Case management of COVID-19 is critically important to save lives and reduce the fear and anxiety evoked by this disease in communities. However, most healthcare systems have been overwhelmed in many parts of the world. It is also essential to screen patients to be able to identify those who are likely to suffer from severe disease, to ensure more effective use of medical resources. Disease severity can be determined based on simple vital signs; some laboratory markers are useful adjuncts.Treatment for COVID-19 is largely supportive although a number of repurposed drugs have been evaluated for its efficacy in clinical trials. For example, dexamethasone has now been considered as a standard therapy for severe cases, while remdesivir and tocilizumab are promising agents in selected patient groups. Its first case was reported in Wuhan, China, in December 2019 and, since then, has spread across the globe. As of March 7, 2021, about 116 million confirmed cases of COVID-19 have been recorded worldwide, including around 2.6 million deaths, as per the reports of the World Health Organization (WHO) (3). The clinical picture is broad, ranging from asymptomatic infection to upper respiratory tract infections, pneumonia, acute respiratory distress syndrome, and multiple organ failure (4). Majority of the fatal cases have been observed among older people or people who have underlying diseases (5).Coronavirus disease 2019 (COVID-19) is defined as an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (6). Treatment for COVID-19 is largely supportive although a number of repurposed drugs have been evaluated for its efficacy in clinical trials (7). Through these concerted efforts, dexamethasone has now been considered as a standard therapy for severe cases who require supplemental oxygen although many clinicians raised safety concerns for its use in the initial phase of the pandemic. In this paper, the clinical management of COVID-19 patients with mild to moderate disease is discussed focusing on Japanese clinical settings (8).Thus, case management of COVID-19 is critically important to save lives and reduce the fear and anxiety evoked by this disease in communities. However, most healthcare systems have been overwhelmed in many parts of the world Patients suspected of having COVID-19 are required to undergo diagnostic testing for confirmation of the disease (polymerase chain reaction or antigen test). After confirming a diagnosis, it is also essential to screen the patients to identify those who are likely to suffer from severe disease, to ensure more effective use of medical resources. During epidemics, it is vital to quickly and thoroughly take one’s medical history to obtain pertinent facts such as date of onset of symptoms, age and sex of the patient, and presence of any underlying disease.(8). However, some patients with COVID-19 do not complain of dyspnea despite the presence of respiratory failure (silent hypoxia); thus, it is desirable to measure percutaneous oxygen saturation (SpO2) using a pulse oxymeter (9). Patients with SpO2 lower than 96% are suspected of having pneumonia and should thus be assessed further (8). Blood tests and imaging modalities add benefits to verify disease severity. Computed tomography (CT) is the method of choice for the early detection of pneumonia findings (ground-glass opacities) (10). Once the disease severity is identified based on SpO2 and the patient’s clinical condition, a treatment policy is decided on ((12).Disease severity can be determined based on simple vital signs, including level of consciousness and respiratory rate as that of community-acquired pneumonia cided on . As rega(8).The indication for hospitalization according to disease severity is based on the judgment of the physician, but even if the patient is asymptomatic or has only mild symptoms, a recommendation for hospitalization and isolation measures may still be given to patients to prevent further spread of the disease in the community (8). If SpO2 is still below 93%, high-flow nasal oxygen (HFNO), noninvasive positive pressure ventilation (NPPV), or invasive mechanical ventilation (IMV) is required to maintain appropriate oxygen level. However, HFNO and NPPV potentially produce more contaminated aerosol in the environment, resulting in the nosocomial transmission of SARS-CoV-2 (13). Thus, a stepwise process of decision-making is helpful to choose the best possible respiratory support modality during an overwhelming situation .Supportive care is still the mainstay of treatment for COVID-19 while there has been a desperate need for drugs to improve prognosis in severe cases. Potentially antiviral, anti-inflammatory, and antithrombotic drugs have been under investigation in clinical trials. In addition, many kinds of medications seem to be utilized on off-label or compassionate use in many countries including Japan (7).As of March 2021, no antiviral or potentially active repurposed drugs are recommended for use in patients with mild COVID-19. For patients with moderate to severe disease who require oxygen administration, the combination of remdesivir and steroids (such as dexamethasone) or steroids alone is considered as the standard therapy as well as the best available supportive care (3).Before the COVID-19 epidemic, no anti-coronavirus drugs have been developed; thus, attempts have been made, since the start of the pandemic, to repurpose existing antimicrobial or other potentially effective drugs for this condition. Clinical trials are currently being conducted on drugs that are effective against SARS-CoV-2 in vitro. However, as of March 2021, the number of drugs with established effects remains minimal. In Japan, remdesivir, which received urgent approval from the US Food and Drug Administration in May 2020, has been given special approval. Compassionate use of favipiravir, an anti-influenza drug stockpiled as a countermeasure against a novel strain of influenza virus, is being offered at the physician’s request. Some of the major drugs that are in clinical trials in Japan and overseas are also listed in (17). In an international joint clinical trial (double blind) led by the National Institutes of Health in the USA in patients with COVID-19 (moderate to severe), clinical symptoms improved faster in the group administered remdesivir than in the placebo group (10 days vs. 15 days). However, in an international joint clinical trial (single blind) led by the WHO, no reduction in case fatality rate was reported . Another open-label randomized clinical trial conducted in India demonstrated a shorter time to resolution of clinical signs and symptoms although it did not show a shorter time of virus shedding compared with the control group (19). Compassionate use of favipiravir has been permitted in Japan and other countries. There have been few adverse reactions reported in patients with COVID-19 treated by this drug. However, it is potentially teratogenic and is, thus, contraindicated in pregnant women.This is an RNA-dependent RNA polymerase inhibitor. This drug has been licensed for pandemic influenza due to a novel strain and stockpiled as a part of pandemic preparedness in Japan. In vitro efficacy has also been shown for SARS-CoV-2, and an investigator-initiated clinical trial on mild cases and a sponsor-initiated clinical trial for NDA on moderate cases (both single blind) have been conducted so far in Japan. In the former, the number of days until a negative PCR test for SARS-CoV-2 was not different from that in the group not administered favipiravir. However, the treated group showed an early tendency of reduced fever though the difference was not statistically siginificant (20).The HIV protease inhibitor, lopinavir/ritonavir, has been studied in small clinical trials against severe acute respiratory syndrome (SARS) and MERS (Middle East respiratory syndrome) before the COVID-19 pandemic and showed potential efficacy. However, this drug did not reduce the case fatality recorded in patients with moderate to severe COVID-19 in well-designed clinical trials (21). Moreover, the WHO has not recommended this drug for COVID-19 in the recent guideline.It was revealed that the antimalarial drug hydroxychloroquine is ineffective in reducing mortality and preventing the onset of the disease. Furthermore, well-designed clinical trials have failed to show its efficacy in treatment and post-exposure prophylaxis (22). A significant improvement in prognosis was seen in those requiring invasive mechanical ventilation. The case fatality rate within 28 days of enrolment was 29.0% in the treatment group as against 40.7% in the control group. Also, 21.5% of the dexamethasone-treated group who required oxygen administration at the time of enrolment died within 28 days after enrolment, compared with 25.0% of the control group. However, no prognostic improvement effect was observed in the group that did not require oxygen administration at the time of enrolment. Therefore, this drug is recommended to be administered as the standard therapy for patients with moderate to severe COVID-19 requiring oxygen therapy. The dose is 6 mg of dexamethasone once daily for 10 days. Other steroids probably have the same effect (hydrocortisone 160 mg once daily and methylprednisolone 16 mg every 12 hours) (23).Steroids have been used in patients with MERS and SARS, but there have been concerns that they may delay virus clearance and increase case fatality rates. A well-designed clinical trial (RECOVERY) conducted in the UK demonstrated the efficacy of dexamethasone against severe COVID-19 (24). As of March 2021, at least five clinical trial results have been published as peer-reviewed articles. A clinical trial on critically ill patients in the ICU conducted in the USA showed that tocilizumab (8 mg per kilogram of body weight) and sarilumab (400 mg) produced shorter organ support-free days and improved survival (25). However, another clinical trial on severe cases conducted in the USA revealed no efficacy (26).As significantly high serum CRP levels are noted in patients with severe COVID-19, the recombinant anti-IL-6 monoclonal antibodies, tocilizumab and sarilumab, have been expected to have immunomodulatory effects and improved prognosis (27). An observational study which included over 3,000 patients with COVID-19 in the USA supported the survival benefit of high-titer convalescent plasma (28).Clinical trials are being conducted under the hypothesis that the anti-SARS-CoV-2 antibody in the plasma of recovered patients will suppress viral growth and have immunomodulatory effects in patients with the disease. The limited number of studies has been published as of March 2021. One clinical trial conducted in the USA showed that early administration of high-titer convalescent plasma against SARS-CoV-2 to mildly ill infected older adults has reduced the progression of Covid-19 (29). High D-dimer levels are identified as a vital marker of suspected thrombosis. In these patients, anticoagulant therapy, such as heparin, is administered. Low-molecular heparin has not been approved for this indication in Japan.Pulmonary thromboembolism can cause respiratory failure and, ultimately, death in patients with COVID-19, with frequent coagulation abnormalities seen in those with moderate and severe diseases requiring oxygen administration (7). Most recovered patients do not require any further treatment. However, if symptoms are prolonged, such as fatigue, dysosmia, and dysgeusia, the patient should be monitored closely in an outpatient setting (30). These clinical manifestations called post-COVID syndrome or long COVID are emerging issues and should thus be further examined under a prospective cohort study.Hospitalization recommendations and work restrictions to prevent the spread of the disease will be lifted 10 days after the onset and 3 days after the symptoms improvement It seems that many patients infected with COVID-19 could not receive appropriate medical care because of limited bed availability in hospitals in most countries. Thus, creating a system to observe mildly ill patients, who may suffer from severe disease at later stages, at home or in designated accommodations in the community is a topic that requires consideration. To reduce the case fatality rate, it is vital to ensure standard treatment at an appropriate time. The coordination of healthcare resources in the community is key to reduce overall mortality.Treatment for COVID-19 is largely supportive, but it is worth noting that many clinical trials have been conducted globally during the pandemic, and some medications have been proven to be effective against COVID-19. There is hope for more effective therapeutics available in the near future.Yasuyuki Kato received consultant fee from Fujifilm Toyama Pharmaceutical, Co.This work was supported by Health Labour Sciences Research Grant (2020HA20).I appreciate the board members of JAPAN COVID-19 Case Management Guide Review Committee for their contribution to this work.https://www.med.or.jp/cme/jjma/newmag/15002/15002.html. The Editors-in-Chief of the Journal of the Japan Medical Association and JMA Journal have permitted the publication of this manuscript.The original version is available at (31) published in Japanese on 1 May 2021.This is the secondary version of the previous article


After relaxing social distancing measures, South Korea experienced a resurgent second epidemic wave of coronavirus disease 2019 (COVID-19). In this study, we aimed to identify the transmission dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and assess the impact of COVID-19 case finding and contact tracing in each epidemic wave.Rt) was estimated using the illness onset of the cases. Furthermore, to identify the characteristics of each epidemic wave, frequencies of cluster types were measured, and age-specific transmission probability matrices and serial intervals were estimated. The proportion of asymptomatic cases and cases with unknown sources of infection were also estimated to assess the changes of infections identified as cases in each wave.We collected data on COVID-19 cases published by local public health authorities in South Korea and divided the study into two epidemic periods (19 January–19 April 2020 for the first epidemic wave and 20 April–11 August 2020 for the second epidemic wave). To identify changes in the transmissibility of SARS-CoV-2, the daily effective reproductive number  in both waves. The proportion of asymptomatic cases at presentation increased from 22% (in the first wave) to 27% (in the second wave), while the cases with unknown sources of infection were similar in both waves .In early May 2020, within 2-weeks of a relaxation in strict social distancing measures, Our study shows that relaxing social distancing measures was associated with increased SARS-CoV-2 transmission despite rigorous case findings in South Korea. Along with social distancing measures, the enhanced contact tracing including asymptomatic cases could be an efficient approach to control further epidemic waves.The online version contains supplementary material available at 10.1186/s12879-021-06204-6. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection . South KSustained increases in cases were observed as the strict social distancing measures were further relaxed by opening public facilities on 6 May 2020 when the first epidemic wave had already ended. Increase of the cases can be observed by the changes of public health efforts of extensive COVID-19 case finding and contact tracing . HoweverTo characterize the transmission dynamics of SARS-CoV-2 in South Korea, we identified the major cluster types of COVID-19 cases, and we estimated the time-varying effective reproductive number, serial interval distribution, age-specific infector-infectee matrices for the two COVID-19 epidemic waves. Furthermore, to assess public health efforts to find and trace cases in South Korea, we estimated the proportion of cases that were asymptomatic and the proportion of local cases with an unknown origin of infection at the time of first clinical assessment.South Korea is comprised of a special city, seven metropolitan cities and nine provinces, with a total population of 51.4 million. Among the 14,700 confirmed COVID-19 cases in South Korea to date (16 August 2020), 8358 (57%) cases were reported from the Daegu-Gyeongsanbuk region (5.1 million population), including 5564 cases from a large initial cluster linked to a religious community , 14.We collected data on COVID-19 cases that had been confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in South Korea. Nasal swab samples from all suspected contacts of COVID-19 confirmed cases were tested at the KCDC or national designated laboratories, following a consistent test protocol . Data inLocal public health authorities provided daily updates of all laboratory-confirmed COVID-19 cases on their webpage and we compiled daily updates from the public dashboard in different local authorities. Information included national and local case number, age, sex, symptoms, symptom onset date, source, exposure date, and location (within or outside South Korea) of infection were provided. We also compiled a database of COVID-19 infector-infectee transmission pairs where precise symptom onset dates and source of infection were available. Here, we focused on the period from 19 January through 11 August 2020, after which the specific information of cases was not yet released by the local authorities under the reinforced regulation of privacy protection .Based on the epidemic curve and the timing of relaxation of social distancing measures, we defined the first epidemic wave of COVID-19 as the period between 19 January 2020–19 April 2020, and the second epidemic wave between 20 April – 11 August 2020. Furthermore, we defined community clusters as five or more linked COVID-19 cases , excludiRt), which defines the mean number of secondary infectious cases generated from a typical primary infectious case at time t [Rt. We estimated Rt using the EpiEstim package in R [Rt is 1, where the epidemic becomes under control if Rt falls below this threshold sustainably. Rt was estimated based on the respective serial interval distributions, estimated from the data for the first and second epidemic waves in this study and the daily incidence of COVID-19 cases over time with a 7-day smoothing window [Rt after 1 February 2020, because a stable estimate of Rt was not available due to the low number of cases. We computed the serial interval, which represents the time between a symptom onset of successive cases in a transmission chain, as the number of days between infector and that of infectee in the transmission pairs. We estimated the serial interval distribution during the first and second epidemic waves by fitting a normal distribution [We compared the differences in age groups between two different waves using chi-squared tests. We described the epidemiological characteristics of COVID-19 cases and the epidemic curve was constructed by stratifying the data into local and imported cases. To identify the potential changes in SARS-CoV-2 transmissibility of local cases after relaxing social distancing measures, we estimated the time-varying effective reproductive number , including 1189 imported cases (547 and 642 cases in the first and second wave), have been reported outside of Daegu-Gyeongsanbuk region in South Korea. There were no statistically significant differences in age groups between the cases from the two epidemic waves , workplaces , institutional homes for the elderly , and a number of other settings  . In the Overall, 634 cases  reported no symptoms at the time of the first clinical assessment. The mean proportion of asymptomatic cases at presentation in those aged 20–39 years increased from 14.7 to 21.8% over time, and the overall mean proportion increased from 21.7 to 27.1% Fig. . There wIn South Korea, the combined public health measures implemented early in the COVID-19 epidemic reduced the spread of SARS-CoV-2 . As mobiA large number of imported cases in the first epidemic wave challenged efforts to control COVID-19 in South Korea. In late March 2020, many Koreans in Europe and the United States traveled back to Korea due to the lockdowns. Public health authorities implemented mandatory laboratory screening at the port of entry and a 14-day mandatory self-quarantine program for travellers from Europe (22 March) and then all overseas travellers (1 April) . Local hRt around 1 more than a month.We identified many clusters associated with religious activities where individuals spend prolonged periods of time in close proximity . These wWe found that a large proportion of transmission occurred between individuals of the same age, and transmission between children was rare. However, school closure in the first wave may have contributed to reduced opportunities for transmissibility between children . After rWe identified that 16.6% of the local cases were asymptomatic at the time of presentation, which is similar to those in Shenzhen (20%) and Hong Kong (21%) in China , 31. TheIn South Korea, all close contacts of laboratory-confirmed cases were quarantined, and public health authorities diligently traced the source of infection. The presence of unidentified cases indicates hidden and uncontrolled transmission in the community. Therefore, a low proportion of unlinked local cases is an indication of effective case finding . In our A study in China demonstrated that serial interval distributions can be shortened by active case finding and enhanced public health measures . In our A modelling study demonstrated that control measures, including contact tracing, testing, and self-isolation, would be less effective if asymptomatic infections are higher . FurtherRt indicated that even when rigorous public health efforts were in place, relaxation of certain social distancing measures in the community, in our case further reopening of public facilities, may allow resurgence of COVID-19 within days. Further research on how social distancing and other public health measures should be relaxed is warranted. Furthermore, simulation studies based on empirical data with the counterfactual scenarios to predict a potential resurgence of COVID-19 would help public health authorities prepare for future outbreaks.Recent studies in Hong Kong, China , and SouOur analysis has several limitations. First, we excluded the Daegu-Gyeongsanbuk region where the epidemic was mainly driven by large superspreading events in a religious group at the very beginning of the first epidemic wave. The outbreak occurred mainly in Daegu-Gyeongsanbuk region and well before the major control measures were implemented, and uncooperative attitude of the members of the religious group was reported during the epidemiological investigation ; hence iRt of SARS-CoV-2 in South Korea from illness onset data, whereas previous studies [Rt using the serial interval distributions, which is evaluated by constructing transmission pairs on this illness onset data. Whereas, the earlier estimates were based on the approximation of serial interval distributions, evaluated for different data and locations, mostly used the early finding of serial interval in China which did not even include the pre-symptomatic transmission [Rt to better characterize the changes of transmissibility after relaxing social distancing measures which did not include earlier study [Our study has several strengths including the estimation of daily  studies , 43, 44  studies . Furthersmission  and mighsmission . Third, er study . Finallyer study , providier study , and incSouth Korea has implemented public health measures to reduce the transmissibility of SARS-CoV-2. However, despite rigorous public health efforts, relaxing social distancing measures allowed the reemergence of COVID-19 in South Korea. This study is the first epidemic analysis of the two different epidemic waves of COVID-19 using empirical data based on the illness onset and serial interval which analysed in our transmission pairs. Our findings suggest that enhanced contact tracing including asymptomatic cases is an efficient approach to control further epidemic waves together with social distancing. A more conservative approach to control COVID-19 should be considered together with close monitoring of social mobility.Additional file 1.


Drosophila larvae and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish for a prolonged duration (5 days) were carried out to understand muscle structuring , primitive Macrophages (in yolk-sac) and inter-dependent lipid and protein-based metabolism. The volume-based study, intensity line-plots and inter-dependence ratio analysis allowed us to understand the transition from lipid-based metabolism to protein-based metabolism during early development . A co-planar multicolor light sheet PSF is introduced to illuminate the tissues labelled with spectrally-separated fluorescent probes. The detection is carried out using a dual-channel sub-system that can simultaneously record spectrally separate volume stacks of the target organ. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged the  Drosophila) to understand organ growth and regeneration beginning from embryonic to adult period. Such studies in Zebrafish inform us about the onset of the formation of the dorsal-ventral stripes, pectoral fins and the circulatory system . In addition, these organisms have aided our understanding of cardiovascular and neuronal diseases and their progression3. A major advance in quantifying these studies requires a non-invasive optical fluorescence microscope with rapid volume imaging capability, large field-of-view, diffraction-limited resolution and minimal photobleaching. Compared to conventional point-scanning technique, the light sheet based plane-scanning technique is faster and has the advantage of minimal photobleaching and phototoxicity6. In addition, such microscopy technique may facilitate inter-organ communication studies and prolonged study of the entire multicellular organism in its natural physiological state.The field of developmental biology routinely uses model organisms  have emerged. Some of these include thin light-sheet microscopy12, ultramicroscopy13, objective coupled planar illumination microscopy (OCPI)14, confocal light-sheet microscopy16, multiple light-sheet microscopy17, dual-inverted selective-plane illumination microscopy (diSPIM)18, light-sheet theta microscopy (LSTM)19, open-top light-sheet (OTLS)21, coded light-sheet array microscopy (CLAM)22 and lattice light sheet microscopy23. The advantages offered by LSFM and its variants have seen an upsurge in the studies related to large biological specimens26. Recently few groups  have shown impressive time-series of entire embryo31. However, the standard protocol requires visualization of a specific specimen plane at a time and thus suffers from missing information from other sections of the whole specimen. The large data acquisition required in traditional light-sheet microscope (LSM) slows-down the volume reconstruction process. LSM techniques require both translation and rotation of specimens, whereas proposed OVSS employs only rotation for data collection. This substantially reduces data acquisition time and thereby fastens volume imaging. So there is a need to accelerate light-sheet based volume imaging and screening system with the capability of simultaneous multichannel imaging and long-time monitoring. This will ensure real-time volume interrogation of drug-receptor interaction and fluorescence label-based assays32. An advanced version of such a system would be able to track and locate target proteins complexes and drug molecule clusters in the organs during early development.The light-sheet-based microscopy is quickly gaining importance as the scope-of-choice due to its large field-of-view (FOV) imaging, high contrast  and diffraction-limited resolution33. In another application, a high-speed volumetric calcium imaging was demonstrated by Prevedel et al using light-field microscopy34. A fast version of light sheet microscopy called SPED was recently demonstrated that show fast sub-cellular resolution imaging of clarity mouse brains and volumetric Ca(2+) imaging of entire zebrafish nervous systems35. Recently, Greer et al. has presented a new speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy for fast calcium imaging of the larval zebrafish brain36.Fast imaging using light sheet is demonstrated in a variety of techniques. Truong et al, showed faster imaging using light sheet when compared to point scanning techniquesTo overcome the limitations  of existing light-sheet microscopy, it is necessary to develop a rapid multicolor volume imaging system. Existing LSM systems require light-sheets to be scanned throughout the specimen in a translation-rotation configuration, but this process demands large scanning time and big data-storage device when compared to rotation-based OVSS system. It is thus imperative to design advanced light-sheet microscopes that are simple in geometry and readily adaptable to the existing systems. A simple yet efficient route to address these challenges is to eliminate translational scanning altogether and rely solely on rotational scanning integrated with reconstruction techniques for volume imaging. Rotation ensures isotropic data collection as compared to linear scanning employed in traditional LSM and its variants.38, we have used cubic interpolation which is more precise. However, note that, the image treatment (interpolation) can improve the visual appearance but it cannot recover missing information. So, the real-time advantage comes with a balance between the speed (data acquisition time) and information content. Using sampling theory, the ideal sampling is found to be approximately 3 degrees for 7.5 micron thick light sheet. Hence 60 2D-angular image are enough to reconstruct volume-stack. From the data acquisition timing diagram, it is found that OVSS method takes 42 ms/frame and a total of 60 images whereas, traditional LSFM takes a much larger number of images for volume reconstruction. In general, OVSS is faster than traditional SPIM. Moreover, it was found that high rotation speed of stepper motor results in mechanical errors due to the vibration.Rotation-based isotropic scanning is attractive as this boosts-up the speed by many folds. This comes with the fact that OVSS requires interpolation to determine data on the rectangular-grid  Microscopy. To demonstrate the versatility of our OVSS system, we performed studies on muscle development and lipid accumulation in the Zebrafish from pharyngula period to larval period. Our OVSS system facilitates the study of multiple organs and abnormalities during development, aiding non-invasive probing with great potential in health-care and therapy.39. Detailed photobleaching characteristics of the fluorophores  used in the present biological study can be found in Supplementary The prolonged data acquisition time and severe photobleaching prohibits real-time visualization of rapidly-occurring biological processes during early development and so there is a necessity to develop fast volume imaging system that can image an entire organism with minimal optical artifacts and are free from photobleaching effect. During preliminary investigation, we observed that organs labeled with fluorophores (Yolksac stained with BODIPY) result in faster bleaching due to short bleach-time constant. So the saturation is avoided during data acquisition. It is however important to note that photobleaching remains a serious concern for rapid volume imagingThe proposed OVSS imaging has a simple geometry that enables simultaneous multi-organ volume imaging and long-time interrogation. Few new features are introduced in LSM microscopy that includes, multicolor lightsheet illumination, rotation-based angular 2D data collection and simultaneous multicolor detection. This led to faster multichannel data acquisition for volume reconstruction  imaging, OVSS provides an alternative system for rapid multicolor scanning, monitoring and whole volume visualization. As an example, light-sheet techniques (MuVI-SPIM) require 41, and (2) Direct measurement of light-field by translating a camera in the beam-path43. First technique requires incorporation of key experimental parameters  and refractive index of objective immersion liquid) as variables in the computational study. Using vectorial theory of light44, the field was calculated at each point within a grid-size of The quality of images acquired is strongly dependent on the electric field distribution at the focus and in the neighborhood. A well-focused lightsheet reduce the plane thickness and thereby greatly improves signal-to-background ratio. Two approaches were adopted: (1) Computationally determining the light-field from the first principle41, and  Direct mz-increment . Figure As a first step towards multicolor OVSS imaging it is essential to calibrate the system and validate it with a known sample. We have used a random mix of 473 nm and 532 nm excitable fluorescent micro-spheres for which the emission spectra peaks at 515 nm (Green window) and 575 nm (Red window), respectively. The beads were embedded in the Agarose gel-sample as per the developed protocols described in the Methods section. Figure Drosophila larvae  is performed. Figure 45. Natural crawling behaviours include turns, head sweeps, pauses, hunching, bending, burrowing, rolling (escape) and forward and backward locomotion46. Such a wide repertoire of behaviours requires coordinated interactions between the motoneuron and the muscle. During Drosophila development, the motor neurons and muscles differentiate synchronously, offering an excellent model system for studying intercellular communication between the two cell-types during synapse formation47. We examined the existence of such interactions by interrogating the thoracic region of the third larval instar. Hence, the larval somatic body wall muscles and the motor neurons that innervate them are chosen for the present study. Muscles were visualised using the localisation pattern of Zasp52, a core component of the Z-disc in Drosophila muscles, which are organising centres that establish and maintain myofibril structure and function. On the other hand, we visualised neurons by driving the expression of the reporter construct CD8-mRFP throughout the nervous system using nSyb-Gal4.Drosophila larvae are in a continual state of crawling, foraging for food to meet the demand of a nutrient-dependent critical weight in order to undergo metamorphosisFigure 48.The parallel study of multiple organs and their interdependence during development is not well understood. A little is known about the metabolic cycle as the development of multiple organs take place in the early stages of organism development. We follow development of organs and even entire organisms in detail, beginning from early pharyngula stage to larval stage of the Zebrafish using OVSS, which is a non-invasive imaging technique to study the development of tissues in Zebrafish. This technique requires separate fluorescent labeling of two different organs (muscle and yolk-sac) with specific fluorescent probes. We have stained muscle using phalloidin-TRITC and yolk with BODIPY. Phalloidin is a bicyclic toxin peptide, commonly used to identify filamentous actin  present in the muscle Dystrophin whereas BODIPY is a fluorescent dye used to stain the lipid-rich yolk-sac (primitive Macrophages) (see Methods section for specific protocols)Figure 49. So, the growth-trait of muscle and lipid are strongly inter-linked. This is still ambiguous and calls for further investigation. Using OVSS, we wish to simultaneously visualize and observe the development of muscle and lipid growth in the early stages of Zebrafish.Understanding development of multiple organs at different phases of Zebrafish is critical in Developmental Biology. Muscle development in Zebrafish is particularly interesting as the embryos begin to move very soon after gastrulation. Specifically, muscle development is known to occur somewhere at the interface of Segmentation and Pharyngula period. Moreover, it is reported by Gao et al., that Myostatin is directly responsible for muscle formation as demonstrated by their studies in Myostatin-deficient Zebrafish, thereby indicating a transition to protein-dependent from lipid-dependent energy metabolism51. Figure Rapid volume imaging of Zebrafish at different phases (from day 0– 5) was performed and simultaneous visualization of Muscle and yolk-sac were carried out. Note that, yolk-sac mainly provides the food for the early development of embryos. It acts as the storage of protein and fat apart from Cyanocobalamin (present in yolk-sac), which is essential for fat metabolism and muscle development. Yolk-sac provides the major metabolic fuels for initial embryonic developmentTo understand the development of organs and determine the characteristics energy metabolism at different developmental stages of Zebrafish, we carried out detailed studies in each period. Figure 7 shows the rate of tissue (muscle and lipid) development across all periods. Interdependence Ratio . So the availability of fast computing engines and an order-less sectional 2D data may enable volume reconstruction in real-time53.In contrast, OVSS technique uses quantized rotation to collect angular 2D image data to reconstruct volume-stack. This eliminates the need for translation and consequently the data recording time is reduced substantially. Since the data obtained is sparse as compared to LSM (that employs both translation and rotation), the reconstruction quality may suffer . This makes the OVSS system faster than the existing LSM system clocking with a volume acquisition temporal rate of 0.4 Hz. By increasing the step-rotation speed one can expedite the process substantially. In comparison, IsoSPIM and diSPIM have reported comparable temporal resolution but they are complex, bulky and often require expertise to use the system to its full potential56. Moreover, the use of multiple CPUs and optionally GPU has accelerated the reconstruction process in less than a 15 min on a NVdia P6000 graphics board57. Specifically, Biobeam computational software based on GPU implementations has substantially reduced volume reconstruction time56. Current state-of-the-art light-sheet microscopes requires a lot of precious data-acquisition time that severely slows down the imaging process59. Another issue is related to chromatic abberation introduced by light of different wavelengths used for excitation. OVSS overcomes these limitation by employing overlapping dual-color light-sheet for illumination and using discrete-rotation based isotropic data acquisition. We anticipate that OVSS system integrated with GPUs can reconstruct volume image in a record time never realized before.The addition of GPUs and GPU clusters along with the proposed quantized rotation-based OVSS system may make the system even faster. It is reported that, GPUs can further accelerate volume imagingDrosophila. Both single and multi- color OVSS imaging were carried-out to determine the functioning and development of organs during different phases. Imaging of Drosophila larvae is demonstrated using multicolor OVSS microscopy . High resolution requires objectives with high numerical apertures (NA), but the associated large angular uncertainty results in a limited range of depths that can be put in sharp focus.The multicolor advantage coupled with parallel data acquisition is probably the best choice for simultaneous study of multiple organs during development. With the aid of multicolor OVSS imaging, we followed the growth of muscle and yolk-sac in Zebrafish beginning from embryo to adult stage. We found an interesting correlation and interdependence between the growth pattern of filamentous actin (reflecting in muscle growth) and lipid depletion (in the yolk-sac) as the Zebrafish transition from one to another period  with minimal photobleaching, an order-less memory requirement and simultaneous multi-organ volume interrogation. Such a system is helpful for long-time monitoring of rapidly occurring biological processes and interdependent organ study during early development.OVSS system has three major sub-systems: multicolor lightsheet illumination, automated sample chamber and simultaneous dual-color detection :Multicolor lightsheet illumination Simultaneous illumination of multiple organs  require separate light sources. Depending on the fluorophores  used in the present study, two different lasers  and 473 nm Laserglow, LRS-0473-TSM-00100-10, Avg power=124.1 mW) were used. The beams were directed by mirrors and combined using beam-combiner dichroic mirror (DM). Linear graded neutral density filters  were used to match the beam intensity at the specimen. The laser intensity used at the specimen was about 3.4 mW. The green light was made to pass through an unit-magnifier (that is placed on a linear-translator) in order to co-align the diffraction-limited lightsheets. The combined beam was expanded and passed through a cylindrical lens to form a lightsheet. A 10Motorized sample chamber The sample was embedded in a Agarose gel-matrix and placed inside a capillary tube. The tube is placed on a pre-fabricated hole on the rotational stage  which is motor-controlled  and made to pass through an Iris to cut off the spherical-aberrated rays. Notch filters [Green notch filter  and Blue notch filter ] were used to remove scattered and reflected excitation light. Subsequently, a combination of tube lens  was used for multicolor imaging. Zebrafish is remarkably similar to the human in its body plan, organ composition and physiology, and is easy to manipulate geneticallyFor multicolour staining of Zebrafish, two distinct region were selected which is biologically known to be active during early developmental. We chose BODIPY 493/503 (Invitrogen-D3922) a green fluorescent lipophilic dye which stains for neutral lipids and can be used as a tracer for oil and other nonpolar lipids. Yolk is the major region of Zebrafish which is stained by this dye and gives distinguishable pattern. BODIPY  . The other part is muscles which are stained with TRITC-phalloidin that binds actin of muscles in Zebrafish embryos.62. Embryos were stained with 1:300 BODIPY and incubated for 1–1/2 h. After this, the embryos were washed with PBS. Zebrafish embryos were transferred into the imaging chamber/capillary tube  of volume For multiple staining, first embryos were fixed in In OVSS imaging system, raw image data are recorded using a dual CCD camera based detection system  of size For accurate registration of multi-channel data correlation function along with fluorescence bleedthrough mechanism was exploited. Fluorescent microbeads , Indian Institute of Science. We declare that, all methods were carried out in accordance with relevant guidelines and regulations approved by Institutional Committee (IC), Indian Institute of Science.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Information.


Background: The World Health Organization has recommended that individual governments identify vaccine hesitancy areas. It is proposed that the governments, with the aid of local organizations, educate and implement social insights on the vaccination so that high population levels are covered with this safe immune program. Methods: A longitudinal online survey covered 3000 adults from India. We examined the demography, behavioral (socio-economic) attitude, vaccine hesitancy, vaccine resistance for the COVID-19 vaccine. The specific reasons for the COVID-19 vaccine decline were also evaluated. Results: Our survey revealed 59% definite response, 29% low-level response, and 7% high-level response for vaccine hesitancy, while 6% had resistant views on the COVID-19 vaccine. Individuals who had higher income levels, lived in a society, maintained social distancing, had downloaded the COVID-19 update app, showed a positive attitude towards their government, and more confidence in their healthcare system were more inclined towards the vaccination. On the contrary, individuals who had overstated the risks of COVID-19 and had a religious and populistic attitude towards vaccination were more hesitant/resistant to vaccination. The respondents who declined vaccine administration were further evaluated for their specific reason for their response. The most common reasons for declining the COVID-19 vaccine were post-vaccine scare of adverse health effects and accepting the information spread by social media. Conclusions: The results in our study show that by identifying population “hot spots” that have negative or unclear information on the COVID-19 vaccination, these “hot spots” can be addressed by involving friendly organizations that can clear their strong disbeliefs and increase the percentage of vaccine-definite people within the population. The role of government-induced COVID-19 vaccine policy measures can always be beneficial to cause this shift from disbelief to confidence within the population. Vaccines have been the foremost confronting factor against viral illness. However, historically vaccine hesitancy has been a challenging factor all over the world. Reasons for vaccine hesitancy worldwide have varied from religious aspects to its association with adverse health hazards . DenmarkIndia has a population of over 1390 million people. The government has presently recommended all its citizens within the inclusion criteria to be immunized by the two vaccines, namely the Covishield -approved Astra Zeneca vaccine) and Covaxin  ,8. The IThe WHO declared COVID-19 as a pandemic in March 2020 and, later during the year, stated that COVID-19 vaccine coverage is the only long-term solution for the fight against the COVID-19 pandemic . This saLiterature studies and the available research data suggest that vaccination intent varies significantly in each country . When anThe aim of this study is to present the formal analysis and results of a national survey that represents the importance of vaccination coverage within the country to fight COVID-19. We present the demography, behavioral (socio-economic) attitude, vaccine hesitancy, and vaccine resistance to the COVID-19 vaccine. We distinguish vaccine-hesitant people from people who are sure to get the vaccine and examine the vaccine-resistant population. Given the Indian government’s active role in providing vaccines at a subsidiary cost, we hypothesize the confidence in the government and healthcare sectors, their attitudes, and adherence towards the delivery of COVID-19 vaccines to the population will play a very vital role in a successful vaccination outcome ,17,18. WThis study used a Minimetric poll that collected data from 3000 respondents across four states in India. The mean age of the participants was 46.64 (Standard Deviation (SD) = 14.3) years. The data collection was performed predominantly online (94%), and a minor portion was obtained using the phone. The phone respondents completed the survey over a period, ranging from 17 respondents on the first day to 1222 respondents on the two other days (12 and 13 January 2021).The individual respondents were initially sent an email invitation comprising of the aim of the study and consent for their involvement in the study. Once accepted, the respondent was provided with a survey questionnaire.The Minimetric poll utilized participants from the India Life online web portal. This portal allowed the recruitment of respondents using email IDs . During The offline members of the India Life online portal were contacted using SMS and frequent call-cycle over a period of 14 days. A weekly SMS reminder was sent on Fridays. The recent accumulative response rate was 7.8%, a minor decline as compared to the previous response rate in January 2021. Base weight is computed for every respondent using two weight categories.The initial selection chances and follow-up post-stratification for their enrolment weight.The available information from respondents and non-respondents to the present wave for their propensity weight.The recent population benchmark was satisfied by the adjustment to the base weights. This was done for the demographic characteristics.The India Life online portal used the following methods to be uniform with the national population benchmark:The Minimetric poll device, its contents and the research methods were submitted for consideration by the ethical board. The board members sought further clarification on societal factors, respondents’ identifiers, and political variables. This was re-edited and submitted. Afterwards, the Human Research Ethics Committee (#22/2021) was approved at RRN hospital and research center, India, for this study. Informed consent was obtained either online or verbally.This study included questions regarding vaccine acceptance, hesitance, and resistance. The questions were provided in English; however, respondents who had language understanding difficulty were guided with a translator upon request to the research team. The questions had independent variables stratified into demographic, socio-economic, political, employment, COVID-19 anxiousness, and adherence to government advocated safety norms. The dependent variables were of their opinion on vaccine acceptance, hesitance, and resistance, as shown in the “Your view on COVID-19 vaccine”;“Are you willing to be vaccinated”?The study of vaccine intention was measured using the following questions: Definitely no (5.5%);Probably no (7.2%);Probably (28.7%); andDefinitely yes (58.5%).The response would be categorized into four weighted sections, namelyVaccine resistance is defined as those people who will definitely not be willing to get vaccinated . VaccineVaccine hesitancy (low level) is defined as people who are uncertain of their decision on being vaccinated .Based on the vaccine acceptance literature, the definitions for vaccine resistance and hesitancy are as follows:The variables included in this study are provided in Demographic variables used in our study are age, gender, birthplace, socio-economic status, education, employment, and income. Health-related variables were the presence of disability, chronic illness, and self-rated health status. The COVID-19-related variables were if they were tested for COVID-19, were worried about a family member contracting COVID-19, agreement to maintain social distancing, and downloaded COVID-19 safe app. Social and political-related questions were attitudes towards social trust, confidence, altruism in government and the health care system, religiosity, and voting intentions.The statistical analysis was performed using the ordinal probit model. This model was used from STATA 15.1 using the oprobit command. The survey included information from 3000 respondents. The number of the respondents varied on the rate of survey completion. The inclusion of numerous independent variables was analyzed using several models.Model 1—demographic variable; completed survey respondents from January 2021; respondents had absolute vaccination intention data.Model 2—demographic and health variables including disability measure from February 2021 Minimetric Poll.Model 3—demographic and COVID-19 variable from April and May 2021 Minimetric Poll.Model 4—demographic and socio-political variable from February and April 2021 Minimetric Poll.p < 0.05) variables from Models 2–4.Model 5—demographic and statistically significant  software.The respondents who were categorized as vaccine hesitant and vaccine resistant were further asked to fill up the reason for their decline response to the COVID-19 vaccine, as shown in One in two Indians (58.5%) were definitely getting vaccinated. The vaccine hesitancy was divided into low levels and high levels. Low-level vaccine hesitancy was respondents who were probably likely to get vaccinated (28.7%). High-level vaccine hesitancy was respondents who were probably unlikely to get vaccinated (7.2%). Vaccine-resistant individuals were respondents who were not taking the vaccine (5.5%).Statistical data from the various models were analyzed. Variables were marginalized from Model 1 and included demographic, socio-political, economic, and education variables, as presented in Society variation showed that respondents living in the fourth disadvantage quintile were more vaccine hesitant and resistant than those living in the third quintile. Respondents living in high-income households were more likely to be vaccinated. Health and disability status, the remaining demographic variables, were statistically insignificant.The respondent survey also revealed that individual and family concerns on contracting COVID-19 were unrelated to vaccine intentions. Respondents who were adherent to social distancing, followed COVID-19 norms, and had the COVID-19 update mobile app were more likely to get vaccinated (+10.8 percentage points). Respondents who felt that COVID-19 was an overtly unnecessary exertion by the public and government constituted a high proportion of the vaccine-resistant (8.1 percentage points) or vaccine-hesitant (4.2 percentage points) groups and were less likely to get vaccinated (−14.9 percentage points).The demographic and socio-political variable was applied in Model 4. There were no statistically significant results from this variable. However, the respondents who were appreciative of the government efforts to fight COVID-19 and those who trusted their local healthcare system intended or were more likely to get vaccinated . These respondents showed lower vaccine resistance (−3.4 percentage points) and vaccine hesitancy (−2.8 percentage points).p < 0.05) variables from Models 2–4. This model revealed that respondents over 55 years of age living in high-income households were more definite of being vaccinated. Females and respondents living in a low socio-economic neighborhood were less likely to get vaccinated. Respondents with a higher sense of social distancing who had downloaded the COVID-19 update app were more intent on vaccination. The respondents who considered COVID-19 an exaggerated disease were less definite for vaccination. Those who appreciated their government and healthcare system were less likely to be vaccine hesitant or resistant. In Model 5, we included demographic and statistically significant  respondents was 1077, and the total number of vaccine-resistant (N) respondents was 162. However, approximately 61.2% of these respondents agreed to consider/change their opinion after a 6-month observation period. This mainly depended on the availability of reasonable information on vaccine safety and vaccine-associated adverse effects.The vaccine-resistant respondents rendered firm disbelief in vaccine efficacy and cited multiple reasons for declining. The vaccine-hesitant respondents were more ready to seek more information about vaccine efficacy and safety from reasonable sources, showed belief in pharmaceutical vaccine research, and were waiting for the government and healthcare facility to upgrade their public service towards COVID-19. Fifty-five percent of vaccine-hesitant respondents were interested to be part of survey research studies in the future, but a striking 98% of vaccine-resistant respondents were unconcerned about being part of any future survey studies involving COVID-19 vaccines. Rumors spread through social media occupied 87.6% of vaccine-resistant respondents’ reasons for COVID-19 vaccine denial. The percentage is astonishingly high compared to 4.6% among vaccine-hesitant respondents.SARS-CoV-2 induces pneumonia by viral replication and virus-mediated direct tissue damage. The infected host experiences a modified immunological response with cytokine recruitment, causing a classical “cytokine storm” within the host. The ability of SARS-CoV-2 to induce COVID-19 within the host and the rapid spread of the infection across the world vindicated the WHO to announce COVID-19 as a pandemic . With thBased on our survey analysis and results, in June 2021, 36% of Indians are COVID-19 vaccine-hesitant and 6% COVID-19 vaccine-resistant. The variables and factors related to vaccine hesitancy and resistance were disparate. We analyzed the individual variables, namely: age group, gender, social attitude, political views, like/dislike of available government strategies and healthcare facilities to fight COVID-19, education, and income within the household.These individual variables showed an essential association with vaccine hesitancy and resistance. As discussed in previous relevant articles, respondents with low socio-economic status, unemployment, and a lack of education constituted the primary reason for vaccine hesitancy and resistance ,17. In oPrevious studies have shown the importance of societal disagreements and anti-vaccine groups mainly associated with vaccine hesitancy or resistance ,23,24. TStudies on vaccine hesitancy or resistance always target the probable respondents who can be converted into an agreeing group by adequately providing them with positive information on the vaccination. The information should mainly include the efficacy and safety of the available vaccine . The govStrong anti-vaccine beliefs are mainly held by vaccine-resistant individuals. They also have lower compliance levels in yielding to government-provided health information about COVID-19 preventive measures . This group of vaccine-resistant or hesitant individuals should be streamlined with serious government-induced COVID-19 regulations and monitored vigilantly as they form the main sector for COVID-19 vaccine denial in the healthcare system ,28.Our study further extended identifying reasons for vaccine hesitancy and resistance among the respondents. As indicated in the results section of our study, the most common rationale for vaccine denial was panic-stricken post-vaccination adverse effects, the presence of co-morbidity, and a lack of clarity in the provided vaccine information . These sIn India, as the COVID-19 pandemic raged during April–May 2021, the healthcare facility was in severe crisis, and this was further exaggerated by the spread of false information and statistical reports by social media. This provoked the Indian government to request the social media companies to seriously regulate their displayed content and check for the spread of such misinformation against COVID-19 and its vaccine. The government also inflated its internet regulation law and enforced arrest and severe punishment to the law defaulters. Few social media platforms were banned in certain states in India ,34.Our study shows that quorum of vaccine hesitancy depends on the individual’s complacency towards vaccination (its effectiveness), a lack of confidence , and a lack of convenience . The individual’s decision on receiving the vaccination entails the composition of cultural, spiritual, political, and socio-economic factors. The vaccine information provided by the internet and social media messages have been a significant concern for inaccurate and anti-vaccine knowledge circulation. The spread of misinformation can be avoided by contacting a reliable physician within the community or family to clarify such negative thoughts or queries on vaccination content.However, identifying this group throughout the country would only be possible if large-scale studies are held with respondent identifiers, causing a more sophisticated scheme of the research study . Such stVaccine hesitancy and resistance have shown to be consistently high across the globe during the COVID-19 pandemic. The factors are categorized into demographic, socio-economic, attainment of educational qualification, and then the actual rational reasons for vaccine denial. As herd immunity mainly depends on the effectiveness of the administered vaccine and the percentage of the population covered by the vaccine, COVID-19 vaccine coverage in India remains dubious, as, with our study, only 59% of respondents were likely to get the vaccination. As these data are limited to the survey population in four states in India, the results extracted from this study may be limited in application to the entire country due to the differences in the population demography, socio-political, economic, and education variability among the other states in India. However, in comparison to similar studies in the literature, the inferences in our study may potentially apply to other areas of the world as well. Our study also suggests that vaccine-hesitant respondents should be addressed rigorously with positive information about the COVID-19 vaccination and public health information to convert them into the vaccine-definite sector. Propaganda and strategies should be in place by the government and healthcare sector in identifying vaccine-resistant individuals to avoid the spread of misinformation among the public.


The epicatechin-3′-glucuronide (E3′G) and epicatechin-3′-sulfate (E3′S) were the most abundant metabolites in all subjects. A high intra-individual correlation  between SREMs concentrations after single-dose and sustained supplementation was observed. The antioxidant capacity of plasma did not change in response to the intervention and was not correlated with any of the SREMs. Conclusion: The individual SREMs profile and concentrations after a 28-day supplementation are comparable to those after a single dose.Background: While the bioavailability of cocoa polyphenols, particularly of the monomer (-)-epicatechin, has been investigated after a single-dose intake, the effect of sustained cocoa consumption on the metabolic profile of the structurally related (-)-epicatechin metabolites (SREMs) has not been investigated. Methods: A randomized, controlled crossover clinical trial in healthy young adults (18–40 year) was conducted to evaluate SREMs after consumption of a single-dose and after daily consumption of 1.3 g of polyphenol-rich cocoa powder for 28 days. The circulating SREMs were measured by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results: Twenty subjects  were enrolled. The SREMs concentrations increased to 1741 ± 337 nM after a single-dose and to 1445 ± 270 nM after sustained supplementation. Sulfate conjugates showed higher levels in females ( Cocoa-derived products are widely consumed and appreciated thanks to their sensorial properties and high content of bioactive polyphenols ,2. The bApproximately 20% of (-)-epicatechin is absorbed in the small intestine by passive transport and is metabolized by phase II enzymes in enterocytes and hepatocytes . StructuAccording to previous studies ,16,17, tThe presence of these metabolites has been correlated with changes in the expression of human microRNA in in vitro studies , and the2, non-smokers, and classified as sedentary to moderately active according to the International Physical Activity Questionnaire (IPAQ) (-)-epicatechin suggested that SREMs are detected in circulation 15 min after the consumption of cocoa polyphenols and reaches a maxC at 1–2 h and that their concentration falls below the detection limit at 8 h post-consumption [The half-life of circulating SREMs is relatively short. This finding has been consistently reported ,27,28. Tsumption . In the sumption ,15,16,17i.The production of phase II metabolites of (-)-epicatechin in the liver and small intestine remained relatively stable after repeated doses with no significant differences in the production of the biotransformed metabolites.ii.The continuous excretion of SREMs does not allow for accumulation in the body, albeit biological effects have been observed after chronic interventions ,29,30,31iii.Long-term interventions using the same vehicles and doses may predispose to a more homogeneous response by reducing the initial variability across individuals, which is influenced by former dietary habits . Future The SREM concentrations after sustained supplementation were similar to those observed after a single-dose intake. Slight reductions in inter-individual variability and mean abundance of SREMs were observed after 28 day of supplementation (1445 ± 270 nM), although the differences were not statistically significant. The results from daily supplementation suggest the following:The identification of new SREMs has evolved in parallel with the development of more sophisticated methods for the identification and quantification of SREMs . OttaviaThe bioavailability and pharmacokinetics of (-)-epicatechin are influenced by the study design  ,32. PrevmaxC is observed varies according to subject characteristics and the food matrix. Maximal concentrations have been identified 1 h post-consumption of cocoa drinks [The complexity of the food matrix used -epicatechin, encapsulated cocoa powder, water-based and milk-based beverages, and chocolate bar) may elicit a differential gastrointestinal response depending on the nutrient content ,34. The a drinks  or at 3 a drinks . This sup < 0.05) and 3′ME4S  were found in females than in males, although non-significant differences in the total concentrations were observed between sex groups. A higher abundance of glucuronides has been previously reported in females after supplementation with resveratrol, possibly associated with sex-specific UDP-glucuronosyltransferase isoenzyme expression profiles regulated by sex hormones [High inter-individual variability was observed in the SREM profiles. Variability has been previously reported and linked to genetic and non-genetic factors such as age, body composition, sex, gut microbiota, and lifestyle, which could influence the enzymatic activity of the host ,32,35. Phormones . Future hormones .Low intra-individual variation in the abundance and SREMs profile was observed after exposure to a single-dose and 28-day supplementation with high polyphenol-content cocoa powder, suggesting that data from single-dose interventions may be reflective of the abundance and metabolic profile of SREMs in the longer-term. Similar results have been observed on circulating SREMs and urinary E3G, E3S, and 3′ME5S after the consumption of two single-doses of cocoa flavanols (CV = 12–13% and Pearson’s correlation coefficient: 0.83)  and afteIn the present study, the consumption of a high-polyphenols cocoa powder did not influence the antioxidant capacity of plasma. The antioxidant activity of cocoa and other flavonoid-rich products has been controversial since different results have been reported across studies . HoweverThe strengths of the present study are the direct measurement of circulating flavan-3-ols metabolites in plasma, the comparison between the effects of a single-dose and sustained intake for 28 day, and the inclusion of male and female participants. It is important to mention that the study design allowed for a comparison of the effects within each participant, at least for the single-dose effect. Among the limitations of the study is the lack of analysis of a larger number of metabolites, the use of a different compound as the control  instead of a placebo, as well as the absence of sample collection at more time points after intake to analyze the absorption and metabolism of polyphenols during a longer period.In conclusion, the abundance and profiles of SREMs after sustained supplementation with high-polyphenols cocoa powder for 28 days are comparable with those obtained after a single dose. The abundance of neither single SREMs nor the sum of them changed the antioxidant capacity of plasma, although their presence may influence the redox response by modulation of the intracellular signaling pathways. While high inter-individual variability was observed at the beginning of the study, it decreased after sustained supplementation, suggesting that individuals exposed to the same long-term treatment may elicit a similar response. However, the factors that will determine the subject’s response to a greater extent will depend on the individual characteristics. Longer interventions that evaluate not only SREMs but also 5C-RFM are recommended in future studies. These findings will contribute to elucidating the effects of chronic exposure to cocoa polyphenols and their impact on human health.


It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac O-GlcNAc modification of serine and threonine residues of intracellular proteins is ubiquitous in eukaryotic cells . The proMetabolic chemical reporters (MCRs) of sugar metabolites with bio-orthogonal functional groups have become a strategy widely used to visualize and identify glycoproteins. This strategy involves synthesizing non-natural monosaccharide analogues with bio-orthogonal functional groups, such as alkyne or azide analogues . These svia the Leloir pathway. Moreover, defects in the breakdown of Gal-related glycans result in several lysosomal storage diseases. Metabolic oligosaccharide engineering provides a powerful tool to investigate the roles of various monosaccharides through the copper-catalyzed azide-alkyne cycloaddition (CuAAC) in combination with appropriate tags. However, few documents disclose the roles of Gal including its analogues in the glycosylation changes in development and disease progression due to the strict substrate specificity. Recently, Baskin’s group developed 6-alkynyl UDP-Gal for imaging N-glycans in developing zebrafish in vivo  and analyzed by Western blot using streptavidin-HRP for biotin labeling. The results of Western blot indicate that dose dependence of labeling is observed with a similar pattern and remarkable labeling at the concentration of 500 μM  for different lengths of time; then, performed cells were lysed and reacted with the Biotin-PEG4-Alkyne tag, and Western blot analysis was performed  for 16 h. The cells were then lysed and reacted with CuAAC. Western blot analysis results show that Ac46AzGal enables efficient labels in different cell lines with a diversity pattern and intensity of modified proteins  in the hexosamine biosynthetic pathway (HBP) and OGA  in the presence of Ac46AzGal for 16 h and then treated CuAAc with azide biotin-alkyne, and Western blot analysis was performed  vehicle for 12 h. The corresponding cell lysates were then incubated with H2O or PNGase-F at 37°C for 6 h, followed by CuAAC with Biotin-PEG4-Alkyne and Western blot analysis. The results demonstrate that no significant loss in the signal is observed, suggesting that Ac46AzGal is not incorporated into N-glycans  to provide further insight into the tolerance of biosynthetic enzymes and glycosyltransferases to Ac46AzGal. We demonstrate that Ac46AzGal is able to label intracellular proteins by treatment with a variety of mammalian cells. Notably, the pattern of this labeling is conducted by OGT but is not related to OGA, which further reveals the substrate promiscuity of OGT. However, substrate specificity of OGT is detected for UDP-6AzGal, and the results demonstrate that UDP-6AzGal cannot be recognized by OGT. Therefore, we infer that the protein labeling of 6AzGal intracellular is incorporated in the form of UDP-6AzGlc instead of UDP-6AzGal, and the two donors are interchangeable via the enzyme in the Leloir pathway. Moreover, the enzymes of galactose-1-phosphate uridylyltransferase (GALT) and UDP-galactose 4′-epimerase (GALE) are also confirmed to regulate the efficiency labeling of Ac46AzGal, suggesting that the enzymes in the Leloir pathway, at least partially, participate in the metabolic biosynthesis of Ac46AzGal. These results support the conclusion that galactose analogues can modify endocellular proteins, and is significant on intracellular protein labeling under certain conditions that involve deficiency of the galactose metabolism.In conclusion, galactose is a naturally occurring monosaccharide used to build complex N-/O-glycans on the surface of vertebrates and is metabolized predominantly


The ongoing SARS-CoV-2 pandemic has tested the capabilities of public health and scientific community. Since the dawn of the twenty-first century, viruses have caused several outbreaks, with coronaviruses being responsible for 2: SARS-CoV in 2007 and MERS-CoV in 2013. As the border between wildlife and the urban population continue to shrink, it is highly likely that zoonotic viruses may emerge more frequently. Furthermore, it has been shown repeatedly that these viruses are able to efficiently evade the innate immune system through various strategies. The strong and abundant antiviral innate immunity evasion strategies shown by SARS-CoV-2 has laid out shortcomings in our approach to quickly identify and modulate these mechanisms. It is thus imperative that there be a systematic framework for the study of the immune evasion strategies of these viruses, to guide development of therapeutics and curtail transmission. In this review, we first provide a brief overview of general viral evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion, and pinpoint the shortcomings in the current paradigm with its focus on overexpression and protein-protein interactions. Finally, we provide a recommendation for future work to unravel viral innate immune evasion strategies and suitable methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases. Seasonal strains of Influenza and periodic epi/pandemics, such as the latest SARS-CoV-2 pandemic are a grave concern for public health. With our developments encroaching into natural habitats, it is unavoidable that the barrier between reservoir hosts for viruses and the human population is slowly eroded, increasing risk of emerging zoonotic viruses . This isvia various pattern recognition receptors (PRRs). For example, viral RNA is recognized by Toll-like receptors  and RIG-I-like receptors (RLRs), while foreign DNA is recognized by cytosolic DNA sensors . The activation of innate immune sensors triggers a signaling cascade ultimately leading to the induction of type I and/or type III IFNs. The type I IFN (consists mainly of IFNα/β) binds to IFNα receptors 1 and 2 (IFNAR1/2) and type III IFN  binds to IFNλR/IL-10Rβ, activating expression of hundreds of ISGs to inhibit the viral replication cycle. Many studies have also shown the potency of IFN pretreatment prior to viral infection in inhibiting viral replication  efficiently inhibits viral replication through production of type I/III interferons (IFNs) and their respective interferon stimulated genes (ISGs). The unique molecular patterns of viruses are detected in host cells lication . Howeverlication . The dellication . This hade novo identification  and virus-host interaction studies .With the ever-looming threat of the next pandemic, understanding how viruses evade the immune response may allow us to devise novel antiviral treatments and strategies, or repurpose existing drugs as a rapid countermeasure to emerging threats; especially with a monumental case study such as the SARS-CoV-2. Furthermore, identifying the viral genes required for host immune suppression may aid in the development of a recombinant virus with key deletions as a vaccine candidate. Recent developments in methodologies have allowed for higher throughput analyses to be performed, providing new tools for In this review, we first provide a brief overview of general evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion. Finally, we provide a recommendation for future work to unravel the innate immune evasion strategies by viruses, and additional methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases.in bold.Viral evasion of the innate immune system can be broadly summarized into a few categories: (a) interference of host pathogen sensors and antiviral factors, usually affecting the type I (IFNα/β) and/or type III (IFNλ) interferon signaling pathways, (b) modification of 5′-end of viral mRNA, and (c) formation of replication organelles. Some examples of common viruses are given, while SARS-CoV-2 will be covered in the next section. Methods used for identification of viral-host interactions in each study will be included in parenthesis The viral genome consists of structural, non-structural, and accessory proteins. These proteins, and even viral RNA, can perturb the innate immune response in the host cell. Below are examples of each class of molecules interfering with the function of the innate immune system, leading to viral evasion.ε complex, thereby interfering with the activation of the type I IFNs (Co-Immunoprecipitation). Influenza M1 protein has been found to interact with C1qA of the complement system, inhibiting its interaction with IgG, thereby protecting the Influenza virus from complement mediated neutralization in vitro .SARS-CoV M protein has been found to interfere with formation of the TRAF3.TANK.TBK1/IKKe I IFNs  .Influenza NS1, perhaps a prime example of innate immune evasion , perturbImmunoprecipitation-Mass Spectrometry [MS]). Influenza A PB1-F2 has been shown to inhibit interferon induction downstream of the pattern recognition receptor pathway, RIG-I-TRIM25, by interacting with MAVS .MERS-CoV ORF8b interferes with the interaction of IKKϵ and HSP70-1A by competing with IKKϵ for HSP70-1A, preventing signaling through the IRF3 pathway from proceeding, thereby inhibiting the production of IFNβ  (Immunopith MAVS  (FunctioRNA-Immunoprecipitation).DENV (Dengue virus) subgenomic flavivirus RNA is known to interfere with the innate immune system through its direct interactions with TRIM25 and interfering with its deubiquitylation, thus interfering with the RIG-I signaling and consequently, type I IFN production  .Influenza has been known to incorporate CD59 into the membrane of the virion, allowing it to inhibit the formation of the membrane attack complex by the complement system   .Influenza viruses have been known to engage in a process known as “cap snatching.” Viral RNA bound RNA-dependent RNA polymerase (RdRp) associates with the C terminal domain of RNA polymerase II, allowing interaction of polymerase basic protein 2 (of RdRp) with the 5′ cap of host RNA. Host RNA is then cleaved 10–13 nucleotides down, producing a capped oligonucleotide that is used to prime viral RNA transcription  (FunctioScreens) . The polScreens) . NSP16 oScreens)   and invaginations, are appropriated intracellular membranes that may, speculatively, shield viral PAMPs from the innate immune sensors present within the cytoplasm . These sImmunofluorescence Assay, RT-qPCR). It has, however, been shown that at least in coxsackievirus (an enterovirus), replication organelles do not seem to result in enhanced innate immune evasion . With diverse observations, the impact of replication organelles on innate immune evasion may be unique to each viral infection and mechanisms behind this phenomenon are still relatively unclear, requiring further research for each virus of interest.DENV, for example, conceals its dsRNA within DMVs, reportedly diminishing the impact of the host innate immunity (as determined by IFNβ expression), as compared to Japanese encephalitis virus, whose dsRNA is detectable in the cytoplasm much earlier than DENV  , spike (S), envelope (E), membrane (M), and nucleocapsid (N)-3′ genes. The ORF1ab polyprotein is comprised of 16 NSPs , but ORF10 was not detected . AnotherAs with all newly emerging pathogen, our understanding of how SARS-CoV-2 evades the immune system, and the functional roles of its genes are limited. Gene overexpression studies are commonly performed to further characterize genes of unknown functions . In suchSeveral studies have utilized IFNβ-promoter firefly luciferase reporter assay as a readout of IFN suppression to screen for IFN antagonists. A study by Taken together, the overexpression of at least NSP1, NSP3, NSP11, NSP12, NSP13, NSP14, NSP15, ORF3, ORF6, ORF8, ORF9b, ORF10, N, and M have been implicated to antagonize IFN induction, though these results were not consistent across studies. In a study by It is expected that more proteins could play a role in viral innate immune evasion owing to the large genome size of SARS-CoV-2 and the presence of numerous non-canonical transcripts with unknown function. Overexpression, while with its caveats, is a relatively simple and quick approach as a first line of screening to search for viral antagonists for antiviral factors.via promoting K48 ubiquitination , protein-protein interaction (PPI) studies are necessary to determine the host factor involved and the pathway affected. Comprehensive PPI maps using AP-MS have been performed by tination . The SARtination . An impoAs an RNA virus with a relatively large genome (∼30 kb) and multiple subgenomic RNAs, it is expected that various RNA-RNA interactions may exist between the virus and host. Indeed, a comprehensive mapping of RNA-RNA interactions in infected Vero E6 cells revealed ∼300 host RNAs that interact with the SARS-CoV-2 genomes, with mitochondrial RNAs and snoRNAs being strong interactors . The autCurrent methods of investigating host-viral interactions rely greatly on overexpression, such as luciferase reporter screens or single protein overexpression followed by pull-down to identify PPIs. As discussed in the previous section, the overexpression of viral proteins may lead to false positive findings due to the artificially elevated levels of viral proteins not found in normal infection. Viruses, utilizing their repertoire of DNA/RNA and proteins from their small genome, adapted multiple means of subverting the innate immune response to ensure their replication at the detriment of the host. The identification of these mechanisms is important to devise strategies to dampen the virulence of a virus and prevent severe infection and loss of life during an outbreak. Thus, future studies should move toward the use of whole virus and biologically relevant cell systems to elucidate the multipronged strategies employed by viruses to evade the innate immune system.Reverse genetics (RG) is a powerful method which allows the study of viral proteins in the whole virus context which more accurately reflects the virus-host interactions during normal infection. The method utilizes an infectious clone generated from full-length cDNA of the virus and allows mutagenesis to be performed to investigate the functional roles of certain genes of interest. Many groups have developed RG systems for SARS-CoV-2, with some safe for use in Biosafety Level 2 laboratories, opening SARS-CoV-2 related studies to more researchers . Howeverex vivo) grown in 3D air-liquid interface cultures have been shown to exhibit the anatomical and physiological processes found in vivo  it originates from monkeys, and (ii) it is known to lack type I IFN genes , which makes it difficult to resolve the complexity of host innate immune responses to SARS-CoV-2 in humans . Other c in vivo  and coul tissues . As prim tissues . Recent  tissues . Further tissues . This ca tissues .In terms of human viral infection, protein-protein interaction between viral and host proteins remains the mainstay of where we identify viral innate immune evasion mechanisms. To study the large permutation of interaction between viral and host proteins, high-throughput methods followed by targeted validation are the main methods widely used to elucidate such interactions.In silico methods are mostly based on the mRNA sequence, structural similarity, and phylogeny and can be used to predict interaction between viral proteins and host proteins These methods can be used to guide verification with in vivo and in vitro methods. In vitro methods can be further categorized into targeted and non-targeted methods. In non-targeted methods, a general screen is done to identify all possible interaction pairs in a set of proteins, which in the case of viral-host interaction studies can be performed on the cell lysate of infected cells. In targeted methods, a protein of interest (bait) is expressed and used as the focal point to identify all PPIs with that protein. In vivo methods mainly make use of two-hybrid systems in simple model organisms. Two-hybrid systems are usually based in yeast, where interaction between proteins (one bound to a DNA-binding domain and the other to an activation domain) recombinantly expressed in the yeast host is required for reporter gene expression , tandem AP, and proximity labeling. Co-immunoprecipitation utilizes immobilized antibodies on beads/surfaces against a protein of interest (bait). The infected cell lysate/protein cocktail is then allowed to interact with the beads/surface, resulting in the binding of the bait to the immobilized antibody. With a robust interaction between the bait and any prey (proteins that are able to interact with the bait), the bait-prey complex will remain associated with the immobilized antibody when the non-specific proteins are washed off, leaving the bait-prey complex bound to the beads . FurtherProximity dependent labeling is based on labeling proteins in close proximity to the protein of interest. This method is useful for determining transient interactions in the protein interactome, as the extensive washing steps required to eliminate non-specific proteins for AP and co-immunoprecipitation may result in the destabilization of such transient interactions. BioID, the first proximity labeling strategy, is based on BirA, a biotin ligase that has been adapted to biotinylate proteins in close proximity (∼10 nm) upon exposure to biotin and ATP . The bioNon-targeted methods include crosslinking investigations, which are based on the premise of forming covalent bonds between amino acid residues that are in proximity. The parameters of the crosslink  depend heavily on the crosslinker used, with each having its own set of advantages and disadvantages. Crosslinking is followed by purification of crosslinked proteins and finally identification by MS .While viral-host RNA-RNA interaction is uncommon in mammalian viruses, the advent of RNA-RNA interaction platforms can serve to uncover novel interaction at the RNA levels between virus and host. For example, studies of small RNA plant viruses have shed light on RNA silencing . Much woin vivo) (in situ conformation sequencing)  , PARIS , 2018, Sin vivo)  and RIC-uencing)  are someThese methods follow a general framework. Psolaren or one of its derivatives, AMT, is used to crosslink interacting RNAs. In context, infected cell lysate/an infected cell can be used to pinpoint viral-host RNA interactions. This is followed by a purification step, such as gel purification, to isolate the crosslinked interacting RNA molecules. Each individual interacting RNA duplex is then subjected to proximity ligation to ensure that their interaction is preserved. Interacting complexes are then isolated before the crosslink is reversed, releasing both RNAs (though they remain associated due to proximity ligation). This preserved interaction allows sequencing methods, such as PORE-cupine  , to pinpThese methods are applicable to both Viral (Protein)—Host (RNA) and Viral (RNA)—Host (Protein) interactions. The methods may be divided into RNA-targeted (using RNA as bait) and protein-targeted (using protein as bait).For RNA-targeted methods, they share a commonality in that a tag is expressed on in vitro transcribed RNA, or a probe is bound to the RNA of interest. This tag, whether biotin  or an apThese methods can be further differentiated by the need for a crosslinker. Crosslinkers commonly used in RNA-protein interaction studies are UV light  and formin vivo biotin-tagged RAP) (A range of methods are available for exploitation: RAP (RNA antisense purification) , PAIR p, MS2-Bioged RAP) , TRIP (tged RAP) , ChIRP (ged RAP) , CHART (ged RAP)  and VIR-ged RAP) .One method that does not require crosslinking makes use of proximity labeling instead. RaPID (RNA-protein interaction detection)  utilizesFor protein-targeted methods, CLIP (crosslinking and immunoprecipitation) can be utilized . Interacvia high-throughput sequencing  can be further investigated for impact on the innate immune response by perturbing and/or enhancing them. Pathogen sensors, IFN and ISG expressions are some of the metrics that can be used to approximate the impact of the interactions.In conclusion, the emergence of novel viruses like SARS-CoV-2 is an inevitability with the constantly expanding global population. The COVID-19 pandemic has demonstrated the lethality of such emerging pathogens which we have limited information about and the impact that they can have on our globalized society. Identifying viral evasion strategies is, thus, an important component of viral research to guide the development of therapeutics and halt transmission within the population, for both emerging and seasonal viruses. There are overlapping and common viral evasion strategies which provide a starting point to uncover these mechanisms and identify viral/host factors involved. From the studies reviewed in this paper, we see that the focus of the viral-host interactions studied are ones that have some form of homology to related viruses with known effect on the innate immunity. Furthermore, the focus seems fixated on overexpression studies (which is not representative of whole virus infection) and protein-protein interactions, with a lack of studies focusing on the RNA-RNA and RNA-protein interactions that emerging viruses may have. Here, by consolidating the current studies on SARS-CoV-2, a virus with strong antiviral evasion capabilities, we have identified areas that require integration of new approaches  to identify biologically relevant viral factors that can be targeted for management of viral infections (summarized in DT, ZL, and KT contributed to the initial conceptualization of the manuscript and writing of the manuscript. DT and ZL contributed to literature search. DT, ZL, KT, and JC contributed to literature selection, review, and finalization of the manuscript. All authors contributed to the article and approved the submitted 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Coronavirus disease 2019 (COVID-19) is recognized as a multisystem disease affecting the whole body, with new complications from the disease being described on an almost-daily basis.Case Report: We report the case of a 50-year-old female with a medical history of diabetes and silicone breast implants who developed right-sided, multicentric breast masses after a prolonged hospitalization for COVID-19 infection complicated by renal failure requiring dialysis. The patient noted an onset of breast pain and masses, and subsequent imaging demonstrated multiple similar oval masses. She underwent biopsy and operative debridement of the lesions and recovered appropriately. Results were consistent with sterile abscesses that were considered secondary to a vasculitis-like process associated with COVID-19 infection.Conclusion: To our knowledge, this case is the first account of breast pathology associated with a diagnosis of COVID-19 in the medical literature and encourages systematic evaluations of patients with coronavirus infections, including breast examinations. We present a case of multicentric breast abscesses complicating COVID-19 infection.Coronavirus disease 2019 (COVID-19) is associated with numerous systemic manifestations, including various vasculitis and vasculitis-like processes, coagulopathies, and other conditions.A 50-year-old female presented to the clinic in August 2020 with a complaint of multiple palpable nodules of the right breast. The patient had a complex medical history, including hypertension, hyperlipidemia, diabetes, and tetralogy of Fallot with a bioprosthetic valve. She had had bilateral subpectoral silicone breast implants in 2013, with no postoperative complications. The patient had no personal history or family history of breast pathology.In spring 2020, she experienced a 68-day hospitalization for respiratory failure secondary to COVID-19 infection. During this initial COVID-19 hospitalization, the patient required paralytics for ventilation, continuous renal replacement therapy for acute kidney failure, broad spectrum antibiotics for staphylococcal bacteremia and ventilator-associated pneumonia, and treatment of an upper gastrointestinal bleed. The patient was never proned. She was transferred to a long-term acute rehabilitation facility and eventually was discharged to home in good condition in late May 2020.When the patient presented in August 2020, the nodules were tender to palpation without drainage, warmth, or erythema. Right breast mammography  and ultrThe patient desired excision of some of the most symptomatic lesions. She was taken for surgical excisional biopsy, and a cluster of nodules on the right lateral breast was removed. Grossly, the nodules had the appearance of caseating granulomas with liquid cores. The creamy exudate was not malodorous. Cultures were sterile for bacterial, viral, and fungal sources. Pathology  showed mThe patient returned to clinic 1 month postoperatively, pleased with her results and with well-healing scars without any additional concerns for reformation of the abscesses.2 Vessel thrombosis can result from endothelial cell damage with subsequent dysfunction and oftentimes apoptosis. COVID-19 can be associated with a hypercoagulable process reflected by elevated levels of factor VIII, von Willebrand factor, and fibrinogen 1. A number of published cases report COVID-19–related thrombotic events involving the central nervous system, renal system, pulmonary system, aorta and major arterial vascular system, and skin lesions in the lower and upper extremities.16 The majority of reports on this subject involve youth and adolescents. A concurrent but separate process can also occur in patients with COVID-19 related to inflammatory lymphocytic vasculitis or other vasculitis-like process.16 Suggested mechanisms include increased complement activation or disseminated intravascular coagulation.16 Iba et al discuss a 30% increase of deep venous thrombotic events that are secondary to these vasculitis processes in patients with COVID-19.1 These processes can result in necrotic lesions that Galván Casas et al observed in approximately 6% of patients with skin lesions.15 Galván Casas et al also reported that the incidence of necrotic changes corresponded to increasing age and severity of COVID-19 and that necrosis may be associated with an increased mortality risk by up to 10%.15Published evidence in the COVID-19 era demonstrates both microvascular and macrovascular thrombosis involving vessels in multiple organs.17 No available research provides a definitive explanation regarding the abscess formation. While our patient's course would not likely have been altered by the knowledge that the abscesses were not infected, clinicians should consider sterile abscesses as a possibility in patients presenting with breast masses following a recent COVID-19 infection. An awareness of previous abscesses following COVID-19 may allow surgeons to confidently delay surgery and its associated risks and instead monitor breast masses in patients who have no other indications of malignancy on examination or imaging.In this case report, we describe one of the first examples, to our knowledge, of a breast pathology likely secondary to COVID-19. We suggest that the observed abscess formation was associated with a similar thrombotic process as described in the literature, likely a vasculitis-like process. The areas of necrosis are consistent with reported findings in other systems, particularly in cutaneous skin reports and cardiac cells undergoing necrosis.Research is needed to elucidate the exact mechanism in the development of these sterile breast abscesses. Conclusions are limited by the fact that ours is the only case reported in the literature. In addition, follow-up with this patient has been short, with only one visit one month postoperatively. Nevertheless, recognizing the possibility of breast involvement in patients after prolonged hospitalization for COVID-19 is important.In this case, we describe one of the few known breast complications related to COVID-19 infection, specifically, the development of sterile abscesses. We report this case to highlight the associated imaging and pathologic findings. Because of the uncertainty of the etiology of these lesions and concurrent symptoms, the patient desired elective surgical excision for both diagnostic and therapeutic reasons. However, avoiding such interventions in patients with a similar imaging or biopsy pattern may be possible.