COVID-19 which is causing high morbidity and mortality worldwide, a special focus on elderly patients with specific comorbidities in current COVID-19 guidelines should be implemented.This study retrieved a multi-country dataset of 1075 COVID-19 patients, including 232 survivors and 183 non-survivors in China, 208 survivors and 258 non-survivors in European regions, and 128 survivors and 66 non-survivors in North America . None ofWe analyzed the age distribution of the 1075 patients in our dataset . The youOur gender analysis suggested that COVID-19 killed more males than females across China, European regions, and North America . The proWe analyzed the proportions of those with pre-existing comorbidities in a subset of 359 survivors and 239 non-survivors whose comorbidities were recorded . As expeWe next analyzed baseline findings of computed tomography (CT) scans. Incidences of ground-glass opacity, pleural adhesions, and pleural effusion were significantly higher in non-survivors than in survivors . Death-related complications were recorded in a subset of 102 non-survivors. The most common complications were respiratory failure (51%), acute respiratory distress syndrome (28.4%), multiple organ failure (27.5%), shock (22.5%), and hypoxia (13.7%). Males and females had similar incidences of death-related complications .To reveal mortality-associated factors, we examined a subset of 242 survivors and 157 non-survivors whose records contained complete information regarding age, gender, comorbidities, symptoms, clinical outcomes, and survival time from symptom onset to clinical outcomes . As showWe next used Cox proportional hazards models to evaluate time-dependent hazards of baseline factors for the clinical outcome of death. Univariate Cox regression analyses revealed seven risk factors: age, male gender, shortness of breath, any comorbidity, cerebrovascular disease, cardiovascular disease, and COPD  High mortality risk of COVID-19 was consistently found among elderly males from China, Europe, and North America. (ii) Risk factor and survival analyses revealed mortality-associated risk factors of age, male gender, shortness of breath, and chronic comorbidities such as cerebrovascular disease and COPD. (iii) Mortality-associated risk factors exert an impact on the survival time and hospital stays of elderly patients. To reduce mortality rates and hospital burden, we carried out a multi-country study on COVID-19-induced mortality, and the results support the early prevention and critical care of elderly males with specific comorbidities.In agreement with previous findings , 10, 11,Why are elderly adults more vulnerable to COVID-19? Although the exact mechanisms remain unclear, several hypotheses have been proposed . First, http://globalhealth5050.org/covid19/).In agreement with previous studies –23, our Why does COVID-19 kill more males than females? Although the exact mechanisms remain unknown, several hypotheses have been proposed to explain gender disparities. (i) The X chromosome encodes the human receptor angiotensin-converting enzyme 2 , which is the human receptor mediating SARS-CoV-2 entry into human cells. Expression of ACE2 might vary in different populations due to genetic differences . (ii) MaIn agreement with previous studies –41, we oWhat are the impacts of comorbidities on the morbidity and mortality of COVID-19? Although the possible mechanisms are still under investigation, answers likely depend on the type of comorbidity. First, our analyses revealed that the comorbidities of cerebrovascular disease and COPD are time-dependent factors associated with a fatal outcome of COVID-19 , 4. Of iFirst, we initially intended to collect all available online records of COVID-19 cases worldwide, but detailed medical records of individual patients were mostly lacking probably because the privacy of patient data is protected and detailed records are often not published in an emergent outbreak. Nevertheless, our major findings of elderly males at high risk for COVID-19 have been consistently confirmed by single-country studies –23. FutuGiven the rapid spread of COVID-19 worldwide, a better understanding of high-risk populations is important for decision making in the treatment and prevention of the disease. The clinical features of non-survivors inside and outside Wuhan are similar , supportst, 2020, in the Sino-French New-City Tongji Hospital in Wuhan. At hospital admission, all patients were diagnosed with COVID-19. Nasal and pharyngeal swab specimens were collected to assess the presence of SARS-CoV-2 using real-time RT-PCR tests according to WHO interim guidelines. All survivors, who fulfilled discharge criteria, were discharged for 14-day home quarantine based on the New Coronavirus Diagnosis and Treatment Guidelines in China  retrieved a total of 1267 patients, including 666 survivors and 601 non-survivors. Because remdesivir and dexamethasone significantly reduce COVID-19 mortality , 9, our A total of 568 survivors and 507 non-survivors with basic information regarding age, gender, country, and clinical outcomes were included in our age-gender analysis . AdditioOur datasets included demographic features , signs and symptoms, pre-existing comorbidities, complications before death, and other available data  for COVID-19 survivors and non-survivors. Symptoms, comorbidities, and complications were treated as categorical variables (yes or no). Clinical outcomes were defined by the status of death (non-survivors) or hospital discharge after the clearance of COVID-19 (survivors). For non-survivors, the survival time was defined by days from symptom onset to the date of death. Since death was not observed before hospital discharge, survivors were treated as censored data in which the observation time was defined by days from symptom onset to hospital discharge.2 tests or Fisher’s exact tests as appropriate. Survival analyses were performed using Cox proportional hazards models. Hazard ratios with the 95% confidence interval were assessed to reveal the effects of risk factors on survival-time outcomes: either death for non-survivors or hospital discharge for survivors. Kaplan-Meier curves were built to show survival time among patient groups (e.g. elderly versus young patients), and significant differences were examined by log-rank tests. A common approach called pairwise deletion was applied to handle missing data. Since no random sampling was conducted, all statistical analyses were descriptive. To reduce the confounding effect in this nonrandomized study, the propensity-score matching method in SPSS V26.0 was applied with default settings  to assess whether any observed confounding was adequately reduced. The effect size of risk factors was estimated by Cohen's d [In comparisons of COVID-19 survivors and non-survivors, continuous variables were analyzed using Mann-Whitney U tests, while categorical variables were analyzed using χohen's d . In all This retrospective study was performed in accordance with the Helsinki Declaration and was approved by the Ethics Committees of The Second Xiangya Hospital, Central South University (ID: LYF2020060), and The First Hospital of Changsha (ID: KX2020002). Written informed consent was waived for analyzing archived records in this retrospective study.Supplementary FiguresSupplementary TablesSupplementary Method 1Supplementary Dataset 1


Prior cost-effectiveness studies of post-mastectomy radiotherapy (PMRT) only compared conventional radiotherapy versus no radiotherapy and only considered tumor control. The goal of this study was to perform cost-effectiveness analyses of standard of care (SOC) and advanced PMRT techniques including intensity-modulated radiotherapy (IMRT), standard volumetric modulated arc therapy (STD-VMAT), non-coplanar VMAT (NC-VMAT), multiple arc VMAT (MA-VMAT), Tomotherapy (TOMO), mixed beam therapy (MIXED), and intensity-modulated proton therapy (IMPT).Using a Markov model, we estimated the cost-effectiveness of various techniques over 15 years. A cohort of women (55-year-old) was simulated in the model, and radiogenic side effects were considered. Transition probabilities, utilities, and costs for each health state were obtained from literature and Medicare data. Model outcomes include quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratio (ICER).For the patient cohort, STD-VMAT has an ICER of $32,617/QALY relative to SOC; TOMO is dominated by STD-VMAT; IMRT has an ICER of $19,081/QALY relative to STD-VMAT; NC-VMAT, MA-VMAT, MIXED are dominated by IMRT; IMPT has an ICER of $151,741/QALY relative to IMRT. One-way analysis shows that the probability of cardiac toxicity has the most significant impact on the model outcomes. The probability sensitivity analyses show that all advanced PMRT techniques are more cost-effective than SOC at a willingness-to-pay (WTP) threshold of $100,000/QALY, while almost none of the advanced techniques is more cost-effective than SOC at a WTP threshold of $50,000/QALY.Advanced PMRT techniques are more cost-effective for breast cancer patients at a WTP threshold of $100,000/QALY, and IMRT might be a cost-effective option for PMRT patients. About 1 in 8 US women will develop invasive breast cancer over the course of her lifetime, and the number of women being diagnosed continues to increase (seer.cancer.gov). A mastectomy is highly recommended for patients with locally advanced primary breast cancer and extensive lymph node involvement, and post-mastectomy radiotherapy (PMRT) has been shown to improve the overall survival for invasive breast cancer patients by reducing the risk of tumor recurrence and cancer mortality [The current standard of care (SOC) PMRT technique in the US is using conventional parallel-opposed tangent photon fields to treat the lateral chest wall plus oblique electron fields; for patients with advanced disease, supraclavicular and axillary nodes are treated with additional photon fields . In the Among all cancer sites, expenditures for female breast cancer remain the highest and will continue to rise to $20.5 billion by 2020 . It is cThe goal of this study was to perform cost-effectiveness analyses of various PMRT techniques including conventional SOC, fixed-beam IMRT, STD-VMAT, NC-VMAT, MA-VMAT, TOMO, MIXED, and intensity-modulated proton therapy (IMPT). Besides tumor coverage, late side effects (cardiac toxicity and secondary cancers) after PMRT were also included in the analyses.A Markov model Fig.  was builThe transition probabilities for SOC PMRT were taken from literature , 22–27. For advanced PMRT techniques, the transition probabilities were largely lacking in the literature. In this study, probabilities of tumor coverage including local recurrence and metastasis after advanced PMRT were assumed to be the same as those after SOC PMRT, while probabilities of radiogenic side effects after advanced PMRT were calculated based on normal tissue complication probability (NTCP) , lifetimThe annual mortality rates due to breast cancer were derived from Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) . The norTable CancerMath is the latest web-based breast cancer prognostic tool that can predict mortality rate in each year for the first 15 years after the current SOC treatment, and the external validity of our model was assessed by comparing 15-year overall survival and breast cancer mortality of patients who received SOC PMRT with the predicted results from CancerMath.We performed a series of one-way sensitivity analyses to determine the variability in the ICER as a function of the probabilities, utilities, and treatment costs of contralateral breast cancer, lung cancer and heart toxicities for seven advanced PMRT techniques versus SOC PMRT.We also performed probability sensitivity analyses (PSA). The probabilities, utilities and costs were varied simultaneously across their distributions using a second-order Monte Carlo simulation. Transition probabilities and utilities were modeled using a beta-distribution and cost was modeled using a gamma distribution as recommended in the literature . The cosFor 55-year-old women with breast cancer, our model predicted a 15-year overall survival rate of 69.7% and breast cancer mortality rate of 19.2%, whereas CancerMath estimated an overall survival rate of 70.5% and breast cancer mortality rate of 18.0%. These comparisons suggest that our model’s predictions are similar to real clinical outcomes.Treatment cost, QALY, and ICER values for all seven advanced PMRT techniques are shown in Table WTP acceptability curves for seven techniques are shown in Additional file To the best of our knowledge, this study evaluated the cost-effectiveness of seven advanced PMRT techniques compared with SOC for the first time. Both tumor coverage and radiogenic late effects were considered in our model. The uncertainty of probabilities, utilities and treatment costs of radiogenic late effects were analyzed using one-way analysis and PSA.We found most of the advanced techniques would be more cost-effective than SOC PMRT at a WTP of $100,000/QALY. IMRT exhibits the lowest ICER relative to SOC $27,310/QALY) among all techniques for the patient cohort, which is possibly because fixed beam IMRT can significantly reduce contralateral breast doses and lower the probability of developing secondary cancer in contralateral breast. IMPT has the highest ICER relative to SOC , which is mainly due to the high cost of the initial treatment  IMRT, (b) STD-VMAT, (c) NC-VMAT, (d) MA-VMAT, (e) TOMO, (f) MIXED and (g) IMPT. Bars indicate range of costs per QALY for given range-specific model input variables. P_: probability of developing certain radiogenic side effect using certain PMRT technique. Figure S3. Cost-effectiveness acceptability curves from PSA that comparing the cost-effectiveness of SOC and (a) IMRT, (b) STD-VMAT, (c) NC-VMAT, (d) MA-VMAT, (e) TOMO, (f) MIXED and (g) IMPT at different willingness to pay (WTP) thresholds. Two dashed lines represent WTP thresholds of $50,000/QALY and $100,000/QALY.


Taken together, our findings provide further insight into the cell-autonomous signals in post-pregnancy MECs that underpin the regulation of gene expression, cellular activation, and resistance to malignant development.Pregnancy causes a series of cellular and molecular changes in mammary epithelial cells (MECs) of female adults. In addition, pregnancy can also modify the predisposition of rodent and human MECs to initiate oncogenesis. Here, we investigate how pregnancy reprograms enhancer chromatin in the mammary epithelium of mice and influences the transcriptional output of the oncogenic transcription factor cMYC. We find that pregnancy induces an expansion of the active cis-regulatory landscape of MECs, which influences the activation of pregnancy-related programs during re-exposure to pregnancy hormones in vivo and in vitro. Using inducible  Mammary epithelial cells are epigenetically modified during pregnancy, these changes can influence the pre-disposition to cancer. Here, the authors examine the epigenetic landscape of mammary epithelial cells pre and post pregnancy and identify changes to the epigenetic landscape, which can protect mice from Myc induced cancer. Substantial tissue remodeling, including the expansion of epithelial cells and ductal structures, is followed by the accumulation of milk droplets as gestation progresses. During lactation, milk production is synchronized with milk release by a series of transcriptional and mechanical events in luminal and myoepithelial cells. As lactation ceases, the mammary gland returns to a nonsecretory state and adopts a tissue organization that resembles the prepregnancy one6. Several reports have also suggested that post-pregnancy mammary glands from several mammalian species respond robustly to the signals of consecutive pregnancies10, suggesting a molecular memory of prior pregnancies. In fact, whole-genome bisulfite sequencing has revealed that pregnancy induces stable and specific changes to DNA methylation in MECs12. These epigenetic alterations correlated with the enhanced kinetics of gene reactivation in a subsequent pregnancy, and suggest that loss of DNA methylation may underlie epigenetic memory in the post-pregnancy mammary gland12.However, post-pregnancy mammary epithelial cells (MECs) are distinct from their pre-pregnancy counterparts, with transcriptional networks being differentially regulated in post-pregnancy mammary tissue from mice/rodents and humans15, there is a long-term reduction of breast cancer risk for women completing a full-term pregnancy before the age of 3019. In several rodent systems, the inhibition of carcinogen-induced and genetic-induced mammary tumorigenesis has also been reported post pregnancy or upon pre treatment with pregnancy hormones22. Given the stability of the molecular programs instated by pregnancy in MECs, and the longevity of cancer preventive effects in rodents and humans, it is likely that these protective effects have a nongenetic basis.Pregnancy signals exert functions in the MECs that go beyond the primary function of milk production and secretion, and include modifying the risk of breast cancer in rodents and humans. While there is an increase in breast cancer risk for roughly 5–10 years after parturitionTo evaluate this hypothesis, we characterized the dynamics of gene reactivation and enhancer organization in murine MECs as they respond to pregnancy signals or early oncogenesis. Analyses of the active enhancer landscape (using H3K27ac ChIP-seq), revealed stable epigenomic alterations that influence the transcriptional output of post-pregnancy MECs in response to pregnancy signals in fat-pad transplantation assays and in organoid systems.cMYC, an inducer of mammary tumor development23, is driven in a doxycycline (DOX)-dependent manner. Post-pregnancy MECs did not undergo malignant transformation in response to cMYC overexpression under in vivo or in vitro conditions, in marked contrast to pre-pregnancy MECs, which engaged in abnormal, carcinoma-like growth. Transcriptomic and epigenetic analysis illustrated that cMYC overexpression drives post-pregnancy MECs into a senescence-like state, and perturbations to such state increased malignant phenotypic changes. Overall, our studies provided new insights into the role for pregnancy in altering epigenomic landscapes and in suppressing the malignant transformation of MECs, and suggest that the influence of pregnancy on breast cancer risk can occur, at least in part, via epigenomic reprogramming.To characterize the influence of a pregnancy-induced epigenome on the response to oncogene expression, we used a transgenic mouse strain (CAGMYC), in which overexpression of the oncogene 12. To test this hypothesis, we mapped global gene expression (RNA-seq) of FACS-isolated luminal MECs from nulliparous (pre-pregnancy) and parous  Balb/c female mice, as well as MECs harvested from female mice during exposure to pregnancy hormones (EPH). For the first and second EPH time points, nulliparous or parous female mice, were treated with slow-released estrogen and progesterone hormones for short-term exposure (6 and 12 days) , in contrast to pre-pregnant MECs  , and a 53-fold gain at intergenic regions , over the same regions in pre-pregnancy MECs, suggesting that pregnancy-induced changes may expand the MEC enhancer landscape  Fig. . Regulatn = 2263), however more PIEs (2-fold) were active only during the second EPH, suggesting that such elements play a role during re-exposure to pregnancy hormones , consistent with pregnancy expanding the active enhancer landscape , in comparison with those cells harvested from mice exposed during first EPH Fig. . These u27. Recipient female mice (2-month post transplantation) were exposed to pregnancy hormones for 6 days, followed by histological analysis of their mammary glands  and progenitor cells could be involved in the epigenetic memory of pregnancy. Transitions during pregnancy cycles could also result in alterations to the mammary microenvironment, involving cell autonomous and non-autonomous regulatory cues to sustain MECs response to consecutive pregnancy signals. To dissect the microenvironment’s role in post-pregnancy MEC response to pregnancy hormones, we used mammary fat-pad transplantation assays. Cleared fat-pads from pre-pubescent, virgin female mice were transplanted with either pre- or post-pregnancy CD1d+ MaSCs, which have increased mammary reconstitution activity in fat-pad transplantsCsn2 mRNA levels (10-fold) and increased CSN2 protein levels (approximately 4-fold), in post-pregnancy organoids cultured with pregnancy hormones compared with pre-pregnancy organoids grown under the same hormone conditions  displayed a 2.3-fold greater number of branching organoids compared with pre-pregnancy cultures Fig. . FurtherA levels 0-fold anons Fig. , our res22. Several of these studies utilized mammary gland-specific promoters, such as MMTV and WAP-CRE, to drive oncogene expression and tumor development. However, these promoters are enhanced by signals present during pregnancy and lactation30, thus potentially masking epigenomic and transcriptomic changes associated with early oncogenesis and pregnancy-induced protection. To overcome this problem, we utilized a mouse model overexpressing cMYC using the CAG promoter, which is independent of pregnancy/lactation signals, under the control of DOX  or 5 days (DD5) of DOX treatment. DOX treatment induced substantial histo-pathological alteration to the mammary gland, including flattening of ductal structures and moderate (DD2) to severe, diffuse (DD5) epithelial hyperplasia with atypia, alterations frequently observed in premalignant mammary lesions in mice32  and analyzed using RNA-seq. In DD2 MECs, we observed enrichment for pathways involving cellular metabolism, such as mitochondrial function and gene splicing, in contrast to pathways upregulated in DD5 MECs, which are associated with control of cell communication processes  of total CAGMYC MECs. Many of the H3K27ac peaks present in DD5 CAGMYC MECs (96%) were also present in DD2 CAGMYC MECs, suggesting that development of premalignant mammary lesions largely rely on programs activated during the initial response to cMYC overexpression 36 in response to cMYC overexpression. Roughly, 4500 H3K27ac peaks were detected at e-boxes in wild-type (WT), non-transgenic MECs and in CAGMYC MECs, suggesting that a set of cis-regulatory elements are activated in MECs independently of cMYC overexpression  also failed to induce the development of premalignant lesions in mammary glands transplanted with post-pregnancy CAGMYC CD1d+ MaSCs, in contrast to glands transplanted with pre-pregnancy CAGMYC CD1d+ MaSCs, which progressed from epithelial hyperplasia to undifferentiated carcinoma lesions  and 3.1-fold (DD2) reduced in post-pregnancy CAGMYC organoids Fig. . Pre-precMYC overexpression, we carried out unsupervised gene expression analysis on DOX-treated, pre- and post-pregnancy CAGMYC MECs  regulatory landscape of total pre- and post-pregnancy CAGMYC MECs. cMYC overexpression induced a 6-fold increase in H3K27ac signal intensity at promoter regions in pre-pregnancy CAGMYC MECs, compared with promoter regions from pre-pregnancy WT MECs  mapping to promoter regions in pre-pregnancy, compared with 6% (n = 295) of promoter regions in post-pregnancy CAGMYC MECs , which were associated with biological processes controlled by pregnancy signals  in response to 39, and found it to display decreased H3K27ac intensity in response to cMYC overexpression in post-pregnancy CAGMYC MECs , compared with those found in pre-pregnancy CAGMYC MECs (n = 1127 regions), supporting that cMYC overexpression is less efficient in altering the epigenome of post-pregnancy CAGMYC MECs , and downregulation of Bcl2L12 and Tbx3 mRNAs, factors whose low expression has been correlated with a senescence-like state45.To analyze the genome-wide distribution of cMYC-chromatin occupancy in pre- and post-pregnancy CAGMYC MECs, we utilized Cleavage Under Targets and Release using Nuclease (Cut&Run). Unbiased transcription factor (TF) DNA-motif analysis revealed enrichment of e-box motifs within the chromatin peaks co-immunoprecipitated with cMYC, suggesting concordance between the observed regions of cMYC-chromatin occupancy (cMYC peaks), and DNA motifs recognized by cMYC , as demonstrated by our gene expression analyses Fig. . Thus, t27. However, we cannot exclude that, after pregnancy, these cell-surface markers recognize a more diverse cell population compared with those existing prior to pregnancy. Previous studies have reported parity-induced MECs53, and recent single-cell RNA-seq analyses demonstrated alterations to MEC populations throughout gestation, lactation, and involution stages of mammary gland development54. However, DNA methylation analyses of several mammary cell types from fully involuted, post-pregnancy mammary glands have demonstrated that alterations to the epigenome were, to some extent, shared by most mammary cell types12, suggesting that pregnancy-induced epigenomic alterations may not be restricted to MEC lineage identities.Our epigenomic and transcriptomic analyses utilized a cell isolation strategy previously applied to isolate-defined MEC populationscMYC overexpression, pregnancy-induced epigenomic alterations, and tissue/cellular abnormalities. cMYC, a potent oncogene, is overexpressed in ~50% of human breast cancers55. In addition, several oncogenic signaling networks  converge on the oncogenic potential of deregulated cMYC expression56. Furthermore, chromosomal abnormalities found in cMYC overexpressing mouse models are syntenically and developmentally comparable to those of human breast cancer57. Therefore, an inducible cMYC overexpression model system to understand mammary malignant initiation may help identify molecular targets for validation in human breast specimens, improving understanding of mammary oncogenesis.Our present study also revealed the relationship between cMYC overexpression timeline, supporting the notion that longer exposure to cMYC overexpression may reprogram breast epithelial cellular identity58. Nonetheless, we have shown that cMYC-driven signals do not fully induce epigenomic and transcriptomic alterations that support malignant transformation of post-pregnancy mammary glands. This is also seen in transplantation assays, where cMYC overexpressing post-pregnancy CAGMYC MECs did not progress into malignant transformation.It is important to note that signals driving early oncogenesis may differ from those present after disease establishment. Our analysis of pre-pregnancy CAGMYC MECs suggests that transcriptional programs and the epigenome are differentially regulated across the 21, and those accompanied by MMTV-driven cMYC overexpression22. Conversely, lack of active p53 is associated with the development of mammary tumors in murine59 and human association studies60, suggesting that a decrease in p53 dosage in post-pregnancy MECs may promote cancer initiation. Our study revealed a substantial increase of p53 protein in post-pregnancy CAGMYC organoids, possibly promoting a senescent state that could block the development of malignant phenotypes.But how can pregnancy decrease mammary oncogenesis? Previous studies established that both a full pregnancy cycle, and an induced pseudo-pregnancy decreased the frequency of mammary tumors in several mouse strains, including in chemically induced mammary tumorigenesis models63. Alterations to immune composition during post-pregnancy mammary gland involution have also been suggested to influence mammary tumor progression64. Interactions among the stroma, immune system and the MEC epigenome should be explored to define their roles in blocking the early onset of oncogenesis in a post-pregnancy setting, and to reveal their intersection with the parity-induced epigenomic changes we describe.It is also possible that pregnancy may induce alterations that influence the mammary gland stroma and/or its immune composition. Pregnancy-induced alterations to the mammary gland ECM have been suggested to play a role in preventing the progression of established cancer cellscMYC overexpression. Using this system, we confirmed post-pregnancy MECs are less responsive to cMYC overexpression. Our analysis also demonstrated the utility of the organoid system for perturbing signals present in post-pregnancy MECs. Utilizing combinations of small-molecule inhibitors and genetic manipulations in this system will enable the identification of signals that either promote or block responses to oncogenic signals in a target-specific manner.Importantly, our mammary organoid experiments confirmed the cell-autonomous characteristics of pregnancy-induced changes, and their ability to block responses to Finally, our data revealed the stability of the pregnancy-induced epigenome, both in in vivo and in vitro pregnancy-naïve environments, emphasizing the cell-autonomous nature of the altered post-pregnancy epigenome. Such in vivo and in vitro strategies will be required to monitor the dynamics of enhancer activity and transcriptional regulation in a pregnancy-hormone dependent fashion. Ultimately, the use of in vitro organoid strategies may allow for better understanding of enhancer activation, transcriptional regulation, and responses to oncogenes in breast tissue obtained from women with various reproductive histories, with the goal to identify and characterize the human-specific features of pregnancy-induced developmental dynamics in cancer predisposition.Balb/C and C57/BL6 female mice were purchased from Charles River. CAGs-rtTA3 mice (B6N.FVB(Cg)-Tg(CAG-rtTA3)4288Slowe/J, the Jackson Laboratory) and tetO-MYC mice (FVB/N-Tg(tetO-MYC)36aBop/J, the Jackson Laboratory) were crossed for the establishment of CAGMYC transgenic mouse strain (129/C57BL6 background). All animals were housed at a 12 light/12 dark cycle, with a controlled temperature of 72 °F and 40–60% of humidity. All experiments were performed in agreement with approved CSHL Institutional Animal Care and Use Committee.Mammary glands were harvested and processed as fully described in “Supplementary Methods.” In short, mammary glands were harvested and digested into a single-cell suspension. MECs were separated from immune cells (CD45+), red blood cells (TER119+), endothelial cells (CD31+), and fibroblasts (CD34+) using lineage depletion antibodies and MACS magnetic column (Miltenyi Biotech). Lineage-depleted (LIN−) MECs were utilized as described in “Supplementary Methods.”FACS-isolated MECs were utilized for the preparation of NextGen Illumina libraries, as described in “Supplementary Methods.”Tissue histology and immunofluorescence staining (IF) were performed as described in “Supplementary Methods.”Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


We investigated whether “late-onset” ADHD that emerges in adolescence/adulthood is similar in risk factor profile to: 1) child-onset ADHD, but emerges later because of scaffolding/compensation from childhood resources; and 2) depression, because it typically onsets in adolescence/adulthood and shows symptom and genetic overlaps with ADHD.We examined associations between late-onset ADHD and ADHD risk factors, cognitive tasks, childhood resources and depression risk factors in a population-based cohort follow-up to age 25 years (N=4224-9764).Parent-rated late-onset ADHD was like child-onset persistent ADHD in associations with ADHD polygenic risk scores and cognitive task performance, although self-rated late-onset ADHD was not. Late-onset ADHD was associated with higher levels of childhood resources than child-onset ADHD and did not show strong evidence of association with depression risk factors.Late-onset ADHD shares characteristics with child-onset ADHD when parent-rated, but differences for self-reports require investigation. Childhood resources may delay the onset of ADHD. The DSM-5 currently conceptualises ADHD as a neurodevelopmental disorder that onsets in childhood, prior to age 12 years . HoweverFindings that late-onset ADHD is associated with higher cognitive ability than child-onset ADHD  could suInterpreting findings on late-onset ADHD is complicated by methodological issues. One important consideration is who reports the symptoms. In childhood, ADHD symptoms are usually reported by a parent, but this typically changes to self-report after the age of 18 years. Agreement between parent- and self-reported ADHD symptoms is typically low. In clinical studies, affected individuals even in adult life tend to under-report symptoms e.g.  comparedA second methodological consideration is how late-onset ADHD is operationalised. Most longitudinal studies have defined late-onset ADHD in individuals who surpass validated thresholds for ADHD symptom counts . An alteOur study aimed to investigate the nature of late-onset ADHD, utilising different informants , operationalisations  and measures . We tested two hypotheses: (1) late-onset symptoms reflect a similar disorder to child-onset ADHD, but with delayed symptom onset. If this hypothesis is correct, we would predict that young-adults with late-onset and child-onset ADHD symptoms will show similar ADHD risk factor profiles and cognitive impairments, but that those with late-onset would have higher levels of childhood internal and external resources that may allow compensation for underlying ADHD liability or obscure the presence of symptoms in childhood (i.e. “compensation” or “scaffolding”). (2) late-onset ADHD symptoms reflect a variant of depression: in this case, compared to those with child-onset ADHD symptoms, young-adults with late-onset symptoms will show stronger associations with depression risk factors.We analysed data from the Avon Longitudinal Study of Parents and Children (ALSPAC), a well-established prospective birth cohort study. Pregnant women resident in Avon, UK with expected dates of delivery 1st April 1991 to 31st December 1992 were invited to take part in the study. When the oldest children were approximately 7 years of age, an attempt was made to bolster the initial sample with eligible cases who had failed to join the study originally, resulting in a total of 14,901 study offspring alive at 1 year of age. Where families included multiple births, we included the oldest sibling. Full details of this study are provided in the ADHD symptoms were measured using the 5-item ADHD subscale of the Strengths and Difficulties Questionnaire (SDQ) and the Genetic risk for ADHD was indexed using polygenic risk scores (PRS). PRS were generated using PRSice version 1.25  based onPreterm birth (<37 weeks gestation) and low birth weight (<2500g) were included as risk factors for ADHD  for singletons (99% of the primary sample).We investigated cognitive tasks that index characteristic features of ADHD: attention and response inhibition.Sustained attention was assessed using the Tests of Everyday Attention for Children (TEA-Ch) Sky SearInhibitory control was assessed using the TEA-Ch Opposite Worlds task, which is a type of Stroop task  at age 8We examined internal and external resources in childhood that may compensate for, or scaffold symptoms. These included (i) verbal IQ, given evidence that this may facilitate compensation in relation to autistic behaviours , (ii) chChildhood verbal ability was assessed using the Wechsler Intelligence Scale for Children  at age 8Family income was measured by mother-report when the child was approximately age 11 years as the average household income including social benefits each week on a 10-point scale from <£120 to ≥£800. Maternal education was assessed by mother-report during pregnancy as the highest educational qualification on a 5-point scale from CSE or no qualifications to university degree.Genetic risk for depression was indexed using PRS based on GWAS of major depression . GenotypHistory of depression in the mothers was assessed during pregnancy. Assessment was by self-report of a lifetime ever history of severe depression.Late-onset ADHD was defined using different informants, operationalisations and measures to assess consistency of findings across definitions.child-onset persistent ADHD if they had high symptoms at either age 17 or 25 years, otherwise they were classified as having child-limited ADHD. Individuals with low ADHD symptoms at ages 7, 8, 9 and 12 years, but who had elevated symptom levels at ages 17 or 25 years were classified as having late-onset ADHD. Those with subthreshold symptoms in childhood, but high symptoms at ages 17 or 25 were categorised as subthreshold late-onset and are presented for descriptive purposes only. Individuals without high symptoms at any time-point were classified as having low symptoms. The definitions of all groups are shown in ADHD symptoms were categorised using a similar procedure to our previous work , using SGrowth mixture modelling (GMM) was used to derive trajectories of ADHD symptoms from ages 4 to 25 years in Mplus  separateWe report the means or proportions for each examined variable for each ADHD group. Our primary analyses compared late-onset versus child-onset persistent ADHD. We used multinomial logistic regression to test associations. We used child-onset persistent ADHD as the reference group for the primary analyses and ran secondary analyses using low ADHD symptoms as the reference. For trajectory analyses we used a bias-free three step approach which accounts for measurement error in class assignment . OrdinalEstimates of late-onset ADHD across definitions are shown in ADHD groupings based on self- and parent-reported SDQ cut-points in adulthood are shown in Comparing parent- and self-rated late-onset definitions, 44.6% of those with late-onset ADHD according to parent-reports were also categorised as late-onset based on self-report. In contrast, only 16.6% of those with late-onset ADHD based upon self-report also had late-onsetADHD by parent-reports. Additional details of overlap between self- and parent-reported adult symptoms are given in the Post-hoc analyses suggested 3.4-6.6% of the sample experienced fluctuating (temporarily-remitted) symptoms; results are presented in the For trajectory analyses, only parent-reports were used to enable a consistent informant across time points. As shown in The means and proportions for examined variables by ADHD group are shown in Regardless of informant or classification system, male sex was consistently associated with a decreased likelihood of having late-onset compared to child-onset persistent ADHD. ADHD PRS were similar in those with late-onset and child-onset persistent ADHD when using parent-reported symptoms but were lower in those with late-onset ADHD when using self-reported adult symptoms. We did not find strong evidence that prevalence of preterm birth or low birth weight differed in the late-onset compared to child-onset persistent group.ADHD cognitive task performance did not consistently differ between late-onset and child-onset persistent ADHD when using parent-reported symptoms but was higher in those with late-onset ADHD when using self-reported adult symptoms.Higher levels of childhood verbal and reading ability, family income and maternal education were associated with late-onset compared to child-onset persistent ADHD.There was not strong evidence for an association with late-onset ADHD compared to child-onset persistent ADHD for depression PRS or maternal depression. There was also not a female preponderance in the late-onset group.This study aimed to investigate the nature of late-onset ADHD using different informants, operationalisations and measures in a prospective, longitudinal population sample spanning childhood to young-adulthood. We tested two hypotheses on the nature of late-onset ADHD symptoms: (a) late-onset symptoms reflect a similar phenotype to child-onset ADHD, but with delayed symptom onset, and (b) late-onset ADHD symptoms reflect a variant of depression.Regardless of ADHD informant, operationalisation or measure, we found evidence for a group of individuals with high levels of ADHD symptoms that start for the first time after childhood. In late-adolescence/young-adulthood, the prevalence of late-onset ADHD was higher for self-reports than for parent-reports. The majority of late-onset ADHD identified was adult-rather than adolescent-onset: while for categorical definitions this could have been due to the lower cut-point used in young-adulthood compared to adolescence, this would not apply to trajectory analyses which used continuous symptom scores. Adult-onset being more common than adolescent-onset may explain why previous trajectory analyses in this same cohort with follow-up to age 17 years did not identify a late-onset trajectory  whereas Our analyses investigating associations with ADHD and depression risk factors, and internal and external resources generally supported the hypothesis that late-onset ADHD reflects a similar phenotype to child-onset ADHD, rather than a variant of depression when parent-ratings were used. However, findings for self-rated ADHD were not clear. We did not find evidence of a female preponderance in the late-onset group, which would have been expected for depression , althougOur “alternative disorder” hypothesis focusses specifically on depression, because this typically onsets at the same age as late-onset ADHD (adolescence or early adulthood)  and deprWe found similarly elevated levels of genetic risk for ADHD (indexed by PRS) in those with late-onset ADHD as those with child-onset persistent ADHD when defined using parent-reports, but found lower PRS in those with late-onset ADHD when using self-reported adult symptoms. This PRS finding for parent-reported late-onset ADHD was consistent across different operationalisations and measures of ADHD. Previous studies have not found evidence of elevated ADHD PRS in those with late-onset ADHD: these have either used self-reports  or have One explanation for why the same underlying disorder (ADHD) may onset later in development for some is that higher levels of childhood internal and external resources may allow individuals to compensate for underlying ADHD difficulties or obscure/delay the presence of symptoms (i.e. compensation or scaffolding). This is supported by reported associations between late-onset ADHD and higher IQ  and is sOur findings should be considered in the context of study limitations. Like many population cohorts, ALSPAC suffers from non-random attrition, whereby individuals at elevated risk of psychopathology are more likely to drop-out of the study . Thus, pIn summary, across different operationalisations and measures, we found similar levels of ADHD genetic liability (PRS) in those with parent-rated late-onset compared to child-onset persistent ADHD, suggesting that parent-rated late-onset ADHD is indexing ADHD genetic liability. Those with late-onset ADHD had higher levels of individual-based and family-based resources. Self-ratings however defined a different late-onset ADHD group which did not show the same profile as child-onset ADHD. Taken together our findings suggest that at least for some, ADHD may onset later in development in the context of childhood compensation or scaffolding resources and further challenges the assertion that ADHD always first onsets before age 12 years.Supplementary Material


This review intends to provide an overview of the current knowledge on neurologic sequelae of COVID-19 and their possible etiology, and, based on available data, proposes possible improvements in current medical care procedures. We conducted a thorough review of the scientific literature on neurologic manifestations of COVID-19, the neuroinvasive propensity of known coronaviruses (CoV) and their possible effects on brain structural and functional integrity. It appears that around one third of COVID-19 patients admitted to intensive care units (ICU) for respiratory difficulties exhibit neurologic symptoms. This may be due to progressive brain damage and dysfunction triggered by severe hypoxia and hypoxemia, heightened inflammation and SARS-CoV-2 dissemination into brain parenchyma, as suggested by current reports and analyses of previous CoV outbreaks. Viral invasion of the brain may particularly target and alter brainstem and thalamic functions and, consequently, result in sensorimotor dysfunctions and psychiatric disorders. Moreover, data collected from other structurally homologous CoV suggest that SARS-CoV-2 infection may lead to brain cell degeneration and demyelination similar to multiple sclerosis (MS). Hence, current evidence warrants further evaluation and long-term follow-up of possible neurologic sequelae in COVID-19 patients. It may be particularly relevant to evaluate brainstem integrity in recovered patients, as it is suspected that this cerebral area may particularly be dysfunctional following SARS-CoV-2 infection. Because CoV infection can potentially lead to chronic neuroinflammation and progressive demyelination, neuroimaging features and signs of MS may also be evaluated in the long term in recovered COVID-19 patients. •Acute neurologic symptoms are observed in one third of COVID-19 patients.•Neurotropism of SARS-CoV-2 is highly suspected.•Brainstem damage may be a crucial driver for COVID-19-associated respiratory failure.•COVID-19 is likely to trigger demyelination similarly to multiple sclerosis.•Monitoring patients using Auditory Brainstem Responses and neuroimaging is warranted. Unlike recent severe acute respiratory syndrome (SARS)-CoV-1 and Middle East respiratory syndrome (MERS)-CoV outbreaks, the recent SARS-CoV-2/COVID-19 pandemic has spread quickly and has been reported in more than 200 countries worldwide. The ongoing epidemic has already caused over 240,000 deaths worldwide, mostly due to respiratory failure, the death rate being around 3–4% of infected individuals . HCoV are known to affect several organs including the brain  and may The neuroinvasive potential of SARS-CoV-2 and its consequences on brain function are currently a subject of great debate. Unfortunately, there is a great paucity of information available on the effects of SARS-CoV-2 on brain integrity at the present moment. Nevertheless, current clinical and preclinical data warrant improvements in patient care practices. In particular, non-invasive evaluation of neurologic functions and of brain inflammatory status may be implemented in routine clinical protocols. In this review article, we expose the current knowledge on how HCoV infection can lead to cerebral dysfunction and how it may relate to disease. We also discuss few complementary clinical examinations that could potentially alleviate COVID-19 patient monitoring and care.22.1In one of the first retrospective studies published, 36.4% (78/214) of Chinese COVID-19 patients exhibited neurologic symptoms, i.e. headache, dizziness and loss of consciousness . There i2.2The etiology of these neurologic manifestations is still unclear. Some of the symptoms reported, especially headache, dizziness and loss of consciousness, are not specific, as they could possibly be due to the severe hypoxia associated with respiratory failure. Other symptoms could result from the dysimmune state caused by the infection, in particular from the abnormally high inflammation that is referred to as “cytokine storm”. For instance, cytokine storm may be involved in acute neurovascular pathologies observed in COVID-19 patients without pre-existing vascular risk factors . Moreove33.1Even if there is still sparse evidence of neuroinvasion by SARS-CoV-2, viral particles have been detected in the CSF of one infected patient with meningoencephalitis  and in t3.2As previously described for SARS-CoV-1 and HCoV-OC43, SARS-CoV-2 may also reach the brain via an hematogenous route . It is s3.3As for SARS-CoV-1 virus, the cellular receptor for SARS-CoV-2 is the ACE2 . The enz44.14.1.1Several mechanisms may contribute to CNS damage after HCoV infection: through direct viral replication in infected cells (neuropathology) or through an indirect process involving neuro-immunopathology. Different studies have shown that systemic inflammation along with massive release of cytokines (i.e. “cytokine storm”) contribute to brain damages induced by SARS-CoV-1 . SimilarFew reports suggest that the cytokine storm may reach the CNS. Indeed, elevated cytokine concentrations have been detected in the CSF of HCoV infected patients . GranuloTo further investigate the cerebral effects of SARS-CoV-1 infection, a transgenic mouse line expressing the Human ACE2 was engineered . Express+ and CD8+ T lymphocytes can invade the CNS when the BBB is permeabilized by metalloproteinases secreted by neutrophils and macrophages  proteins in microglial cells (+ regulatory T cells (Treg) are recruited when inflammatory response reaches a peak, and modulate the extend of the immune defense response  may verwBesides pathophysiological aspects, experimental studies raise the possibility that cerebral infection by SARS-CoVs may be one the main trigger of death among patients. Animal studies have demonstrated that intracranial injection of very low SARS-CoV-1 titers leads to rapid death without noticeable lung deterioration . In addiUnfortunately, involvement of any brain lesion or dysfunction in the pathophysiology of SARS-CoV-2, and especially in respiratory failure, is still very speculative given the current state of knowledge. Nonetheless, the existing evidence warrants systematic evaluation of the physical and functional integrity of the brain, especially of the brainstem, in patients diagnosed with SARS-CoV-2, at least those presenting with moderate to severe respiratory failure along with other neurologic symptoms.55.1Although the level of evidence indicating a role of microglial priming in post-infectious neuroinflammatory processes is quite low at the moment, extrinsic factors such as pre-existing history of brain injury  and stress-related diseases may be systematically assessed at admission to complement clinical diagnosis of COVID-19 patients. In addition, long-term neuropsychiatric follow-up of COVID-19 patients exhibiting these potentially predisposing conditions may be recommended.5.25.2.1Because it may spread into the brainstem through peripheral nerves and the olfactory pathway, SARS-CoV-2 could not only affect neural respiratory centers, but also disturb the auditory pathway. Therefore, ABR could be used to assess brainstem injury caused by SARS-CoV-2. ABR belong to the toolbox of ICU and is used to monitor the integrity of brainstem function because they resist well to sedation and muscle paralysis .Brainstem dysfunction affects the aspect and the latency of wave V and is associated with prolonged III-V interval, weak (<0.5) amplitude ratio between waves V and I, and depressed wave V . In the 5.2.2Besides acute aspects, potential sequelae of CoV infection may include multiple sclerosis (MS) in patients with genetic predisposition . Post-mo5.3Brain inflammatory processes are very difficult to detect and measure. In severely or critically ill patients, lumbar puncture could be performed to measure cytokine levels in CSF. However, this is an invasive and technically challenging procedure. By contrast advanced neuroimaging could be a non-invasive technic to monitor brain inflammatory processes, particularly microglia activation state. Among other techniques, gadolinium-enhanced magnetic resonance imaging (Gd-MRI) or translocator protein-positron emission tomography (TSPO-PET) could be used to detected abnormal activation of brain inflammatory processes in SARS-CoV-2 infected patients . Gadolin6Despite the fact that there is still only few evidence, to date, demonstrating that SARS-CoV-2 has potency to invade the brain and induce neurologic defects in patients, previous clinical and preclinical studies conducted on homologous CoV warrant detailed evaluation of brain status in patients infected with SARS-CoV-2. Improvement of the monitoring protocols of patients with ongoing symptoms and those that were, apparently, successfully treated may be implemented in clinical practice . First, None.None.


Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level.Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bern Vaccination programs are one of the most effective means of controlling infectious diseases  and withorig) vaccine virus strain, a successor of the ERA strain [ORV programs in foxes are aimed at increasing herd immunity in the target population using oral rabies vaccines distributed into the environment. Over the past four decades several oral rabies vaccines, mainly live replication-competent attenuated rabies virus vaccines, have been successfully used in ORV campaigns . In CanaA strain ,8. WhileA strain ,10,11,12A strain , Poland,A strain , such caA strain ,8.While previous analyses using high-throughput sequencing approaches revealed substantial genetic heterogeneity within commercial SAD-derived oral rabies virus vaccines , the subIn this study, we attempted to further analyze and elucidate the mechanisms of genetic selection using a combined deep sequencing, variant, and haplotype analysis of a large set of vaccine-induced rabies cases that included additional SAD- and ERA-induced rabies cases from Poland and Canada alongside vaccine virus batches. With this comprehensive approach, we could demonstrate the utility of this approach for identity and genetic stability (revision to virulence) testing of vaccines with a heterogeneous genetic background. Additionally, we were interested to know whether viruses from vaccine-induced rabies cases differ from virulent field rabies viruses (RABV) at a population level.orig-derived vaccine-induced rabies cases from Canada [In this study, original brain material of additional ERA-BHK21 and SAD Bernm Canada  and Polam Canada .® Ultra™ II Non-Directional RNA Second Strand Synthesis Module . The generated cDNA was fragmented using the Covaris M220 (Covaris) and subsequently converted to Ion Torrent compatible libraries using the GeneRead L Core Kit (Qiagen) and IonXpress Barcode Adaptors  followed by size selection as described [Samples were processed according to two different methodical approaches. Methodology 1 corresponds to the published protocol of Wylezich and colleagues . Brieflyescribed . After qMethodology 2 followed sample preparation as described before . In shorCS, and SAD B19P1  vaccine batches, whereas vaccine batch B/SAD/Bern/0213 was shifted to the vaccine-induced rabies cases  did not cluster with the related vaccines as shown before [orig- and ERA vaccine-induced rabies cases to their progenitor vaccines.Population analysis of the viruses isolated from vaccine-induced rabies cases revealed two distinct patterns. Those viruses that were associated with SAD Bernn before . In contThe ERA-BHK21 vaccine virus population displayed a high genetic heterogeneity represented by diverse single nucleotide variants distributed across the N, P, G, and L genes , supportEach of these groups was characterized by a unique set of differences at the consensus level ranging between three and nine genome positions that were not present in any of the other groups . This suorig rabies cases. This observation seemingly contradicts results from the population-based analysis where the ERA vaccine-induced cases clustered closely together with the ERA-BHK21 vaccine batch. However, differences at the consensus level of the viruses of ERA vaccine-induced rabies cases were already included in the progenitor vaccine population (ERA-BHK21) and likewise, in some variants, the former consensus sequence was present as a minor variant. For instance, in each of two ERA vaccine-induced rabies cases (C/CAN/1992N8944 and C/CAN/1994N6762M) one additional single nucleotide variant  appeared in the viral populations; in both cases, the progenitor consensus was maintained as a minor variant  exchange Thr178Ile at an interferon-antagonist motif in the P-gene [To elucidate whether the induction of disease in these vaccine-induced rabies cases was a result of mutations at known antigenic sites ,30,31,32e P-gene  found ine P-gene . On the When selected field RABV viruses were included in the Manhattan distance matrix analysis, the attenuated vaccine virus strains and viruses from their vaccine-induced rabies cases were clearly separated from them by high distances . FurtherAlthough various studies have focused on oral rabies vaccine-induced cases ,13,18,33orig vaccine-induced rabies cases strongly suggests that only one replication-competent haplotype was present in those cases, despite a higher number of variants. One reason could be the passage history. Both ERA-BHK21 and SAD Bernorig vaccine virus strains descend from the same derivative of the cell culture-adapted field virus strain, namely SAD [orig vaccine virus strain was further developed into the commercial vaccine virus strains SAD Bern and SAD B19. Eventually, increased cell culture adaptation of SAD Bern and SAD B19 vaccine virus strains may have selected for only a single replication-competent haplotype. In parallel, this adaptation towards efficient replication in cell culture is favoring the development of defective interfering particles [While viruses from ERA vaccine-derived cases seem to descend from different replication-competent virus haplotypes that were already present within the progenitor ERA-BHK21 vaccine virus strain , the absmely SAD ,6, but wmely SAD  for a momely SAD ,15. On tarticles  that proAs attenuated live vaccines consist of replication-competent virus particles, a major concern in their application is the potential reversion to virulence as demonstrated by vaccines for the severe acute respiratory syndrome coronavirus (SARS-CoV) , peste dThe results of this study clearly demonstrate the benefits of utilizing deep sequencing followed by a comprehensive stepwise data analysis focusing on consensus, population, and variant level. While each of the analyses may lead to biased conclusions, only the combination allows for a holistic assessment, and thus should be standard in similar scenarios.


Conventional treatments for brain tumors relying on surgery, radiation, and systemic chemotherapy are often associated with high recurrence and poor prognosis. In recent decades, intra-arterial administration of anti-cancer drugs has been considered a suitable alternative drug delivery route to intravenous and oral administration. Intra-arterial administration is believed to offer increasing drug responses by primary and metastatic brain tumors, and to be associated with better median overall survival. By directly injecting therapeutic agents into carotid or vertebral artery, intra-arterial administration rapidly increases intra-tumoral drug concentration but lowers systemic exposure. However, unexpected vascular or neural toxicity has questioned the therapeutic safety of intra-arterial drug administration and limits its widespread clinical application. Therefore, improving targeting and accuracy of intra-arterial administration has become a major research focus. This systematic review categorizes strategies for optimizing intra-arterial administration into five categories: (1) transient blood-brain barrier (BBB)/blood-tumor barrier (BTB) disruption, (2) regional cerebral hypoperfusion for peritumoral hemodynamic changes, (3) superselective endovascular intervention, (4) high-resolution imaging techniques, and (5) others such as cell and gene therapy. We summarize and discuss both preclinical and clinical research, focusing on advantages and disadvantages of different treatment strategies for a variety of cerebral tumor types. Brain tumors are a large class of heterogeneous neoplasms, generally classified as benign or malignant tumors. It is widely believed that uncontrolled proliferation and tissue infiltration of dedifferentiated cells caused by harmful chemical, physical, and biological exposures are the main causes of brain tumor malignancy . Non-malBrain tumors are relatively rare compared to other neurological diseases, and complex pathogenesis of some brain tumor types  often renders existing treatment strategies ineffective in prolonging survival time. Thus, brain tumors are the most life-threatening cancers in humans . The traIntra-arterial administration was first described for the treatment of brain tumors in the 1950s . In compOn the other hand, a number of strategies to improve the therapeutic effects of intra-arterial administration for the treatment of brain tumors have been suggested. This systematic review summarizes the five most important ones. The first option is blood–brain barrier (BBB)/blood-tumor barrier (BTB) disruption using chemical reagents  or penetration drug carriers  to increase the direct entry of drugs into tumor and brain tissues. The second option is intra-arterial injection during transient cerebral hypoperfusion (IA-TCH) or flow arrest (IA-FA). This increases local plasma drug concentration and exposure time by reducing dilution, absorption, and contact with blood components. The third option are microcatheters designed to allow superselective intra-arterial cerebral infusion (SIACI) into the tumor-feeding arteries to reduce neurotoxic side effects while achieving well-targeted drug delivery. The fourth option is to combine imaging techniques, such as MRI, CT, X-ray, SPECT, and PET with intra-arterial infusion of labeled therapeutic agents, to monitor delivery and accumulation in the tumor and brain parenchyma. The fifth option is to use advanced techniques including intra-arterial infusion of gene-edited viruses or cells to achieve targeted molecular or cell therapy for brain tumors. Next to providing an overview, this review also discusses advantages and limitations of these different strategies that became obvious in preclinical and clinical research.Literature search and evaluation were conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) standard. No ex-ante protocol was used.n = 1,835) and updated on June 30, 2020 . One more study was included during the updated search. The publication time in EMBASE was 1963–2020, PubMed 1962–2020, and Web of Science 1946–2020. A keyword-based search strategy was applied as follows: In EMBASE, the keyword terms “brain tumor” and “intraarterial drug administration” of EMTREE dictionary were used. The MeSH-defined keywords “Brain Neoplasms” and “Injections, Intra-Arterial” or “Infusions, Intra-Arterial” were used in PubMed searches, while Web of Science searches were conducted with “Brain tumor” and “Intra arterial.” In addition, other eligible publications selected from the list of references in the included literature were used to supplement the search results. Only peer-reviewed articles published in English were included. The included literature should focus on strategies that aimed at improving the therapeutic effect of intra-arterial treatment. Therefore, articles that simply evaluated the therapeutic effects of intra-arterial administration but without comparing them to alternate approaches were excluded (n = 105). We also excluded conference abstracts (n = 10), papers without available full-text (n = 3), papers not reporting brain tumor treatment (n = 21), and in-vitro studies (n = 5), as well as studies not reporting intra-arterial treatment (n = 4) or being focused on embolization only (n = 5). Reviews were also excluded (n = 28).A systematic literature search was conducted in three databases: EMBASE, PubMed, and Web of Science. The original search date was October 22, 2019 (n = 486), PubMed (n = 534), and Web of Science databases (n = 835). By screening the reference lists of the included papers, one more publication was added to the search results, resulting in a total of 1,856 articles. Screening for duplicates and their removal resulted in a total of 1,070 articles. We next screened titles and abstracts, obtaining 399 articles not meeting any exclusion criterion. Finally, 218 articles published between 1981 and 2020 were included after full-text evaluation . Numbers of studies focusing on BBB disruption gradually decreased from 2001. However, the number of publications on BBB disruption was still higher (2001–2010) than or equal to (2011–2020) the numbers of studies focusing on other strategies. Research on nanoparticles became a main research focus for improving BBB/BTB penetration in the past decade (2011–2020). Several techniques emerging from medical physics were applied in preclinical research on TCH (n = 10). With the improvement of microcatheters, superselective infusion techniques gradually became a routine treatment option in 2011–2020 (n = 14). In parallel, real-time image-based monitoring became increasingly important for optimization of therapeutic procedures (n = 14).As shown in The BBB represents a major obstacle for CNS drug delivery . The ana99mTc-glucoheptonate (TcGH), SPECT scans confirmed BBB opening within 40 min after mannitol injection and returned to the steady state after 6–8 h. This provides a wide time window for clinical applications   , 1-O-penreagent) , or calcreagent)  into ratreagent) . RMP-7 preagent) , which sreagent) , 124. Inreagent) , 125 andreagent) , 126. Shreagent)  (Table 2reagent) . Moreovereagent) . Animal reagent)  and E4 (reagent)  increasereagent) . RMP-7 mreagent) , 112. Horeagent) , 130. ThImportantly, drugs used for tumor treatment may also exert effects on the BBB . Intra-cRecently, it has been found that convection-enhanced delivery (CED) using catheters stereotactically inserted into brain tumors fosters drug delivery into these tumors and surrounding brain tissue by establishing a local positive pressure gradient . In contFocused ultrasound is a novel non-invasive strategy for reversibly disrupting BBB tight junctions. Application of low frequency continuous wave ultrasound  generated well-controllable damage to tumor tissue, but minimal disturbance to surrounding healthy brain tissue . MicrobuThe recent advent of nanotechnology has led to widespread nanoparticle applications in intra-arterial administration . Due to The pharmacokinetic theory of intra-arterial drug administration suggests that a comparable tumor response can be obtained faster by intra-arterial than by intravenous administration due to the first pass effect. Regional slow blood flow can increase tissue drug levels and rapid systemic clearance . In a noAlmost 30 years ago, it was shown that glucose reduces the pH of tumors by reducing blood flow, which in turn promotes thermochemotherapeutic effects . VariousDespite these promising findings, the translation of TCH-promoted intra-arterial drug administration to brain tumors into clinically applicable procedures is still not completed. Although the strategy has been validated in standardized animal models, tumor response and survival time after intra-arterial drug administration in humans were not yet described. TCH has been widely used in neurosurgical procedures for the treatment of arteriovenous malformations and aneurysms, but drug distribution in tumor vessels is complex and blood flow might be variable. Moreover, a relatively high risk of systemic hypotension or regional low cerebral blood flow is a challenge for the clinical application of TCH in intra-arterial drug administration.11C-BCNU are consistent with the metabolic changes captured by PET  (195mPt-labeled cisplatin (99mTc-hexamethyl-propyleneamine oxime (HMPAO) allows one to evaluate the cerebral distribution after either fast pulsatile or slow continuous supraophthalmic carotid infusion (99mTc-hexakis-2-methoxyisobutyl-isonitrile (MIBI), SPECT can also assess changes in tumor MIBI uptake before and after radiochemotherapy  . Glucose18F-FDG) , as well18F-FDG) . Anotherisplatin . Moreoveinfusion . Using 9otherapy .The main limitation of SPECT and PET is the relatively low spatial resolution of these imaging modalities. CT is superior in this respect, has widespread availability, and is easier to apply. The permeability of blood vessels in brain tumors can be assessed in CT images by measuring iopamidol distribution in blood vessels and the extracellular fluid space . Angio-CWith the widespread availability of MRI, the intra-arterial administration of therapeutic drugs to brain tumors is further improved. Due to its high resolution and sensitivity, MRI has the ability to accurately assess the therapeutic response to the tumors. Magnetic resonance spectroscopy (MRS) is used to reveal intracellular pH changes after BCNU treatment for anaplastic astrocytomas or glioblastomas with alkalization in intra-arterial administration and acidification in intravenous chemotherapy . The redMRI usually determines the changes in tumor volume by measuring the size of the contrast-enhanced lesion. However, given that both recurrent tumors and therapeutic drugs may cause BBB destruction leading to heterogeneous enhancement and T2/FLAIR hyperintensity, MRI has limitations in distinguishing between tumor recurrence and long-lasting sequelae of the therapeutic intervention. To overcome this deficiency, hemodynamic changes in tumors are considered an important indicator to accurately predict disease progression. Originally, MR DSA following bolus injection of gadolinium chelates offers a high temporal resolution for showing large cerebral arteries, venous sinuses, and tumor blood vessels. In some doubtful cases, MR DSA was able to identify meningioma and acoustic neuroma by first-pass stain. The tumors were later confirmed by pathological examination . Next, dHowever, use of gadolinium-based agents is associated with a potential risk for brain deposition and nephrogenic systemic fibrosis . ChemicaMRI monitoring and magnetic targeting also play an important role for intra-arterial drug delivery systems. A prominent example is highly selective deposition of magnetic microparticles in brain tumors . In the There are a number of other approaches to improve intra-arterial administration. The first approach is the combination of compounds to enhance the efficacy of chemotherapeutic drugs. For instance, it was shown that the combination of oral 2% D, L-alpha-difluoromethylornithine and intra-arterial injection of BCNU doubled the median survival time of T9 gliosarcoma-bearing rats by reducing polyamine metabolism required for tumor growth and enhancing anti-tumor cytotoxicity of BCNU . SpirohyThe second approach is the combination of multiple therapeutic regimens. For example, intra-carotid administration of cisplatin combined with intravenous doxorubicin injection is an alternative treatment for patients with inoperable meningiomas . Short-tThe third approach is the use of new therapeutic agents. Injection of bromodeoxyuridine (anaerobic radiosensitizer) into the external carotid artery through a catheter has been found to increase susceptibility of glioma cells to radiotherapy and increases survival time of treated patients . MoreoveThe fourth approach is gene therapy. Intra-arterial infusion of a p53-containing adenoviral vector can delay the growth of Gli36 glioblastoma tissue carrying a missense-mutated p53 gene . PhosphoThe fifth approach is cell therapy that may benefit from intra-arterial administration. Intra-carotid rather than intravenous administration of a human cytotoxic T-cell line (TALL-104) in the 9L glioblastoma model and a metastatic xenograft model of epidermoid carcinoma enhanced specific anti-tumor effects and significantly prolonged survival time . Injectiin-vitro culture system. These organoids have greater feasibility in large-scale screening of therapeutic agents and can exhibit their aggressiveness and proliferative ability and establish vascular system in host brains. In addition, some large animals exhibit an anatomical structure of the brain and its blood vessels being similar to that of humans, which may be a good option to optimize intra-arterial procedures. These models can also make excellent use of clinical imaging technologies (This systematic review comprehensively describes existing strategies for promoting the accuracy of intra-arterial drug delivery in experimental and clinical brain tumor therapy . These snologies . Taken tAll datasets generated and analyzed for this study are included in the article/supplementary material.RH, JB, and SL defined the search strategy, conducted the literature search and review, analyzed the data, and drafted the manuscript. All authors approved the final manuscript version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most recently, 18F-NaF coronary uptake was shown to be a robust and independent predictor of myocardial infarction in patients with advanced coronary artery disease. In this review, we provide an overview of the advances in coronary 18F-NaF imaging. In particular, we discuss the recently developed and validated motion correction techniques which address heart contractions, tidal breathing, and patient repositioning during the prolonged PET acquisitions. Additionally, we discuss a novel quantification approach—the coronary microcalcification activity . This new method provides a single number encompassing 18F-NaF activity within the entire coronary vasculature rather than just information regarding a single area of most intense tracer uptake. On. On18F-s03689946 ).18F-NaF uptake have been proposed, one evaluating the whole-heart uptake  and another focusing on the uptake within the coronary arteries (coronary-specific method). The global coronary method employs a whole-heart segmentation, with manual delineation of the cardiac silhouette on consecutive axial slices of fused PET/CT images [18F-NaF activity can be calculated using a volume-corrected summation of the mean standardized uptake value (SUVmean) of each of the regions of interest drawn on consecutive images. A different approach evaluating the uptake in individual coronary plaques (coronary-specific method) was proposed by Dweck et al. and later refined by Joshi et al. [18F-NaF uptake, employing coronary CT angiography to visually identify atherosclerotic plaques. Regardless of the method utilized for 18F-NaF assessment, the detected radiotracer activity in an atherosclerotic plaque is affected by the blood pool activity [18F-NaF activity, providing a maximum target-to-background (TBRmax) output (TBRmax = maximum SUV of the lesion/mean SUV obtained in the background blood pool). The calculation of the TBRmax, however, is affected by the changes in the background blood pool activity which has been shown to change over a 2-hour period ranging from 1-hour postinjection to 3-hour postinjection while coronary lesion uptake remains consistent [Two different quantification techniques of coronary T images . This meT images . The gloi et al. , 20. Thiactivity . To offsnsistent . To corrnsistent .18F-NaF activity detected as higher than the blood pool originates from the coronary vasculature. However, there is a growing body of evidence that cardiac valves and great vessels bind 18F-NaF and, thus, have an impact on the accuracy of the quantitative assessments when using the global heart assessment technique [18F-NaF is often observed in the infarcted myocardium, myocardial scar tissue, and cardiac amyloidosis [18F-NaF reading leading to overestimation of coronary 18F-NaF activity [18F-NaF maximum TBR in culprit compared with nonculprit plaques is only 34% which is severalfold lower than PET tracer uptake reported in oncological settings [Both the global and coronary-specific approaches have their limitations. While the global coronary method is attractive due to its simplicity, the specificity of the method is limited. This is largely due to the small coronary lesion size (which results in low plaque activity) and the fact that it is prone to false-positive findings due to myocardial, valvular, or pericardial uptake as well as background noise . The gloechnique –35. Addiloidosis –38. All activity . The plaactivity . Additiosettings . Finally18F-NaF coronary uptake assessments, it is essential to take full advantage of state-of-the-art image reconstruction approaches [18F-NaF PET as it will enable making direct comparisons between centers/vendors and facilitate establishing large registry datasets [For precise and reproducible proaches . As demoproaches . In a reproaches . Accordidatasets .Imaging of the cardiovascular system is associated with significant motion during the often-lengthy imaging sessions—with acquisition times extending for up to 30 minutes , 34, 44.18F-NaF imaging is being considered a potential noninvasive tool for imaging and identification of high-risk coronary artery disease, the uptake quantification method should ultimately be in line with our current understanding of the coronary atherosclerotic disease. While for decades physicians and researchers have pursued the quest to identify vulnerable atherosclerotic plaques, despite major advancements in imaging technology that allow visualization of rupture-prone plaques, this approach did not translate into an improved prediction of adverse events [18F-NaF uptake can be utilized [Since coronary e events . It has e events . Similare events . Furthere events . In thise events . A compre events . The reae events . It was e events , 59. Imputilized .18F-NaF coronary imaging, reporting only maximum TBR values for a given patient can be suboptimal. To adjust 18F-NaF coronary uptake quantification to the new per patient (rather than per plaque) paradigm, we recently proposed a novel semiautomated approach for image analysis [18F-NaF uptake throughout the entire coronary vasculature using CT angiography-derived centerlines to build 3-dimensional tubular volumes of interest around each of the main epicardial coronary arteries. Extending from the coronary-specific method, the coronary microcalcification activity method permits the evaluation of coronary 18F-NaF activity on a per vessel and per patient basis, providing more global assessments of disease activity in the coronary arteries than the per lesion approach.In view of the shift from a per lesion to a more global per patient approach, it is immediately apparent that for analysis . The cor18F-NaF uptake measure resembles the Agatston method for quantifying coronary CT calcium scores and is based on PET quantification techniques widely employed in oncology and cardiac sarcoidosis [18F-NaF PET activity can be calculated as the integrated activity in SUV units exceeding the background blood pool SUVmean + 2 standard deviations. As a result, in patients and vessels with multiple foci of uptake, CMA provides a measure of disease activity across the coronary vasculature.The coronary coidosis –64. Tort18F-NaF PET within 3 weeks from an acute myocardial infarction. Previous studies suggested that 18F-NaF activity is associated with the presence and correlated with the volume of CT-derived markers of plaque vulnerability [18F-NaF activity with better categorical separation  or absence of activity (CMA = 0) in the coronary tree, there was 100% agreement in the categorization between observers and scans. This is clinically relevant especially in populations without known coronary artery disease if 18F-NaF is to be used as a screening tool where little or no activity is expected. In such a population, the fact that CMA = 0 is perfectly reproducible between observers and scans is of extreme importance as it could reliably inform the physician regarding the coronary microcalcification activity in a given patient.For an imaging biomarker to have clinical utility, it should demonstrate precise and reproducible analytical performance , 70. Mulal scans , 28, 70.al scans . We haveal scans . By leve18F-NaF coronary PET.Given that in the group of patients with established CAD prediction of adverse events has proven challenging , we evaluated whether 18F-NaF could predict myocardial infarction in this population [18F-NaF PET provides a powerful independent prediction of fatal or nonfatal myocardial infarction. In 293 study participants with a high atherosclerosis burden , we observed 20 myocardial infarctions during almost 4 years of follow-up. On receiver operating characteristic curve analysis, the coronary microcalcification activity showed a greater area under the curve for the prediction of myocardial infarction than coronary calcium scores and clinical risk scores. To generate distinct clinical risk groups, we dichotomized the population according to their coronary 18F-NaF uptake. A threshold of 1.56 for CMA (derived from the receiver operating characteristic curve by the Youden index) achieved a specificity and sensitivity of 66% and 80%, respectively, for the primary endpoint. Consequently, on univariable Cox proportional regression, CMA > 1.56 emerged as a predictor of fatal or nonfatal myocardial infarction . Importantly, these associations persisted on multivariable analysis after adjustments for sex, comorbidities, segment involvement score, number of coronary stents, multivessel coronary artery disease, coronary calcium score, and risk scores  for the primary endpoint; While the ability to identify culprit plaques in patients with acute myocardial infarction and lesions with adverse morphology is scientifically attractive until recently, we lacked a meaningful clinical application of pulation . In a po18F-NaF PET has emerged as a robust tool for the assessment of active calcification processes. It has been shown to reliably identify culprit plaques, lesions with adverse morphology, and predict adverse events. Importantly the methods of image acquisition, image reconstruction, and analysis have been investigated and greatly improved over the past decade. In the near future, the outcome implications of 18F-NaF coronary PET will be further elucidated in the Prediction of Recurrent Events With 18F-Fluoride (PREFFIR) study which shall prospectively investigate the ability of 18F-NaF coronary PET to predict recurrent events in patients with multivessel disease and recent myocardial infarction. In regard to coronary artery disease, future studies should evaluate the potential role of 18F-NaF for risk stratification and guidance of therapeutic decisions in subjects with advanced atherosclerosis [18F-NaF coronary PET research was predominantly focused on subjects with established disease, this imaging modality was also shown to identify active atherosclerotic disease across the entire spectrum of patients with subclinical, suspected, and established coronary artery disease. While in patients with advanced atherosclerosis 18F-NaF PET clearly outperforms clinical characteristics and scores in risk stratification, it remains to be studied whether it can add to the already-robust risk prediction in patients with the suspected disease [18F-NaF PET imaging across multiple vascular beds and further improve approaches for data acquisition and analysis.To date, coronary clerosis , 72. Alt disease . Similar


Among 11 patients in Thailand infected with severe acute respiratory syndrome coronavirus 2, we detected viral RNA in upper respiratory specimens a median of 14 days after illness onset and 9 days after fever resolution. We identified viral co-infections and an asymptomatic person with detectable virus RNA in serial tests. We describe implications for surveillance.  During January 2020, persons in Thailand were tested for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection if they had a combination of fever or respiratory illness and a history of travel to Wuhan, China. Persons determined to be close contacts of a laboratory-confirmed coronavirus disease (COVID-19) case-patient also were tested during enrollment into contact tracing. Clinicians were able to request testing if they had a concern regarding persons who were exposed to travelers. During January 8–31, 2020, Bamrasnaradura Infectious Diseases Institute, the national infectious disease referral hospital in Bangkok, admitted 11 patients with laboratory-confirmed COVID-19. We describe clinical features, clinical management, and results of serial reverse transcription PCR (RT-PCR) testing for SARS-CoV-2 RNA for these patients.http://www.fast-trackdiagnostics.com), according to the manufacturer’s instructions. During the study period, Thailand’s discharge criteria for hospitalized COVID-19 patients required resolution of clinical signs and symptoms and 2 respiratory specimens without detectable SARS-CoV-2 RNA collected >24 hours apart.The 11 hospitalized patients had daily nasopharyngeal and oropharyngeal sampling for SARS-CoV-2 RNA testing. Specimens were collected by using synthetic fiber swabs, which were combined and placed into a single sterile tube containing ≈3 mL of viral transport medium. RNA was extracted and tested with conventional RT-PCR and real-time RT-PCR (rRT-PCR). We developed SARS-CoV-2–specific primers and probes by using a protocol from the World Health Organization  days after illness onset, and these patients had detectable SARS-CoV-2 RNA in upper respiratory tract specimens for a median of 14 (9–26) days after illness onset . HoweverAmong persons of Chinese ethnicity in our study, only 3/9 who traveled from China were detected through airport screening. During the study period, <7% of all persons under investigation for COVID-19 in Thailand were detected through airport screening (–Patient 4 had detectable SARS-CoV-2 RNA for 4 consecutive days, but we were only able to follow her for 7 days before she returned to China. Her case is an example of a person without reported symptoms but radiologic evidence of disease and detectable virus over several days. Other studies have described asymptomatic patients with upper respiratory specimens positive for SARS-CoV-2 (Our case series has some limitations. Patients could have recall bias regarding symptom onset before hospitalization. We were unable to complete a 14-day observation for some patients because they returned to China after discharge, including patient 4, who had no reported respiratory symptoms. The relatively long duration of hospitalizations in our study highlights the effects that current surveillance and isolation procedures can have on clinical care surge capacity. Duration of hospitalization was extended by Ministry of Public Health requirements for patients to remain in the hospital until symptom resolution and clearing of SARS-CoV-2 RNA in clinical samples. We observed that it took a median of 9 days to clear SARS-CoV-2 after fever resolution. In addition, we noted serial detection of SARS-CoV-2 RNA in respiratory specimens of an asymptomatic patient. Our observations of possible viral co-infections in COVID-19 patients and the resolution of fever relatively early during clinical course also have implications for surveillance strategies. Specifically, case definitions requiring fever could miss COVID-19 cases, especially later in the clinical course, and surveillance strategies that test only for SARS-CoV-2 could miss co-infections. Clinicians should consider the possibility of co-infection because the presence of other respiratory pathogens does not exclude the possibility of SARS-CoV-2 virus infection. Clinicians also need to better understand the relationship of RT-PCR detection of SARS-Cov-2 via multiple shedding routes (Additional information on hospitalized patients diagnosed with coronavirus disease, Thailand.


Correction to: BMC Med Res Methodol 20, 211 (2020)https://doi.org/10.1186/s12874-020-01097-6Following publication of the original article , the autThe sentence currently reads:The Beta-Binomial model was originally proposed by Ball, and further development of the Binomial model was introduced in his recent safety monitoring paper as well [27].The sentence should read:The Beta-Binomial model was originally proposed by Ball, and further development of bridging blinded and unblinded analysis was introduced in Lin’s recent safety monitoring paper as well [27].