It has been reported that patients diagnosed with COVID-19 become critically ill primarily around the time of activation of the adaptive immune response. However the role of antibodies in the worsening of disease is not obvious. Higher titers of anti-spike immunoglobulin IgG1 associated with low fucosylation of the antibody Fc tail have been associated to excessive inflammatory response. In contrast it has been also reported that NP-, S-, RBD- specific IgA, IgG, and IgM are not associated with SARS-CoV-2 viral load, indicating that there is no obvious correlation between antibody response and viral antigen detection. In the present work the micro-Fourier-transform infrared reflectance spectroscopy (micro-FTIR) was employed to investigate blood serum samples of healthy and COVID-19-ill (mild or oligosymptomatic) individuals . The molecular-level-sensitive, multiplexing quantitative and qualitative FTIR data probed on 1 µL of dried biofluid was compared to signal-to-cutoff index of chemiluminescent immunoassays CLIA and ELISA (IgG antibodies against SARS-CoV-2). Our main result indicated that 1702–1785  It has been recommended that hyperinflammation should be recognized and treated using already approved therapies with proven safety profiles to reduce the increasing mortality1.Accumulating evidence suggests that a subset of patients with severe COVID-19 may have cytokine storm syndrome4. Hoepel et al.2 reported that antibodies play a role in disease exacerbation at the time of seroconversion. They found that the excessive inflammatory response depends on two antibody features which are specific to patients with severe COVID -19: (1) higher titers of anti-spike IgG and (2) the anti-spike IgG of patients with severe COVID -19 is inherently more pro-inflammatory due to different glycosylation, particularly low fucosylation, of the Fc tail of the antibody. Analogously Chakraborty et al.3 suggested that patients with severe COVID-19 have a unique serological signature, including an increased likelihood of IgG1 with afucosylated Fc glycans. On the other hand, Luo et al.4 showed that NP -, S-, RBD- specific IgA, IgG, and IgM were not associated with SARS-CoV-2 viral load, suggesting that there is no obvious correlation between antibody response and viral antigen detected in nasopharyngeal swabs. They analyzed antibody and cytokine responses in COVID-19 from asymptomatic to severe patients (123 serum samples from 63 COVID -19 patients) and evaluated the impact of various risk factors, including comorbidities, male sex, and advancing age on the host immune response COVID-19 patients. These antagonistic data highlight the relevance of investigating structural aspects of serum IgG from COVID-19 patients to establish its rule as a severity marker.It has been noticed that patients diagnosed COVID-19 become severely ill, especially at the time of activation of the adaptive immune response5. Vibrational spectroscopy involves label-free techniques that allow detection of electronic changes in the internal vibrational energy levels of biomolecules. It has been used in the study of cells, tissues and biofluids and provided valuable insights into pathologies8. Barauna et al.9 investigated saliva samples from throat swabs using Attenuated Total Reflectance FTIR (ATR-FTIR). Initial classification of swab samples into negative and positive COVID-19 infections was based on symptoms and PCR testing results. The proposed method is based on ATR-FTIR analysis of the extracted RNA and machine learning modeling. The authors report that this technique achieves 97.8% accuracy, 97% sensitivity, and 98.3% specificity while reducing the testing time from hours to minutes after RNA extraction. The reported area under the ROC curve (AUC) ranged from 0.54 to 1, depending on the statistical model used. Wood et al.12 proposed a portable infrared spectrometer with custom-made transflectance accessories for rapid point-of-care detection of COVID-19 markers in saliva. They tested the system on samples from 29 subjects who tested positive for SARS-CoV-2 by RT-PCR and 28 who were negative, and achieved a sensitivity of 93% and a specificity of 82%. However the work of Wood et al. did not aim to explore the rich biochemical information contained in the spectral data.Vibrational spectroscopic techniques such as Fourier-transform infrared absorption (FTIR) have been successfully used to study biological samples. They provide quantitative and qualitative multiplexed information at the molecular level, even when triggered by subtle changes in a sample13. They used ATR-FTIR to investigate serum samples from 56 patients and revealed that the CoronaVac-Sinovac COVID-19 vaccine administration induced significant changes in some functional groups belonging to lipids, proteins and nucleic acids. FTIR has been used to investigate the secondary structural composition and changes in structural dynamics of IgG upon glycation, oxidation and glycoxidation14. Thus, it is a suitable technology to study structural changes in IgG induced by COVID-19.Some clues concerning immune response and COVID-19 had been considered by Dogan et al.Aiming to investigate possible correlation between antibody response and SARS-CoV-2 infection blood serum samples from adult healthcare workers with COVID-19 (asymptomatic or mild level) and healthy individuals were investigated here by micro-FTIR reflectance spectroscopy. Detailed characterization of the immune response of asymptomatic or oligosymptomatic individuals is important for prospective studies to describe the physiology of these cases compared to cases with higher severity and also to determine the extent of spread of infection by these patients.Serum samples from 82 healthcare workers volunteers of the “Instituto de Infectologia Emilio Ribas”, São Paulo, Brazil were included in this study. These samples are part of a cohort study to evaluate the seroprevalence of SARS-CoV-2 infection in healthcare population. Samples were collected from July to November of 2020, before the beginning of the vaccination program in Brazil. Levels of illness were mild or oligosymptomatic. This study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by Research Ethics Committee of the “Instituto de Infectologia Emílio Ribas”, São Paulo, Brazil, protocol number CAAE 32264120.5.2001.0061. Invited volunteers were informed about the objectives, propositions and conditions of this project, and those who agreed to participate in the research signed the free and informed consent term. A volume of 5 mL of whole blood was collected by peripheral vein puncture and stored in a 10 mL dry tube for each patient. Then it was centrifuged at 2052 g for 10 min to separate the serum. Two mL of serum were stored in a 15. The results were expressed in terms of ratio of the sample signal to a calibrator-assigned cutoff signal with threshold of 1.0. Negative diagnosis were considered for outcomes with reactivity index 16. The test sensitivity was reported to be 90.0%  immunoassay was performed to detect immunoglobulin G anti-Spike protein from SARS-CoV-2. This assay does not differentiate binding IgG antibodies from virus-neutralizing IgG antibodies17.Anti-SARS-CoV-2 IgG ELISA  assay was performed to detect IgG antibodies against SARS-CoV-2. In this diagnostic test IgG antibodies against SARS-CoV-2 spike protein subunit 1 (S1) are detected in human serum or plasma. Following the instructions of the manufactures, serum samples were 1 : 101 diluted, added to wells coated with recombinant SARS-CoV-2 antigen and incubated for 60 min at 18. The final droplet presented an average radius of 2 to set the area of interest for measurement on the sample. The spectral resolution was set to All samples were brought to room temperature prior to preparation for micro-FTIR measurements. Aliquots of 1 µL of serum (1 : 3 in ultrapure water) were deposited in platinum sample holder and dried at 80% relative moisture under a desiccator with NaCl saturated solution in order to avoid coffee ring effect and obtain a homogeneous biofilm19 was performed on mean centered raw data to extract outliers and identify possible experimental bias. All spectral analysis steps were implemented in the ChemSpec vignette available in the software R20. Outliers were identified using the Q and Q statistics indicates how well each observation matches to the PCA model and the The classical Principal Components Analysis (PCA)21. All spectra were normalized and scaled using probabilistic quotient normalization22. Then PLS-DA analysis was performed. It is a multivariate supervised method that uses linear regression of original variables to predict the class membership. In our case the PLS regression was performed using the plsr function provided by R pls package23. The classification and cross-validation were performed using the corresponding wrapper function using the caret package20. A permutation test was performed to assess the performance of class discrimination. In each permutation, a PLS-DA model was built between the data and the permuted class labels using the optimal number of components determined by leave-one-out cross validation for the model based on the original class assignment. The class discrimination performance was measured using classification accuracy, 25. Two quantifiers were used to measure the vibrational band frequency importance in PLS-DA model. The first, Variable Importance in Projection (VIP) is a weighted sum of squares of the PLS loadings taking into account the amount of explained spectral intensity-variation in each dimension. The other importance measure is based on the weighted sum of PLS regression. The weights are a function of the reduction of the sums of squares across the number of PLS components. For multiple-group analysis, the same number of predictors will be built for each group and the average of the feature coefficients were used to indicate the overall coefficient-based importance. The receiver operating characteristic (ROC) analysis was used to evaluate the discriminating performance and the area under the ROC curve (AUC) as its summary index. In general tests with excellent discriminating capability will furnish 27.All spectra were pre-processed to become comparable for statistical analysis. The baseline was corrected using the least-squares polynomial curve fitting method as described by Lieber and Mahadevan-Jansen28 was applied to test the hypothesis that IgG data does not differ significantly from that which is normally distributed. The F-test was used to test the hypothesis of equality of averages of two sets of data populations of unequal size (IgG and demographic data)29The Kolmogorov-Smirnov test of normality30. We notice that CLIA and ELISA tests were 100% concordant about the patient’s diagnosis in our cohort.The main clinical and demographic pieces of information about volunteers are presented on Table 31.Average FTIR spectra for negative (black line) and positive (red line) classes of samples in the fingerprint spectral window  versus 25 was checked  is shown in Fig. 38. The basic H2L3 structures consist of the Fc region  and Fab region , both composed mainly of 38.Thus the increased intensity of 35. Besides, the C36. Also it had been reported that the FTIR spectrum of glycated human serum albumin presents a new peak carbonyl group at 34. Also 39The important vibrational frequencies (VIP) identified by PLS-DA are represented in Fig. 3 which proposed that patients with severe COVID-19 are more likely to have IgG1 with afucosylated Fc glycans as signature. It is well reported that IgG glycosylation can determine whether an antibody glycoform is pro-inflammatory  or anti-inflammatory 37. Since 1768–1786 In our case bands in 1702–1785 AUC are also shown. The ROC curves for positive/negative  data. The discriminating performance of the spectral data alone was not reported. Zhang et al.41 investigated by ATR-FTIR blood samples from 20 healthy donors and 76 patients, of which 41 were confirmed with COVID-19, 15 had respiratory viral infections caused by influenza A/B or respiratory syncytial virus (RSV), and 20 were with inflammation-related diseases. In this case the AUC for COVID-19 discrimination was reported to be 0.9947. To the best of our knowledge only these two exciting articles reports COVID-19 diagnosis using ATR-FTIR on blood or plasma samples. In spite of diverse sample preparation method and amount of individuals included in the cohort, our AUC is very similar to that found by Banerjee et al. and Zhang et al. The key differences between our approach and the ATR-FTIR relies on waiting time for result outcome, amount of samples which would be tested by row, and COVID-19 fatality capability prediction since we explored in deep the biochemical pieces of information in the FTIR spectra. For spectrum acquisition on ATR-FTIR a single drop of sample need the deposited and dried over the surface of ATR crystal. Then only a single patient could be tested at once. This process demands usually > 5 h35. It is not well reported whether the proteins degradation over this time would impact on quality of diagnosis. Moreover the morphological characteristics of the deposited bio-film  is dependent of drying conditions as relative moisture and temperature. The quality of spectrum  is very sensitive to the morphology of the bio-film. These points impose challenges to the standardization of the method35. On the other hand, our approach enables the preparation of drops of several patients over the substrate. Over an equivalent area of a microplate of 96 wells it is possible the deposition of 500 droplets. Using the microscope re-positioning the IR beam over each patient drop is relatively easy which increases the scale of testing. Banerjee et al. reported the fatality prediction only when considering in the statistical analysis a larger set of data of different kinds as spectra and clinical pieces of information which in some aspect would represent a bias in the statistical test. We were able to discriminate 3 possible groups in an independent way of validation. The clinical data were used to compare and discuss the data but not as input.Banerjee et al.42 evaluated the performance of spike protein antigens for SARS-CoV-2 serology compared four spike proteins: RBD, S1, S2 and a stabilized spike trimer (ST). They used indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ROC curves indicate that ST is the best candidate for serological testing with the highest AUC . Factors such as sampling time, viral mutation, inadequate handling, improper storage, transportation of samples, among others had been cited to influence the result. The requirements to perform these tests evolve high workload, skilled personnel for testing and sample collection, special reagent kits, costly centralized infrastructure with specific equipment for process and measured the sample signal, and professional bio safety level (BSL)-2 lab47 imposing elevated costs, relatively longer time to delivery of results (2–3 h) and dependence of international chain of suppliers. This last aspect would be disrupted during the pandemic period. One important advantage of the micro-FTIR method is the minimal need of sample preparation which impacts lower cost per testing and relative independence of external suppliers. The time elapsed pipetting , drying, acquiring spectra is typically 10 min. Another aspect to mention is that ELISA and CLIA tests are able to detect a single protein at once. Nevertheless FTIR has multiplexing advantage enabling comparison of several metabolites which increases the quality of prognosis being a suitable tool for precision medicine48. Our method enables in principle to probe 500 samples per row while ELISA and CLIA usually are limited to 96 samples. Table However as summarized by Liu and RuslingAUC was comparable to the ELISA, CLIA, and other ATR-FTIR methods (Our results showed that micro-FTIR was able to probe key structural aspects of serum IgG from COVID-19 volunteers. The 1702–1785


The recent outbreak of the Coronavirus SARS-CoV-2 has been declared a worldwide pandemic. Within various multi-organ involvement, several ocular manifestations have been described, such as conjunctivitis and retinopathy. The prevalence and severity of retinal lesions and their relation to the severity of the systemic disease are unknown. We performed a prospective, observational study on 172 consecutively hospitalized patients with acute confirmed COVID-19 infection. All patients underwent screening widefield fundus photography at the time of hospital admission. Despite no ocular or vision-related symptoms, we found cotton wool spots (CWS) and/or hemorrhages in 19/172 patients (11%). Diabetes history, overweight, and elevated C-reactive protein were more frequently observed among patients with retinal abnormalities, while a history of systemic hypertension was more frequently observed among patients without retinal findings. At a 3-month follow-up visit, CWS had subsided in all patients. In December 2019, a new disease, Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), emerged from China. The virus quickly led to a pandemic disease named coronavirus disease 2019 (COVID-19) . It has Similarly, alterations of the retina in patients with SARS-CoV-2 have been described. The most frequent are cotton wool spots (CWS) reported in several cross-sectional studies ,10,11,12The objective of this study was to perform a systematic screening for retinal alterations among consecutive patients hospitalized for an acute COVID-19 infection requiring supplemental oxygen at a tertiary university hospital and provide an estimate of retinal involvement prevalence and a description of the observed alterations. Furthermore, we sought to identify clinical features associated with the presence of retinal abnormalities and to study the longitudinal evolution of the identified lesions.This was a longitudinal, prospective, observational study conducted at the University Hospitals of Geneva (HUG), Switzerland, in collaboration with the Clinical Eye Research Centre Adolph de Rothschild (CERC). We prospectively invited into the study all consecutive patients admitted to the Department of Internal Medicine and Medical Intermediate Care Unit of HUG between 15 March and 15 June 2021. Patient recruitment and screening were conducted at the Department of Internal Medicine and Medical Intermediate Care Unit of HUG. Upon discharge, follow-up was performed at CERC.Patients were invited into the study according to the following inclusion criteria: adult patients (age >18 years) with acute SARS-CoV-2 infection (confirmed by a positive RT-PCR test), requiring oxygen supplementation (oxygen saturation of 89%), requiring an inspired fraction of oxygen superior to 50%. In accordance with institutional and international recommendations , all patThis study followed the principles of the Declaration of Helsinki and was approved by the HUG Ethics Committee (ID 202002873). All participants signed the informed consent form prior to enrollment. Patients unwilling or incapable of signing the informed consent form and patients unable to sit to allow for fundus photography were excluded from the study.On first day of admission, all patients fulfilling the inclusion criteria underwent non-mydriatic widefield fundus photography  in both eyes. Detailed ophthalmological history was taken, in particular in regard to any potential vascular retinopathy such as diabetes, systemic hypertension, or others. Vital parameters and biological variables were collected on the day of admission. Concomitant diseases were listed.All fundus photos were analyzed on the same day by two experienced ophthalmologists (AS and GSM). Patients with fundus photographs showing retinal anomalies of any kind were asked, according to the protocol, to undergo a second full eye examination, including fundus photographs and optical coherence tomography (OCT) . Three-month ophthalmic follow-up was scheduled.We performed a detailed evaluation of the presence of retinal lesions, their correlation to possible preexisting comorbidities, and the possible correlation with the severity of COVID-19 infections.The primary aim of our study was to evaluate the early presence and prevalence of retinal lesions in a sample of COVID-19 infected patients suffering from a moderate disease admitted to the ward or intermediate care unit. The secondary aim of our study was to evaluate the possible correlation between retinal anomalies and patients’ clinical or biological characteristics.P-value for statistical comparison is 0.05 for all comparisons. Given the uncertainty regarding the prevalence of retinal abnormalities, uncertain evolution of the epidemic at the time of recruitment, and the limited availability of the fundus photographer, 2-month recruitment was planned without formal sample calculation.Patient characteristics were reported with means and standard deviations as appropriate. Between-group comparisons between patients with or without retinal abnormalities are provided using Mann–Whitney test for between-group values comparison and chi2 for proportions comparisons. The During the 2-month study period, a total of 172 patients were enrolled in the study. Eighty-nine were males, and eighty-three were females; the average age (±SD) was 55 years old (±11.7), and 17 patients reported diabetes history (9%), 52 reported systemic arterial hypertension (30%), 21 reported dyslipidemia (12%), and 11 reported coronary disease history (6%) .Ophthalmological screening was performed after a median of 1 day (range: 1 to 3 days) from hospitalization. All subjects received 6 mg of dexamethasone daily since their admission. On the day of fundus examination, none of the patients reported ocular symptoms.Of the 172 examined patients, all of them had good-quality photos of both eyes. Retinal lesions were found in 19 patients (11%). Cotton wool spots alone (CWS) were found in 10 patients (6%), hemorrhages alone were found in 3 patients (2%), and CWS with hemorrhages were found in 6 patients (3.5%). Among these 19 patients, 6 had a bilateral presentation (3.5%). Typically, CWS were located at the posterior pole .There were no signs of macular edema, optic nerve head, or peripheral retinal involvement. OCT scans showed hyperreflectivity of the inner retinal layers, consistent with CWS .Three months after screening, there was a complete resolution of clinically evident CWS  with conDemographics, clinical features, and laboratory parameters of the 19 patients with observed retinal findings are reported in p = 0.025) and potassium levels .All variables were compared to those of subjects with no observed lesion. As shown in In the present study, CWS and/or hemorrhages were observed in 11% (19/172) of patients admitted to the hospital with acute COVID-19 infection in the absence of ocular or vision-related symptoms. Among the latter, ten presented only CWS (6%), three presented only hemorrhages (2%), and six presented both types of lesions (3.5%). Six patients (3.5%) had a bilateral presentation. Retinal lesions were located in the posterior pole in all cases without evidence of macular edema, optic nerve head, or peripheral retinal involvement. Retinal findings subsided completely 3 months after screening for COVID-19 retinopathy. All affected patients were symptom-free throughout the study period. We also observed that diabetes history and obesity were significantly more prevalent among patients with retinal abnormalities, while hypertension was more frequently reported among patients without retinal lesions.CWS are non-specific, acute retinal lesions representing a focal ischemic process due to an occlusion of a terminal retinal arteriole. The exact physiopathology, incidence, and behavior of the CWS found in relation to COVID-19 is not yet well understood.One explanation could be a direct viral tissue injury and infiltration of the endothelial cells causing vascular obstruction. In fact, it is reported that one of the major players for SARS-CoV-2 entry into the host cell is the angiotensin-converting enzyme 2 (ACE2) receptor, which is expressed in the lung, heart, kidney, intestine, and, most relevantly, the retina ,14,15,16Another explanation could be the occlusion of retinal pre-capillary arterioles caused by a hypercoagulable state in relation to a disproportionate production and deposition of fibrin clots in small- and mid-sized vessels. Such findings have been observed in disseminated intravascular coagulopathy-like syndrome . The sigOne last hypothesis could be that CWS manifest following diffuse or focal vasospasm caused by hemodynamic disturbances. Hypoxia related to acute respiratory distress or to an acute rise in blood pressure in the early phase of the disease could be the cause of such a condition. It is yet undetermined if vascular changes following the acute respiratory failure in COVID-19 may mimic a chronic condition such as sleep apnea. Sleep apnea is often associated with systemic hypertension causing retinal arteriolar vasoconstriction. Thus, focal CWS could be explained by focal anoxia in some susceptible terminal arterioles. Nevertheless, vasoconstriction related to sleep apnea manifests as part of a chronic condition. A recent study supports our results by demonstrating a significant reduction in vessel density of the superficial and deep capillary plexus in patients who had recovered from COVID-19 compared to those in healthy subjects ,23.To our knowledge, this is the first study that performed fundus examination in patients with COVID-19 and subsequently conducted a long-term follow-up of patients with visualized lesions. This study also highlighted a statistically significant correlation between CWS lesions and diabetes history and obesity. Considering that retinal vasculature can be directly evaluated via fundus examination, visualization and quantification of such lesions could represent signs of vascular involvement in other organs besides the eye and, therefore, a new biomarker related to COVID-19. Thus, the above-mentioned comorbidities could be a severe risk factor not only for the retinal vasculature but also for the brain and other organs’ vasculature.The main strength of this study was that we performed a prospective, observational study during which we screened patients at an early stage of their symptomatic disease. Moreover, we performed follow-up investigations on patients with detected lesions in order to characterize the evolution and duration of such lesions.On the other hand, the limitation of this study was that, unfortunately, because of the complexity of executing a fundus examination on intubated patients, we were not able to investigate the vascular impact of SARS-CoV-2 in severely ill patients in the ICU.


Participants living in rural areas compared with those living in the largest cities  had three times higher odds of refusing the COVID-19 vaccination . Respondents who declared willingness to vote for one of the right-wing political parties publicly supporting the anti-vaccination movement in Poland had eight times higher odds  of refusing the COVID-19 vaccination compared with other groups. Moreover, those who had three children or more, respondents who declared passivity towards participating in religious practices as well as active internet users had significantly higher odds of refusing the COVID-19 vaccination.We aimed to assess the factors associated with a lack of willingness to vaccinate against COVID-19 among adults in Poland three months after the introduction of mass vaccination against COVID-19 in Poland. This cross-sectional study was carried out between 8 and 18 April 2021 on a representative nationwide sample of 1131 inhabitants of Poland aged 18 and over. Almost one-third of adult inhabitants of Poland  declared a lack of willingness to vaccinate against COVID-19. Females had higher odds of refusing COVID-19 vaccination compared with males . The lack of higher education was significantly ( On 21 December 2020, the European Medicines Agency (EMA) announced their decision on the conditional marketing authorization for the first coronavirus disease 2019 (COVID-19) vaccine in the European Union (EU) . AccordiPoland is the fifth most populous EU country. The public authorities had ordered 60 million doses of the COVID-19 vaccine from six suppliers . To orgaVaccination against COVID-19 is voluntary throughout the European Union. From 1 July, the EU Digital COVID Certificate  is in force in EU member states . The CerThe introduction of the COVID-19 vaccinations meant that every adult must decide whether to vaccinate against COVID-19. Until now, public debate on adult immunization has been limited to the influenza vaccination. In Poland, influenza vaccination coverage rates do not exceed 5% of the population, as opposed to the Netherlands or the United Kingdom, where the influenza vaccination coverage rates exceed 60% of the population ,8. MoreoWe aimed to assess the factors associated with a lack of willingness to vaccinate against COVID-19 among adults in Poland three months after the introduction of mass vaccination against COVID-19 in Poland.This cross-sectional study was carried out between 8th and 18th of April 2021 on a representative nationwide sample of 1131 individuals aged 18+ in Poland. Computer-assisted personal interviewing (CAPI), computer-assisted telephone interviewing (CATI) and computer-assisted web interviewing (CAWI) were used. Each respondent could choose the preferred method of survey.The surveying process had been carried out by a specialized survey company—Public Opinion Research Center . The Public Opinion Research Center (CBOS) conducts a national cross-sectional survey on the population’s opinion regarding political and social issues in Poland .The respondents were adult inhabitants of Poland aged 18–90 years, randomly selected from the PESEL register . The PESEL database is an official database managed by the Ministry of Internal Affairs and Administration. The sampling frame was based on demographic data included in PESEL register . The results are weighted to the data of Statistics Poland  and demographic data from the PESEL register. The following characteristics of the respondents were used for weighing: gender, city/village class, voivodeship of residence, age group, education level, social and professional group. In total, 1131 individuals were included in this study.The study was conducted in April 2021. Questions concerning the COVID-19 pandemic, perception of anti-epidemic measures implemented by public authorities, attitudes towards the COVID-19 vaccine, and the willingness to be vaccinated had been included in the questionnaire. The questions  also addWillingness to vaccinate against COVID-19 was defined using the question: “Would you like to be vaccinated against COVID 19?”. Respondents who indicated negative attitudes towards COVID-19 vaccination by choosing the answers: “Definitely not” or “Rather not” were classified as those who declare lack of willingness to vaccinate against COVID-19.For the needs of the logistic regression model, quantitative variables describing age was divided into 5 categories and then recoded into series 0–1 variables (dummy variables). In the same way, the variable describing the number of children under 18 was transformed, finally creating a series of four 0–1 variables.When asked about participation in religious practices, 34 individuals refused to answer. For analytical purposes, these participants were added to those who declared that they did not participate in religious practices. Moreover, 9 individuals refused to answer when asked about using the Internet. For the purposes of these analyses, these participants were added to those who declared that they did not use the Internet.In terms of political preferences, the willingness to vote for a candidate of one of the right-wing political parties was analyzed. This right-wing political party is the only political party in the Polish Parliament (Sejm) that publicly supports the anti-vaccination movement in Poland as well as denying the importance of vaccination against COVID-19 and the threat posed by COVID-19 .The data were analyzed with SPSS V.27 . Demographic weighting was applied. A multivariate logistic regression model, crosstabs and descriptive statistics were used for statistical analysis. The chi-squared test was performed for bivariate analyses. The level of statistical significance was set at 0.05.This study was carried out following the principles expressed in the Declaration of Helsinki. This study is a secondary statistical analysis. Data used in this study were collected by a specialized survey company—Public Opinion Research Center (CBOS). Datasets were anonymous and prevented the identification of any individual study subject by the research team at any stage of the study.The analysis is based on responses received from 1131 individuals . Almost one-third of adult inhabitants of Poland  declared a lack of willingness to vaccinate against COVID-19. The highest percentage of respondents, who declared lack of willingness to vaccinate against COVID-19  was observed in the group of people who declared support for one of the right-wing political parties. Negative attitudes towards the COVID-19 vaccine were also relatively frequent in the group of people with three or more children—. Moreover, almost half of respondents aged 18–29 years  declared lack of willingness to vaccinate against COVID-19. The lowest percentage of respondents who declared a lack of willingness to vaccinate against COVID-19 was observed among those aged 60–74 years  as well as inhabitants of the largest cities above 500,000 residents . Details are presented in Of the 491 respondents who have not yet been vaccinated but declared their willingness to vaccinate against COVID-19, 60.7% (95% CI: 56.3–64.9%) indicated that they care about which COVID-19 vaccine they will receive. In this group, the COVID-19 vaccine from Pfizer-BioNTech (70.1%) was the most preferred. The Moderna COVID-19 vaccine was preferred by 27.9%, Johnson & Johnson’s single-dose COVID-19 vaccine was indicated by 16.1% and AstraZeneca was preferred by 6.7% of respondents.p < 0.001) associated with greater odds of a lack of willingness to vaccinate against COVID-19. When compared with those aged 75 and over, participants aged 60–74 years had more than three times lower odds of refusing the COVID-19 vaccination . Females had higher odds of refusing the COVID-19 vaccination compared with males . Moreover, having three children or more, passivity towards participating in religious practices as well as being an internet user were significantly associated with a lack of willingness to vaccinate against COVID-19. Details are presented in The multivariate logistic regression that explained the lack of willingness to vaccinate against COVID-19 obtained Cox and Snell R-Squared of 0.147 and Nagelkerke R-Squared of 0.209. Respondents who declared willingness to vote for one of the right-wing political parties  had eight times higher odds  of refusing the COVID-19 vaccination compared with respondents who support other political parties or do not intend to participate in the elections. Participants living in rural areas had three times higher odds of refusing the COVID-19 vaccination  compared with those living in the largest cities . Lack of higher education was significantly , started operating; therefore, every inhabitant of Poland had the opportunity to get vaccinated against COVID-19 in the vicinity of their place of residence ,6. The oOut of eight predictors of the lack of willingness to vaccinate against COVID-19, respondents who declared willingness to vote for one of the right-wing political parties had eight times higher odds of refusing the COVID-19 vaccination compared with respondents who support other political groups or do not intend to participate in the elections. This political party is the only political party in the Polish Parliament (Sejm) that publicly supports the anti-vaccination movement in Poland . The leaThis study showed that living in rural areas or cities below 100,000 inhabitants was associated with higher odds of refusing the COVID-19 vaccination. This observation may result from the fact that due to the population density, level of urbanization and industrialization, the number of COVID-19 cases and COVID-19-related deaths reported in rural areas and smaller towns was lower compared with large urban centers ,14. Due Currently, there are no conclusive data on the impact of an individual’s educational level on their COVID-19 vaccine trust ,17,18. ANumerous studies showed that females are more hesitant to get vaccinated against COVID-19 ,21. AddiThe impact of age on attitudes towards COVID-19 vaccines was observed only in the case of participants aged 60–74 years, who had more than three times lower odds of refusing the COVID-19 vaccination compared with those aged 75 years and over. This observation is in line with other studies, where older people  were significantly more prone to vaccination than younger groups .In this study, respondents with at least three children had significantly higher odds of refusing the COVID-19 vaccination compared with those without children. We can hypothesize that people with more than two children are more likely to receive anti-vaccine content as they repeatedly decide whether to vaccinate their children, but this hypothesis requires further investigation .Passivity towards participating in religious practices was another factor that significantly affected attitudes towards the COVID-19 vaccine. We can assume that the attitude towards religious practice reflects other social characteristics and behaviors, so it is not possible to unequivocally explain this observation. The positive stance of religious authorities on vaccination against COVID-19 may encourage participants of religious practice to vaccinate, especially among people over 60 years of age .An infodemiological study showed that during the COVID-19 pandemic, the Internet was the major source of misinformation on COVID-19 ,25. MontOur study showed that in April of 2021, 70% of adults in Poland are willing to get vaccinated. However, real-world data are needed in order to verify these declarations with social behaviors. As of 26 August 2021, cumulative uptake of at least one dose of the COVID-19 vaccine among adults in Poland amounts to around 59% , which iThis study has practical implications for policymakers and public health authorities. First, the eight factors that were described in this study and are associated with attitudes towards COVID-19 vaccination can be a source of information on the target group, the persuasion of which to vaccinate will increase the chance of achieving herd immunity. Secondly, our research may be the basis for future research on health literacy among inhabitants of Poland. Moreover, the experience that results from the implementation of the National COVID-19 Vaccination Program can be used to plan communication towards building confidence in vaccination in Poland.This study has several limitations. First, we used secondary data on attitudes towards COVID-19 vaccines, so the scope of the analysis is limited to available data. Secondly, this study was based on the results of a poll conducted in April 2021. Public opinion may change because of media campaigns and vaccination promotions by public authorities and medical professionals. Nevertheless, this is the most up-to-date study on attitudes towards COVID-19 vaccines in Poland, carried out three months after the introduction of mass vaccination against COVID-19 in Poland.In April 2021 in Poland, females, respondents without higher education, those living in rural areas or cities below 100,000 inhabitants, respondents who declared willingness to vote for one of the right-wing political parties, those who had three children or more, respondents who declared passivity towards participating in religious practices as well as active internet users had significantly higher odds of refusing the COVID-19 vaccination.Identifying groups that have negative attitudes towards the COVID-19 vaccination is crucial to developing personalized communication to build COVID-19 vaccine confidence.


Background: We assessed the effect of bisphosphonates (BPs) on breast cancer (BCa) patient survival and explored how long the effect can persist after treatment.Methods: We performed a meta-analysis and trial sequential analysis (TSA) of prospective studies including randomized controlled trials (RCTs) and cohort studies. We performed extensive sensitivity analyses to assess the robustness of the findings.Results: Seventeen RCTs and eight cohorts with 81508 BCa patients were identified. A significant beneficial effect of BPs on BCa survival was found , and the TSA results also suggested firm evidence for this beneficial effect. Both summarized results from RCTs and cohorts provided firm evidence for this effect, although the effect estimates were stronger from cohorts than RCTs . This beneficial effect was confirmed for bone-metastases  and postmenopausal women . Importantly, our results demonstrated that this beneficial effect was retained at least 1-2 years after treatment completion  and could persist for up to more than 4 years after treatment completion . Extensive sensitivity analyses showed the robustness of our results. The GRADE quality of evidence was generally judged to be moderate to high.Conclusions: The present study provides firm evidence for a significant beneficial effect of BPs on BCa survival in patients with early-stage BCa, and this effect was retained at least 1-2 years after BP treatment completion. Breast cancer (BCa) is the most frequently diagnosed cancer and the leading cause of cancer deaths among females worldwide, with more than 2 million newly diagnosed cases in 2018 . ConsequBisphosphonates (BPs) are widely used for the prevention and treatment of osteoporosis and are recommended as the standard care for bone loss or skeletal-related complications caused by malignancies , 6. ReceA recent large meta-analysis by the Early Breast Cancer Trial Collaborative Group (EBCTCG) showed a substantially significant reduction in bone metastases and BCa mortality in postmenopausal patients . Then, aTrial sequential analysis (TSA) is a novel method for improving the quality of information from conventional meta-analysis and providing TSA-boundaries that determine whether the evidence is reliable and conclusive. Therefore, we performed this comprehensive meta-analysis and TSA of data from prospective epidemiological studies to determine and quantify the impact of BPs on BCa survival in women with early-stage BCa. We also explored whether the effect persists, attenuates, or disappears over time. Furthermore, we evaluated the robustness of our findings with extensive sensitivity analyses.This study was registered on the PROSPERO database (CRD42014014699) and was reported according to the PRISMA guidelines. We systematically searched the electronic databases of PubMed, Embase, CENTRAL , and ProQuest through 25 June 2019 to identify relevant publications (detailed information on the search strategy is provided in the The inclusion criteria are as follows: (1) prospective epidemiological studies including RCTs or cohort studies that addressed the effect of BPs versus non-BP control on BCa survival among BCa patients with no evidence of any relapse or metastasis; (2) studies that reported effect estimates, such as hazard ratio (HR), relative risk ratio (RR), or odds ratio (OR), with 95% confidence intervals (CIs), or reported sufficient information to calculate these values; (3) BP users are defined as BCa patients who received BP treatment only before the occurrence of any BCa relapse or metastasis; (4) when multiple articles reporting on the same study but reporting outcomes at different time points, we used the data at the longest follow-up time in the overall meta-analysis and included all of the datasets at different follow-up time points from the same study in subgroup analyses, cumulative meta-analyses, and TSAs by follow-up duration; and (5) when no-event trials were identified, we used an empirical constant continuity correction  and we included no-event trials in the main analysis. There were no restrictions on language, sample size, or participant characteristics.The eligibility determination, data extraction, and quality assessment for each study were performed independently by two reviewers (YPL and YXZ). Discrepancies were resolved by discussion and consensus with another reviewer (SZ). Standard electronic forms specifically created for the present study were used to extract the data from each study. Both the maximally adjusted and unadjusted effect sizes with 95% CIs were recorded, if available. The extracted data from each study were carefully checked before performing the analyses. When necessary, we contacted the authors of studies for missing information. For RCTs, the bias risk was assessed with the domains recommended by the Cochrane collaboration tool. Trials were considered to have a high risk of bias overall  if ≥4 of these domains were judged to have a high or unclear risk. Otherwise, they were considered to have a low risk of bias overall . We used RevMan version 5.3 to assess the risk of bias. We assessed the quality of the cohort studies with a previously described quality-scoring system . BrieflyIn this meta-analysis, the maximally adjusted effect sizes and 95% confidence intervals (CIs) were summarized using random-effects models rather than fixed-effects models to analyse the results in a conservative manner. In the main analysis, we included only the high-quality studies. Predefined subgroup meta-analyses by type of BPs, recurrence site, and menopausal status of participants in individual studies were conducted. To assess how long the beneficial effect would be maintained, we performed subgroup analyses according to the follow-up duration of RCTs, taking into account different BP treatment periods and different follow-up periods across the RCTs; we did not include cohort studies in this analysis because of the uncertain BP treatment duration. Comprehensive Meta Analysis version 2.0  was used for the meta-analyses and conventional cumulative meta-analyses.Q test and the I2 statistic. To investigate potential sources of the between-study heterogeneity, we performed subgroup and meta-regression analyses using random-effects models. To assess potential publication bias, we used funnel plots for asymmetry and formally used Begg’s rank correlation and Egger’s linear regression tests. Furthermore, we robustly adjusted for the summarized results by applying Duval and Tweedie’s trim and fill method.We tested the between-study heterogeneity using the We conducted several prespecified sensitivity analyses. Confounding RRs were used to test whether the underlying confounders could have influenced the results. The confounding RR is defined as the ratio of the summarized results of the maximally adjusted and unadjusted data . To testD2, a heterogeneity-adjustment factor). The diversity adjusted RIS (D2-RIS) was used to reduce type II errors, and the TSMB was used to reduce type I errors. In this study, the incidence of outcome events in the control group and an anticipated relative risk reduction were estimated based on the summarized results of the included studies. TSA was conducted to maintain an overall two-sided 5% risk of type I error and a 20% risk of type II error (80% power), which is the common standard in most meta-analyses. When the z-curve crosses the TSMB, a firm level of evidence has been reached, and if the accumulated information size simultaneously reaches the D2-RIS, one may conclude that no further studies are needed. If the z-curve does not cross any of the boundaries and D2-RIS has not been reached, there is insufficient evidence to draw conclusions. When the accumulated information size reached the D2-RIS, we further conducted a post hoc sensitivity analysis with the intention to maintain a 90% power and to assess consistency and reliability of the observed effect. When several articles were published from the same study, only the data of the longest follow-up period were analysed in the TSA according to publication year, whereas in the TSA for follow-up duration, we included all of the datasets with diverse follow-up durations from the same study, from the shortest to the longest among the analysed RCTs, irrespective of the treatment period. TSA Viewer version 0.9beta  was used for TSA.Conventional cumulative meta-analyses are prone to producing random errors because of few data and repetitive testing of accumulated data when the required information size (RIS) has not been met . We therWe used GRADE methodology to assess the quality of evidence, which was classified as high, moderate, low, or very low based on the evaluation for study design, bias risk, inconsistency, indirectness, imprecision, publication bias, and confounding bias. Summary tables were constructed with the online version of GRADEproGDT software.The flow diagram of study selection is shown in Among these eight cohorts included, two studies were nested case-control studies. All cohorts originated from developed countries and enrolled 66899 BCa patients, with 14731 (22.02%) of the participants recorded as BP users. The sample size varied considerably from 226 to 21190. The mean follow-up period was 78.2 months , and 6670 outcome events were reported . All cohort studies were categorized as high-quality .P<0.001; 81508 participants; D2-RIS was 37932 participants. The TSA showed that the cumulative z curve had crossed the trial sequential monitoring boundary (TSMB) for the beneficial effects of BPs, and the actually cumulative sample size (ACSS) currently exceeded the D2-RIS with a summarized RR of 0.700 , implying that there is firm evidence for an overall protective effect of BPs over controls in women with primary BCa  and 0.892 , respectively. TSA showed that the cumulative z curve crossed the TSMB for beneficial effects in both RCTs . In the TSA, however, only the finding for post-treatment 1-2 years was confirmed  was accrued, suggesting firm evidence for a carryover beneficial effect for up to 1-2 years after treatment completion. For subgroups of post-treatment 3-4 or >4 years, TSA-corrected effects did not reach statistical significance, although the cumulative z curve for the later subgroup crossed the conventional boundary.onfirmed –3E. The z curve crossed the benefit TSMB when the follow-up duration reached 42 months with a final summarized HR of 0.716 , supporting the evidence for this carryover beneficial effect , suggesting firm evidence for a protective effect of BPs against bone metastases (z curve crossed the futility boundary (FB), suggesting firm evidence for no effect of BPs on local-regional relapses. For contralateral BCa, the conventional meta-analysis showed a summarized RR of 0.775 , but the TSA showed that the cumulative z curve crossed the FB  but not for non-postmenopausal women  and postmenopausal women (P=0.056) but not to sample size (P=0.486), number of BP users (P=0.902), outcome events (P=0.439) or follow-up duration (P=0.454). We found no evidence for publication bias (In general, there was obvious between-study heterogeneity, which was mainly from the cohort studies. The between-study heterogeneity among RCTs was very small or nil, whereas it was very obvious for cohort studies. Findings from the meta-regression analyses  revealedion bias .D2-RIS were accrued, further trials are needed. The results from eight ongoing RCTs with a total of 14286 participants that focus on the association between BPs and BCa survival are expected, and our systematic review has to be updated when these additional results are available.For the first time, we combined the data from prospective studies including RCTs and cohorts and confirmed the positive beneficial effect of BPs on BCa survival using the TSA method. The present study provides firm evidence for the beneficial effect of BPs on BCa survival, especially for bone metastases and postmenopausal patients. Importantly, our results demonstrated that this observed effect was retained for at least 1-2 years after treatment completion. Subgroup analyses revealed a statistically non-significant 9.5% improvement in BCa survival in the treatment duration, a significant 22.6% improvement for 1 to 2 years after treatment completion, a non-significant 20.2% improvement for 3 to 4 years after treatment completion, and a borderline significant 9.5% improvement for more than 4 years after treatment completion, which suggested that the beneficial effects may persist for up to 4 years after the treatment completion. Interestingly, this carryover effect is similar to the effect of tamoxifen on BCa survival , 52. GivIn addition, the findings indicated that the benefit of BP treatment seemed to diminish during the follow-up period longer than 4 years after completion of the treatment. The diminution of the beneficial effect may be explained by a significant decrease in BP concentrations in the body. BP concentrations in the urinary excretion sharply decreased beyond 4 years after completion of the BP treatment, although it was still detectable up to 8 years after the end of treatment . It is tOur present study confirmed the results of the EBCTCG study and provided firm evidence for the beneficial effect of BPs on BCa survival, although the overall beneficial effect might be primarily from firm evidence about bone metastases. Bone is the most common metastasis site in patients with BCa . The bonRecently, an increasing body of biological mechanisms has supported the notion that BPs may modify the tumour cell microenvironment and directly affect tumour cells through a variety of pathways, including inhibition of osteolysis  and the Given the global use of BPs among millions of women over the past two decades, it can be concluded that adverse effects related to BPs are relatively low . CommonlBPs should have a substantial and extensive public health benefit in the improvement of BCa survival, since BPs are already widely prescribed for the prevention and treatment of osteoporosis or cancer-induced bone loss, particularly in developed countries , 55, andThe major strengths of this study were the application of TSA and the inclusion of all of the currently available prospective epidemiological studies, including RCTs and cohort studies, in our study. To our knowledge, this is the first meta-analysis using TSA to assess the effect of BPs on BCa survival. TSA, taking into consideration the required information size, the accumulated information size, and the effect size, is more conservative and reliable and probably more accurate because it allows for repetitive testing of accumulating data. We included a total of 81508 early-stage BCa patients enrolled in the analysed studies, including 22521 BP users and 9491 events, which added reliability to our findings. The results of the RCTs and cohort studies are consistent, even though there is obvious heterogeneity between study designs. RCTs tend to evaluate the effect of BPs under ideal conditions among highly selected BCa patients, whereas cohort studies evaluate the effect in real-world settings. Therefore, it is important that new therapies be evaluated using RCTs and assessed using prospective cohort studies before interpreting the real-world effects. Additionally, a high consistency of the results across diverse statistical models and extensive sensitivity analyses and the moderate to high overall GRADE quality of evidence added further reliability and robustness to our findings.However, several limitations of this study should be considered. First, various dosages and durations of BP treatment used in the individual studies could have contributed to the obvious heterogeneity. Second, multiple testing could have affected our results since we conducted post hoc subgroup analyses by follow-up duration and menopausal status and our findings from these subgroup analyses should be interpreted with caution. Despite being post hoc analyses, the findings of subgroup analyses by follow-up duration raised interesting hypotheses: whether the benefits of BPs can exert a carryover beneficial effect for up to 4 years after the treatment completion and whether re-treatment with BPs 3 to 4 years after completion of the first round of medication would maintain a long-term, equally beneficial effect thereafter. Even though we performed the TSA to correct for these random errors, sparse data, and repetitive testing, the results suggest there is insufficient information to confirm this hypothesis. Thus, a confirmatory large sample size RCT is required to explore and confirm this hypothesis, especially if the findings are from subgroup analyses. Finally, we could not explore whether the beneficial effect of bisphosphonates differs across different molecular subtypes of breast cancer due to the lack of available data.In summary, this study provides firm evidence for a significant beneficial effect of BPs on BCa survival in patients with early-stage BCa, and this effect could persist at least 1-2 years after the completion of BP treatment. Several unanswered questions remain, including what is the optimal procedure of BP treatment for women with primary early-stage BCa, which type of BPs would be better, whether the beneficial effects persist or attenuate with time, or become more pronounced if a second round of treatment with BPs is given, and which group of women may derive the greatest benefit from BP treatment.Supplementary MaterialsSupplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Tables 5 and 6Supplementary Table 7Supplementary Table 8-12


The contamination of body fluids by Severe Acute Respiratory Syndrome Coronavirus 2 during surgery is current matter of debate in the scientific literature concerning CoronaVIrus Disease 2019. Surgical guidelines were published during the first wave of the COVID-19 pandemic and recommended to avoid laparoscopic surgery as much as possible, in fear that the chimney effect of high flow intraperitoneal gas escape during, and after, the procedure would increase the risk of viral transmission.The aim of this study was to evaluate the possibility of SARS-CoV-2 transmission during surgery by searching for viral RNA in serial samplings of biological liquids.This is a single center prospective cross-sectional study. We used a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) test to perform swab tests for the qualitative detection of nucleic acid from SARS-CoV-2 in abdominal fluids, during emergency surgery and on the first post-operative day. In the case of thoracic surgery, we performed a swab test of pleural fluids during chest drainage placement as well as on the first post-operative day.A total of 20 samples were obtained: 5 from pleural fluids, 13 from peritoneal fluids and two from biliary fluid. All 20 swabs performed from biological fluids resulted negative for SARS-CoV-2 RNA detection.To date, there is no scientific evidence of possible contagion by laparoscopic aerosolization of SARS-CoV-2, neither is certain whether the virus is effectively present in biological fluids.The online version contains supplementary material available at 10.1186/s12893-022-01571-6. The possible contamination of body fluids by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) during surgery is a recent topic of discussion in the scientific literature concerning COronaVIrus Disease 2019 (COVID-19) [Previous studies on different viruses reported that viruses like hepatitis B virus, HIV, and HPV were previously detected in aerosolizations of biological liquids, during laparoscopic surgery –5.Based on these data, the use of personal protective equipment (PPE) was advocated by national and international guidelines as the best method to reduce the risk of contamination in theatre staff, especially during aerosol-generating procedures.Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) recommending pre-operatory COVID-19 testing for all surgical patients, intubation and extubation in negative pressure rooms, and appropriate filtration and ventilation of the operating rooms in case of suspected or confirmed COVID-19 patients [In May 2020, the European Society of Coloproctology released a joint statement with the European Association of Endoscopic Surgery (EAES) and the patients . Surgicapatients , 8.Although the absence of the virus in laparoscopic fumes seems to be demonstrated, there are still insufficient data concerning the risk of contagion from biological fluids during surgery. Some considerations for and against the possibility of a viral translocation exist: first, it is widely known that peritoneal membranes have a maximum pore diameter ranging from 20 to 40 nm, while the diameter of SARS-CoV-2 virion measures approximately 50–200 nm. For this reason, it is not possible to exclude a theoretical transmigration of the virus across the barrier represented by the peritoneal membrane, especially in case of increased permeability (inflammation) or damage.Furthermore, the cell membrane protein angiotensin-converting enzyme-2 (ACE-2) is the key for receptor-mediated cell entry of SARS-CoV-2. This protein is expressed in pneumocytes  as well as in the gastrointestinal tract  and it may represent “the access door” through the peritoneal membrane during surgery, both open and laparoscopic .Despite the theoretical risk of contamination, only few case reports confirmed the presence of SARS-CoV-2 in abdominal fluids –12, whilThe aim of this study was to evaluate the possibility of viral transmission of SARS-CoV-2 during surgery by searching for viral RNA in serial samplings of biological liquids.This is a single center prospective cross-sectional study, performed in a hospital serving a wide and low densely populated rural area in the province of Ferrara, Italy. We used a real time reverse transcriptase (RT) polymerase chain reaction (PCR) test to perform swab tests for qualitative detection of nucleic acid from SARS-CoV-2 . For abdominal fluids, a first abdominal swab sample was performed at the time of abdominal incision, while a second swab sample was collected at the end of the surgical operation, before washing the abdominal cavity with saline solution. In case of cholecystectomy, a biliary swab test was also collected. On the first post-operative day, a swab sample of fluid from the abdominal drainage (if placed) was collected together with a naso-pharyngeal swab. In thoracic surgery, we performed a swab test of pleural fluids during chest tube placement as well as on the first post-operative day.In order to review the literature on this topic, we used the advanced searching function of PubMed library. The key words used were the following: “Peritoneal Fluid”, “Pleural Fluid”, “COVID” and “SARS-CoV-2”. The research was limited to studies written in English, including adult patients (older than 18 years): we found a total of 13 articles concerning abdominal fluids and 4 about thoracic fluids.3 chest tube placements for pneumothorax 2 open sigmoid Hartmann resections for perforated diverticulitis 1 exploratory laparotomy for massive bowel infarction 1 laparoscopic cholecystectomy 1 open cholecystectomy From February 2020 to May 2021, a total of 8 COVID-19 patients underwent emergency surgery , 13 from peritoneal fluids, and 2 from biliary fluid. 6 out of 8 patients had a diagnosis of COVID-19 related pneumonia, while two of them did not have lung involvement by SARS CoV-2. All subjects were tested positive for SARS-CoV-2 RNA detection through naso-pharyngeal swab. 4 patients died during hospitalization, those with pneumothorax and the one with massive bowel infarction. All 20 swabs performed from biological fluids resulted negative for SARS-CoV-2 RNA detection.This study focused on evaluating the presence of SARS-CoV-2 RNA in a total of 20 peritoneal and pleural fluid samples from 8 COVID-19 patients  who underwent emergency surgery over a period of 15 months.Based on our single center experience, we could not demonstrate the presence of the virus in any of the samples. Overall, we investigated a total of 8 patients with positive SARS-CoV-2 RNA naso-pharyngeal swab undergoing emergency surgery. With the limits of the small size of sampling, we registered a prevalence of females  and a greater number of swabs performed from peritoneal fluid . 4 out of 8 patients died during hospitalization due to the progressive deterioration of their clinical condition.In a recent article, some Authors performed SARS-CoV-2 RT-PCR assay from the peritoneal effluent after dialysis of a symptomatic COVID-19 patient: the assay was performed twice after 6-h duration of dwells without centrifugation, but it resulted negative in both samples . There aSome authors also reported the presence of SARS-CoV-2 RNA within the intestinal tissues during upper gastrointestinal surgery  or insidThe presence of SARS-CoV-2 RNA in other tissues, such as sputum and stool, was already demonstrated .Tests of feces with molecular swabs allowed to detect SARS-CoV-2 RNA in a percentage of infected patients ranging from one third to one half, suggesting the potential local viral translocation in particular favorable conditions . To dateAlthough aerosolization of blood borne viruses was already shown to occurr during laparoscopy, several reports did not demonstrate the presence of SARS-CoV-2 RNA in the peritoneal fluid of COVID-19 patients –28.Our review of literature showed that most studies are in favor of the absence of SARS-CoV-2 RNA in biological fluids  when swab tests are performed during surgery, while positive viral reports are limited to patients with immune system compromission (patients with organ transplantation or with cancer) or with peritoneal lymphatic drainage illnesses (e.g. cirrhotic patients).We tried to hypothesize some possible explanations of these findings. First of all, the virus may be present but undetectable through the available tests. The sensitivity of molecular swabs was already evaluated in some studies that cast doubts on possible false negatives results, due to sampling problems or to thermal damage of the swabs: this could happen in case of patients with low viral load , 30. FurMoreover, it shall be considered that a “dubious” result (a weak positivity or negativity) can also be caused by factors that are usually unrelated to the virus itself or to its load, such as the method of storage or transportation or sampling. The difficulties encountered in the interpretation of some swab tests results can be partially overcome by recognizing such intrinsic disadvantages of the process itself and, at the same time, this goes to explain why some patients can meet discordant results in case of samples from different body fluids. Finally, it is to remind that the only swabs currently certified are those taken from the oropharynx or from bronchoalveolar lavage .The second possibility is about the inactivity of the virus, that may be present (either detectable or not). To date, no certified case of contagion between patient and operator during surgery were reported. This could indicate that the presence of the virus (detected or unrecognized) is not a sufficient element to guarantee the risk of contagion of healthcare workers in the operating room, probably due to environmental conditions that are hostile to the spread of adequate viral loads or due to the presence of inactive viruses. This would mean that the presence of the virus in biological fluids, detected through a RT-PCR assay or not, does not reach the infecting viral load threshold.Our last hypothesis is that the virus could not be always present. In a recent article, we hypothesized that the possible presence of SARS-CoV-2 in the peritoneum may depend on the disease stage and the associated cytokine storm . In the Although the viral spread through the pleural and/or peritoneal fluid seems unlikely, there is urgent need for further evidence to confirm or refute this hypothesis, in order to help surgeons feel more comfortable and safe during their daily activities with COVID-19 inpatients.To date, there is no scientific evidence of possible contagion by laparoscopic aerosolization of SARS CoV- 2, nor it is known whether the virus is effectively present in biological fluids. However, it is important to consider that the methods employed in these studies to detect the presence of SARS-CoV-2, such as the variable number of samples, the different types of such tests and the immune status of patients can be confounding factors in the phase of viral detection. The possible relationship between higher viral loads  in symptomatic patients has not been clarified yet and, thus, needs further investigation. The impact of favoring open over minimally invasive techniques during the pandemic could be a health burden due to the prolonged length of hospitalization and cause of a higher rate of postoperative complications, precluding the gold standard approach  for most patients.On one hand, it is essential to provide healthcare workers with adequate PPE, especially in case of possible complications by unknown agents, and the general surgical community has been taking a cautious approach towards COVID-19 patients.However, we also believe that depriving such patients of timely “gold standard” treatments, in favor of a cautious approach, such as the conservative management or the conventional open surgery, is not always the best choice for these patients and it might be matter of deeper collegial discussion.Additional file 1: Table S1. Body fluid swabs for Covid-19 in patients operated in Lagosanto Hospital, Ferrara, Italy. CCI: Charlson Comorbidity Index. 1st Wave: February 2020-July 2020; 2nd Wave: August 2020-December 2020; 3rd Wave: January 2021-today.


The BNT162b2 vaccine, containing lipid nanoparticles-formulated mRNA encoding the full-length spike protein of SARS-CoV-2, has been employed to immunize health care workers in Italy, administered in two doses 21 days apart. In this study, we characterized the antibody response induced by the BNT162b2 vaccine in a group of health care workers, tested at baseline, after the first dose and after the booster. Thirty-nine subjects without previous exposure to SARS-CoV-2 were vaccinated with the BNT162b2 vaccine. IgM, IgG, and IgA anti-receptor binding domain (RBD) were tested by ELISA. Neutralizing antibodies were evaluated testing the inhibition of RBD binding to ACE2. Antibody avidity was measured by urea avidity ELISA. IgM anti-RBD are produced after the first dose of vaccine and persist after the booster. IgG and IgA anti-RBD antibodies are detected in high amounts in all the subjects after the first dose and further increase after the booster. A few subjects, already after the first dose, produce antibodies inhibiting RBD interaction with ACE2. After the booster, high levels of inhibitory antibodies are detected in all the subjects. Affinity maturation takes place with boosting and IgG anti-RBD avidity increases with the number of immunizations. A less pronounced increase is observed with IgA. These data indicate that the BNT162b2 vaccine can induce high levels of protective antibodies of high avidity in vaccinated subjects; both IgG and IgA anti-RBD antibodies are produced. Further studies are needed to evaluate antibody persistence over time. The COVID-19 pandemic outbreak, caused by the novel severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China and rapidly spread all around the world, still representing a threat for the public health.Social distancing, partial or total lockdowns, and public education about personal protection have been exploited by all countries and helped to limit the virus’ transmission and to relieve the burden on hospital wards, but were far from representing a solution for the challenge launched by SARS-CoV-2.The best strategy to fight the pandemic is represented by a global vaccination campaign able to administer safe and effective vaccines for the protection of individuals, to achieve sufficient herd immunity to ultimately control the COVID-19 pandemic . The antibody avidity in COVID-19 patients is also reported [AI = 4.07 (3.46–4.86)] ] B.T1 = 3.54 (2.77–4.73); AIT2 = 4.05 (2.97–5.42)] and between vaccinated subjects and COVID-19 patients [AI = 3.51 (3.04–5.09)] ] C,D.The data we obtained confirm that the BNT162b2 vaccine is highly effective in inducing both IgG and IgA anti-RBD antibodies, which are produced after the first dose and further increase after the second one. The production of high-titer IgA is of particular interest, for their potential protective role at the mucosal level. In this study, only serum IgA was detected but multiple reports indicate that the amount of antigen-specific secretory IgA is correlated with serum levels in COVID-19 patients ,15. AntiThe amounts of IgG and IgA anti-RBD antibodies far exceed those observed in COVID-19 patients, suggesting a highly efficient antigen production and presentation in all the subjects However, the titer of anti-RBD antibodies does not allow firm conclusions to be drawn on their role in protection from viral infection. It is well established that the immune response to RBD is polyclonal and not every antibody to RBD is expected to play a protective role. Plaque reduction neutralization tests represent the golden standard for the detection of neutralizing antibodies. However, a positive correlation between neutralization assays and inhibition of RBD–ACE2 interaction has been obtained by many authors ,20 and tThus, we measured the potentially protective antibodies of any subclass that inhibits the interaction of RBD with ACE2. The high level of inhibition we observed by means of the SPIA assay indicates that the vaccine elicits protective antibodies, already after the first dose but in higher amounts after the booster.The goal of vaccination is the induction of an immune response endowed with high specificity and high affinity. Thus, we further tested the quality of induced anti-RBD antibodies analyzing antibody avidity.Avidity ELISA is a technique employed in a wide number of situations for the analysis of antibody avidity: during infections , in autoAs expected, affinity maturation takes place with boosting and the avidity assay we employed indicates a clear increase in IgG avidity with the number of immunizations. On the contrary, the increase in IgA avidity is less pronounced.Overall, these results suggest that the two-dose strategy is able to achieve high levels of protective antibodies of high avidity in vaccinated subjects.The main limitations of this study are represented by the number of subjects included and the short observation time, which does not permit any inference on vaccine efficacy over time. Antibody persistence will be analyzed in ongoing longitudinal studies, which allows the evaluation of the factors affecting magnitude and persistence of the immune response such as previous exposure to SARS-CoV-2 virus ,28, age A further limitation is the exclusive focus on humoral immunity, evaluated at the level of antibodies, without analyzing the B cell compartment functionally and phenotypically.Further studies are needed to verify whether patients affected by autoimmune disorders or cancer patients display a similar response to mRNA vaccines and whether immunosuppressive treatment affects the qualitative or quantitative features of the immune response to vaccine.


Non-coding variants have long been recognized as important contributors to common disease risks, but with the expansion of clinical whole genome sequencing, examples of rare, high-impact non-coding variants are also accumulating. Despite recent advances in the study of regulatory elements and the availability of specialized data collections, the systematic annotation of non-coding variants from genome sequencing remains challenging. Here, we propose a new framework for the prioritization of non-coding regulatory variants that integrates information about regulatory regions with prediction scores and HPO-based prioritization. Firstly, we created a comprehensive collection of annotations for regulatory regions including a database of 2.4 million regulatory elements (GREEN-DB) annotated with controlled gene(s), tissue(s) and associated phenotype(s) where available. Secondly, we calculated a variation constraint metric and showed that constrained regulatory regions associate with disease-associated genes and essential genes from mouse knock-outs. Thirdly, we compared 19 non-coding impact prediction scores providing suggestions for variant prioritization. Finally, we developed a VCF annotation tool (GREEN-VARAN) that can integrate all these elements to annotate variants for their potential regulatory impact. In our evaluation, we show that GREEN-DB can capture previously published disease-associated non-coding variants as well as identify additional candidate disease genes in trio analyses. Information on the controlled gene(s), tissue(s), and associated phenotype(s) are provided in GREEN-DB when possible. Here, we present a unified framework that can leverage this new resource to process standard variant call format (VCF) files and generate a comprehensive annotation of non-coding variants. We anticipate that this will aid annotation of regulatory non-coding variants identified from WGS thereby improving variant interpretation and diagnostic yield.To compile an up-to-date, comprehensive collection of regulatory elements in the human genome (GREEN-DB) we collected and aggregated information from 16 different sources, including seven previously published curated databases, six experimental datasets from recently published articles, and predicted regulatory regions from three different algorithms. Five additional datasets were included to integrate region to gene/phenotype relationships. The full list of data sources and references is reported in Original tables from the various data sources were processed to generate a normalized representation of potential regulatory regions organized in a SQlite database containing information on their controlled gene(s), method(s) of detection, tissue(s) of activity and associated phenotype(s). When needed, region coordinates were converted from GRCh37 to GRCh38 coordinates using the UCSC LiftOver tool. We performed several processing and quality control steps to remove unreliable regions and to reduce information redundancy by collapsing regions with large overlap. For this clean set of regions we then used information from GTeX eQTLs to infer additional regulated genes, and large GWAS catalogues and HPO annotations to infer phenotypes associated with each region. Finally, we refined the region-to-gene connections to ensure consistency in gene representation and remove connections with poor support. Additional datasets such as TAD domains, TFBS, DNase clusters, super-enhancer, UCNE, and enhancer LoF tolerance were also included in GREEN-DB with information on their overlap with the regulatory regions. A detailed description of the extensive data processing steps that were undertaken, and the SQlite database organization is provided in Supplementary Methods.U test, we compared the fraction of bases having a PhyloP100 score above 1, 1.5 and 2  and compared the median and maximum score values for ncER, FATHMM MKL and ReMM scores obtained for the GREEN-DB and control regions. We also performed an in-depth analysis of the gene-region connections collected in the database, evaluating the distance between a region and its controlled genes, the occurrence of connections within TAD domains and the specificity of gene-region relationships (see Supplementary Methods). We then compared information in GREEN-DB with information present in each of the 16 individual data sources that were collected in our database and for each dataset we computed the fraction of genome covered and the number of genes present in the dataset annotations.Given that creation of the GREEN-DB regions table involved a complex pre-processing of the original data sources, we first verified if these regions can still capture functional and conservation signals, features which are often reported to provide support for the regulatory role of variants in the original data sources. First, we used Fisher's exact test to evaluate over-representation of functional genome elements in GREEN-DB regions considering ENCODE TFBS, ENCODE DNase hyper-sensitivity clusters, UCNE regions, and a curated set of non-coding disease-associated variants (from ). Then, We used data from gnomAD v3 to evaluate the possible variation constraint across GREEN-DB regions by evaluating the deviation of observed number of variants from the expectation based on a linear regression model including region length, GC percentage and overlaps with segmental duplications, low-complexity regions and exonic regions. Regions above the 99th percentiles of the resulting constraint value were considered as constrained regions. We characterized these regions and the controlled genes by looking at: (i) possible enrichment of true positive variants in the curated set of disease-causing non-coding variants from , Gene OnWith the aim of providing a framework useful for variant prioritization, we reviewed the potential of 28 non-coding variant impact prediction scores to inform the analysis of WGS data from patients with rare diseases. Among these, we excluded: 10 scores not providing pre-computed values, making them difficult to apply programmatically, 2 scores developed specifically for somatic variants, and 1 score providing only disease-specific predictions for a limited set of phenotypes see . We usedZ-scores that summarize the predicted relationship of each gene with the provided HPO profile. Using GADO, we considered genes above the 95th and 99th percentile in the GADO ranking as likely or strongly disease-related, respectively.Our GREEN-VARAN tool ranks variants overlapping GREEN-DB regions from Level 1 to Level 4 by combining GREEN-DB data with population allele frequency, impact prediction scores , and functional elements  as detailed in Table We applied this strategy to analyse 53 WGS trios with recessive mode of inheritance and computed the resulting number of recessive and compound heterozygotes candidate variants when considering only coding variants, only GREEN-DB annotated variants or the combination of both. In each scenario, we computed the number of candidates considering either all genes, genes prioritized based on the HPO profiles using GADO (see prioritization strategy above), clinically relevant genes from ClinVar pathogenic list, and PanelApp disease genes. Details on the WGS cohort and the filtering strategy are given in Supplementary Methods.Z-score. We repeated this analysis assuming either a recessive or dominant mode of inheritance. To better evaluate how GREEN-DB annotations can help identifying regulatory variants in distant control elements, we also applied our annotations to a set of 18 previously published variants involved in human diseases and located in distant enhancers with a validated effect on gene expression   , DNase hypersensitivity peaks (OR 13.13), GTeX significant eQTLs (OR 2.93), ultra-conserved non-coding elements . At the same time, they are, as expected, depleted for difficult-to-address regions such as segmental duplication  and low-complexity regions . Finally, they are also enriched for a curated set of non-coding disease-causing mutations (OR 2.05) . Compare U test)  as well  U test) .For each region in the database we annotated controlled genes based on experimental screens or previously curated collections (directly controlled genes), the closest gene and the closest transcription starting site (TSS). Overall, about 35% of GREEN-DB regions have at least one directly controlled gene, ranging from 22% for bivalent regions up to 45% for promoters Figure . An addiConsidering only the 837 879 regions annotated with directly controlled genes, they interact with a total of 48 230 different genes, covering 67% of all genes and 97% of protein-coding genes from ENCODE v33 basic set. Controlled genes also cover 97, 98 and 100% of clinically relevant genes from PanelApp , ClinVarP < 2.2E–16, P 1.42E–27).We calculated a constraint metric for GREEN-DB regions ranging from 0 to 1, so that regions with higher values have lower than expected numbers of variants. Based on this metric we defined as constrained the 23 102 regions above the 99th percentile of the distribution . Complete enrichment results are reported in We considered 28 previously published prediction scores that can be applied to evaluate the impact of non-coding variants. Of these, 13 do not provide pre-computed values or were developed for somatic variants only and were thus removed from further analyses, while GWAVA and EIGEN provide 3 and 2 possible values respectively. Using a curated set of disease-causing, non-coding variants from , we evalWe created a tool (GREEN-VARAN: Genomic Regulatory Elements ENcyclopedia VARiant ANnotation) to annotate VCF files with information from GREEN-DB. The tool is written in the Nim programming language using hts-nim  and procWe used an independent set of 45 rare curated variants from  Supplem to evaluMost of the above validated variants in non-coding regions are actually located in UTR or intronic regions of the controlled gene; thus we extended our analysis to a set of 18 previously published variants located in distant enhancers with demonstrated regulatory effects on a disease gene. GREEN-DB annotations were able to capture all tested variants, linking them to the expected gene. When applying the proposed prioritization strategy we found 18, 13 and 2 variants ranked at Levels 2, 3 and 4, respectively. Details of each variant are reported in When compared with the individual data sources integrated in the database, GREEN-DB annotations captured a larger proportion of variants from our non-coding evaluation datasets, both the set of 45 rare disease-causing non-coding variants as well as the 18 variants located in distal enhancers .Genomizer and GREEN-VARAN performed similarly when applied to a set of 45 rare disease-causing non-coding variants. When considering all variants identified, both methods were able to capture 40 validated variants and associate them with the correct gene, while 32 and 33 variants were captured when considering GREEN-VARAN level 3 variants and variants at the top of the Genomizer ranking, respectively . When apTo evaluate the impact of adding non-coding annotations in a more realistic scenario, we applied our annotation framework to the analysis of small variants from 53 non-consanguineous WGS pedigrees. The main aim was to evaluate the number and relevance of new candidate variants and genes, considering that the interpretation of VUS and novel genes is time consuming and expensive and thus it is important that new tools do not add too many false-positives. Considering variants identified in each individual, we found a median of ∼75.7k rare variants (population AF < 0.01), including ∼38k in GREEN-DB regions (Level 1), ∼17k also overlapping functional signals (Level 2) and ∼1.5k further prioritized based on prediction scores (Level 3) . When coVariants in non-coding regions of the genome have clearly been implicated in disease risk from both GWAS studies  and WGS We have collected data from published, experimental, and computational sources to create a database providing a standardized representation for ∼2.4 million regulatory elements in the human genome (GREEN-DB) which represents a significant advancement compared to previously published resources. To support the interpretation of the impact of genetic variants, each regulatory region is annotated with a rich set of information including controlled gene(s), closest gene(s), tissue(s) of activity, potentially associated phenotype(s) from GWAS studies and Human Phenotype Ontology, and a constraint metric representing the tolerance to genetic variation. To interpret the biological role of a regulatory region, it is essential to know the genes it controls and in which tissues it is active. GREEN-DB contains experimentally validated region-gene links and tissue information for ∼35% and ∼40% of the regions, respectively. Overall, the database provides regulatory information for 48,246 genes, including most of the clinically relevant genes from PanelApp, ClinVar and ACMG, supporting its usefulness in human disease research.Our analysis confirms the complexity of the relationship between regulatory regions and controlled genes that can not easily be explained by spatial proximity in the (linear) genome as previously demonstrated, e.g., by high-throughput studies of chromatin interactions ,58–60. IWe also integrated information from GWAS studies and HPO databases to provide a possible associated phenotype for ∼15% of the regions. This resource will be useful for the interpretation of new variants found in the regulatory regions, providing hypotheses on their potential biological impact. The fact that only a limited number of regions has an associated phenotype, despite a large number of GWAS hits available , can parWe calculated a constraint metric that reflects the tolerance of each region to sequence variations. The maximum constraint value for regions controlling essential genes and genes involved in human diseases is significantly higher compared to other genes and constrained regions  are associated with genes under variation constraint (low observed/expected ratio for loss of function variants in gnomAD) and strongly enriched for essential genes and genes involved in human diseases. Thus, our constraint metric can be used as a stringent filter to prioritize regions likely to be relevant in controlling disease genes, even if the redundancy of the regulatory network implies that also more variable regions may be involved as suggested by the analysis of median constraint across all regions associated with ClinVar pathogenic genes.To further assist the interpretation of variants located in regulatory regions, we collected pre-computed values from 19 different impact prediction algorithms and compared their ability to classify a curated set of established disease-causing non-coding variants. Overall, we must take into account that such comparisons are limited by (i) the nature of the known variants collected so far, which are mostly variants within the affected gene and poorly capture distant regulatory elements ; and ii, FATHMM We developed a tool (GREEN-VARAN) that integrates information from GREEN-DB, non-coding impact prediction scores, functional elements and population AF annotations, into a 4 level prioritization system to rank the regulatory potential of non-coding variants in a VCF file. The proposed approach can effectively capture variants involved in human diseases, as shown by our ability to recapitulate known disease-associated variants from the literature.When applied to a set of 45 validated non-coding variants, our approach associated 40 of them with the correct gene and classified 32 as likely impacting gene expression (prioritization level 3), while considerably reducing the number of candidate variants to be evaluated for the causative gene (in 25 cases the causative variant was the only one selected). Since the validated variants we considered were mostly located within the controlled gene, we further tested GREEN-VARAN prioritization on 18 variants in distant enhancers from  and confApplication of our new annotation system to a dataset of 53 WGS trios highlighted its potential impact for rare-disease variant analysis. When considering rare recessive variants that segregate with the phenotype in each pedigree, the number of candidate variants is greatly increased by the addition of GREEN-VARAN annotations. However, when considering only variants with stronger support for regulatory impact, the number of candidates is reduced to an amount manageable for downstream analysis  variants even if the interpretation of the regulatory impact for this kind of variants can be challenging. Indeed, the function of a regulatory element is more likely to be disrupted when hit by a structural variation compared with a single nucleotide variant, but structural variants would often overlap many different elements possibly linked to different genes making the interpretation of the actual biological effect challenging. However, being able to annotate regulatory regions affected by structural variants allows the identification of interesting compound heterozygous combinations, as in the case of a disruptive coding variant complemented by a non-coding SV deleting a regulatory element for the same gene.Very few solutions exist to prioritize non-coding variants that as well as associate them with putatively affected genes. Compared with Genomiser, a popular automated annotation framework that can run on non-coding variants, our approach showed similar performances in classifying validated non-coding variants close or within a gene. Genomizer achieved a better ranking for the causative variants when using the combined score that also takes into account the HPO-based prioritization of candidate genes, while GREEN-VARAN was able to better refine the number of candidate variants when only variant-supporting evidence was taken into account. Thus, the better Genomizer performance is likely motivated by a better integration of HPO information for known disease genes which has a particular impact given the nature of the tested variants that are associated with well-established disease genes. Moreover, all the variants in the validated set are located within or in close proximity to the causative gene, making it simpler to associate them with the correct gene using a proximity approach. Indeed, when comparing a set of 18 distant enhancer variants, GREEN-VARAN demonstrated a superior ability to prioritize and correctly retrieve the controlled gene, compared with Genomizer, which failed to correctly annotate these variants.Overall, the proposed framework is not intended for exact variants ranking, but more to provide a summary of evidence for the potential regulatory role of each variant so that they can be more easily interpreted in the context of disease biology. Our approach presents a set of unique features compared to previous methods developed to address non-coding variants or regulatory regions Table . While iIn summary therefore, we have developed a comprehensive dataset of regulatory regions (GREEN-DB) and integrated it with functional elements and prediction scores into a new framework for the annotation and prioritization of regulatory variants in WGS analysis. We provide a complete annotation workflow implemented in Nextflow that uses our GREEN-VARAN tool to prioritize regulatory variants in VCF files from whole genome sequencing data. The resources presented here therefore represent a significant advance for researchers and clinicians engaged in analysing patient genomes and will be useful for rare disease research as well as for the interpretation of common disease variants from GWAS.https://zenodo.org/record/3981033. GREEN-VARAN tool, the annotation workflow and additional data are available from https://github.com/edg1983/GREEN-VARAN.GREEN-DB BED files and SQlite database are available from gkac130_Supplemental_FilesClick here for additional data file.