Ectopic gas in the graft is occasionally encountered upon follow-up computed tomography (CT) after anterior cervical corpectomy and fusion (ACCF). However, most cases lack inflammatory responses and manifestations of infection. Although the clinical significance of ectopic gas in the graft has not yet been established, to the best of our knowledge, no previous studies have described ectopic gas in the graft after ACCF. This study evaluated ectopic gas in the fibular graft upon follow-up CT after ACCF.We reviewed 112 patients who underwent ACCF and follow-up CT, with a minimum follow-up period of 3 years. CT images were retrospectively reviewed to confirm the presence of ectopic gas in the graft and bone fusion. Bone fusion was defined as follows: mobility less than 2 mm between spinous processes on the flection-extension radiograph or a bone bridge on CT images.p = 0.033). Bone fusion was not observed when CT images exhibited ectopic gas in the graft, whereas ectopic gas was not observed when CT images exhibited bone fusion.Of the 112 patients, 30 (27%) patients had ectopic gas in the fibular grafts. Among them, ectopic gas was initially observed 3 months after surgery (early onset) in 23 (77%) patients and 6 months after surgery (late-onset) in the remaining seven (23%) patients. Upon the latest follow-up CT, ectopic gas more frequently remained in late-onset  rather than in early-onset  cases (Ectopic gas in the fibular graft was observed at both early and late-onset after ACCF; late-onset gas remained significantly. The remaining gas was strongly associated with pseudoarthrosis; therefore, pseudoarthrosis should be considered when ectopic gas in the graft is observed on CT images. Anterior cervical corpectomy and fusion (ACCF) and strut bone grafting is an established surgical option for the treatment of ossification of the posterior longitudinal ligament (OPLL) and cervical spondylotic myelopathy (CSM). The ilium and fibula strut bones are used in routine surgical procedures to correct (sustain) corpectomy effects. After such procedures, ACCF has been shown to achieve satisfactory clinical outcomes.Its success in improving pain and disability scores has been well documented , 2. HoweE. coli and Klebsiella spp. Ectopic gas in the graft is occasionally encountered upon follow-up computed tomography (CT) after ACCF, while the majority of cases lack inflammatory responses and manifestations of infection. Although the clinical significance of ectopic gas in the graft has not yet been established, to the best of our knowledge, no previous studies have described ectopic gas in the graft after ACCF. The purpose of this study was to evaluate ectopic gas in the graft upon follow-up CT after ACCF and to determine the clinical significance from the perspective of pseudoarthrosis.Gas within the soft tissues usually indicates gas-producing infection caused by anaerobic bacteria or facultative gram-negative bacilli such as This study was approved by the human research committee of the Institutional Review Board, and it complied with the guidelines outlined in the Health Insurance Portability and Accountability Act of 1996. The requirement for written informed consent was waived due to the retrospective nature of the study. We retrospectively reviewed 112 cases  that underwent ACCF for CSM, OPLL, and cervical disc herniation in our hospital between 2007 and 2018. Patients were included if they had a minimum follow-up period of 3 years. The exclusion criteria included infection, tumor, cases without CT follow-up, and cases requiring reoperation. Bone graft reconstruction was performed using the fibula in 95 patients and the ilium in the remaining 17 patients. The follow-up period after ACCF ranged from 41 to 113 months . All patients underwent postoperative CT examinations 3, 6, 12, and 24 months after ACCF. C-reactive protein (CRP) levels, white blood cell (WBC) counts, and erythrocyte sedimentation rates (ESR) were consistently measured during the follow-up period.After corpectomy, the defect length was measured, and the fibula or tricortical ilium was harvested. During manual gentle traction of the skull, the bone graft was impacted in the defect. In ACCF with 1- or 2-level corpectomies, anterior cervical plates were indicated. Using a Philadelphia collar, the patients were immobilized until osseous fusion was confirmed. In ACCF with 3-level corpectomies, patients were immobilized with halo vests for 3 months; after that, they wore Philadelphia collars until osseous fusion was obtained.Follow-up CT imaging was performed using an 8-slice CT system , a 16-slice CT , or a 64-slice CT system . Unenhanced transverse and sagittal multiplanar reconstruction CT images were reconstructed with bone algorithms using a 2.5-mm section thickness and no overlap.A radiologist with 21 years of post-training experience in musculoskeletal imaging and an orthopedic surgeon with 22 years of post-training experience in spine surgery, individually reviewed all CT images. Any disagreements between the reviewers were resolved by consensus.First, the reviewers assessed the presence of ectopic gas in the graft upon follow-up CT images. Subsequently, a window level and window width were changed to clearly identify ectopic gas using commercially available DICOM viewers. According to the timing of the initial appearance of ectopic gas, patients with ectopic gas were classified as early-onset (3 months after surgery) and late-onset (6 months after surgery).Second, the reviewers also assessed the presence of the bone fusion of the graft. Based on the findings from a previous study , bone fuP-values < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS version 22.0 . Fisher’s exact tests were used to compare the amount of the remaining ectopic gas identified in the latest follow-up CT between early- and late-onset cases. Of the 112 included patients, 30 (27%) patients had ectopic gas in the graft in the follow-up CT images. All 30 bone grafts with ectopic gas were reconstructed using the fibula. Four, 14, 10, and two cases had 1-, 2-, 3-, and 4-level corpectomy, respectively. No signs of infection were found during the follow-up period in any of the patients.p = 0.033).Among 30 patients with ectopic gas, ectopic gas initially appeared 3 months after surgery (early-onset) in 23 patients and 6 months after surgery (late-onset) in the remaining seven patients. Upon the latest follow-up CT, ectopic gas disappeared in 20 (87%) early-onset and three (43%) late-onset cases, whereas ectopic gas remained in three (13%) early-onset and four (57%) late-onset cases. Thus, ectopic gas more frequently remained in the late-onset  than in the early-onset  cases , whereas ectopic gas was not observed when CT images exhibited bone fusion (n = 97).The relationship between bone fusion and ectopic gas in the graft upon the latest follow-up CT is summarized in Table ACCF is an effective surgical procedure for treating CSM, OPLL, and cervical disc herniation , 8–13. TThe presence of ectopic gas in vessels in and around the spine and within the skull usually leads to concerns regarding infections . HoweverThe accumulation of gas within the bone marrow is observed in various conditions such as osteomyelitis, focal ischemia and osteonecrosis, posttraumatic states, and solitary bone cysts . AnotherCoulier et al. speculated the physiological algorithm for the vacuum phenomenon , 26. TheJiang et al. systematically reviewed papers on ACCF and summarized that non-union rates were 5.1 and 15.2% for 1-level and 2-level ACCF, respectively. Non-union rates for three disc levels fused were much higher than those for two disc levels fused . Graft dWe believe that the etiology of ectopic gas in the fibular graft after ACCF is similar to that of the vacuum phenomenon or pneumatocyst. Based on our findings we hypothesize, that gas in cases with early onset is caused by migration of air into the graft during surgery, while gas in cases with late onset is caused by micromotion between the graft and the vertebral body. The fact that ectopic gas disappeared after graft fusion was achieved supports our theory.p = 0.033). This suggests that the presence of ectopic gas in the early postoperative stage could predict the gas remaining at postoperative years 1–3, which is related to the non-union of the graft.Moreover, in this series, ectopic gas more frequently remained in cases with late onset  than in those with early onset  (Ectopic gas in the fibular graft was observed at both early and late onset after ACCF; late-onset gas remained significantly. The remaining gas was strongly associated with pseudoarthrosis; therefore, pseudoarthrosis should be considered when ectopic gas in the graft is observed on CT images. The careful observation of ectopic gas in the graft is a simple and useful method for predicting graft fusion.


A high density of CD8-positive T lymphocytes in the primary site and a high level of CD169 expression in LySMs were independently associated with greater CSS in patients with total and advanced gastric cancer . The expression on CD169 in LySMs is a predictor of a favorable clinical course in patients with gastric cancer, and might be useful for evaluating anti-cancer immune responses.The induction of an anti-cancer immune responses is potentially associated with the efficacy of anti-cancer therapy. Recent studies have indicated that sinus macrophages in regional lymph nodes are involved in anti-cancer immune responses in the cancer microenvironment. In the present study, we investigated the correlation between lymphocyte infiltration in cancer tissues and macrophage activation in regional lymph nodes. We retrospectively identified 294 patients with gastric cancer who underwent surgery from 2008 to 2012. Using immunohistochemistry, we evaluated CD169-expression on CD68-positive macrophages, and the density of CD8-postive lymphocytes in tumor microenvironment. We statistically examined the correlation between CD169 and CD8 expression, and performed Cox regression analysis of potential prognostic factors, including CD169 and CD8 expression, for cancer-specific survival (CSS) in patients with total and advanced gastric cancer. CD169 overexpression in lymph node sinus macrophages (LySMs) was positively correlated to the density of CD8-positive lymphocytes in primary cancer tissues (R = 0.367,  Gastric cancer is one of the most common cancers, with about 865,000 patients with this disease dying worldwide each year . VariousLymph nodes, as immune organs, play an important role in the induction of specific immune responses to cancer , 8. VariCD169, also called sialoadhesin, is the foremost member of the sialic acid-binding lectin (Siglec) superfamily . It bind+ T-cell infiltration in primary lesions. We also examined the relationship between CD169 expression and various clinicopathological factors.No studies have yet examined the role of LySMs in patients with gastric cancer. In the present study, we aimed to elucidate the association between CD169 LySMs and prognosis in gastric cancer, including its pathological stage and subgroups: histology, tumor-stroma ratio, distant metastasis, and LN metastasis. In order to determine the significance of CD169 expression in patients with gastric cancer, we used tissue specimens to investigate the correlation between CD169 expression on LySMs and CD8We conducted a retrospective analysis in accordance with the Declaration of Helsinki and with the approval of the Hospital of the University of Environmental and Occupational Health (UOEH), and Wakamatsu Hospital of UOEH (H30-172). The present study utilized paraffin-embedded specimens of primary lesions and regional lymph nodes (RLNs) resected from 294 patients with gastric cancer who had undergone surgery at the Hospital of the UOEH from 2008 to 2012 and Wakamatsu Hospital of the UOEH from 2011 to 2012. We excluded patients who died from non-primary cancer causes or patients who were no longer available for follow-up within a year after surgery. We also excluded cases in which the lymph nodes were difficult to evaluate and in which no lymph node resection had been performed.2 total area) for TSR evaluation, and excluded areas with necrosis or mucin deposition. TSR evaluation was also conducted by two or three pathologists  who were blinded to clinical outcomes.Samples from 294 gastric cancer cases were stained with hematoxylin and eosin (HE) and evaluated histopathologically by two or three pathologists  who were blinded to clinical outcomes. As previously published, the tumor-stroma ratio (TSR) was calculated as the percentage of stroma relative to tumor area; tumors were subgrouped as having a high (>50%) or low TSR –26. We s+ and CD68+ cells in RLNs and CD8+ cells in tumors, we used the HALO 2.3 system . We selected four random fields  from the primary tumor to count CD8+ T cells. To count CD68+ and CD169+ macrophages in RLNs, we delineated the RLN sinus with a line in two to four fields  in serial sections. After counting cells and measuring these areas, we calculated the density of CD8+ T cell infiltration into the tumor, and the ratio of CD169+ cells in CD68+ LySMs. For double-IHC, HistoGreen substrate  was used for peroxidase-based immunostaining.Tumor tissues and RLNs were fixed in 10% neutral formalin and embedded in paraffin. Anti-CD169 , anti- CD68 , and anti-CD8  antibodies were used as primary antibodies for immunohistochemistry (IHC). Antigen retrieval and IHC were performed as previously published . Lymph n+ to CD68+ LySMs were performed using a χ2-test. The relationship between two numerical factors was analyzed using Spearman’s correlation analysis. The association of multiple prognostic factors with cancer-specific survival was assessed using univariate and multivariate Cox proportional hazard model analysis. Multivariate analysis included age, sex, histology, depth of invasion, LN metastasis, distant metastasis, lymphatic invasion, vascular invasion, tumor-stroma ratio, density of CD8+ T cells, and the ratio of CD169+ to CD68+ LySMs. Survival curves were calculated using the Kaplan-Meier method, and the difference between survival curves was analyzed using the log-rank test. Differences were considered statistically significant at P-values of < 0.05.We carried out statistical analyses using SPSS 25 software . Bivariate comparisons of clinicopathological features between patients with high (n = 135) and low (n = 159) ratios of CD1692 . The ratio of CD169+ to CD68+ cells ranged from 0.00% to 132.78%  indicated a significant association between CD169 expression in LySMs and anti-cancer immune responses. These findings are consistent with our previous research findings in colorectal cancer, melanoma, esophageal cancer, and bladder cancer , 28–30. Immune checkpoint blockade targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) has become a promising approach for anti-cancer immunotherapy . The ATThigh group had greater CSS than the CD169low group in the TSR high group. Because the stromal component of tumors has been proven to have a significant impact on tumor development  and Takeda Science Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this study was to identify clinical risk factors for COVID-19 in a German outpatient fever clinic that allow distinction of SARS-CoV-2 infected patients from other patients with flu-like symptoms.th of April 2020 to 15th of May 2020. Symptoms, comorbidities, and socio-demographic factors were recorded in a standardized fashion. Multivariate logistic regression was used to identify risk factors of COVID-19, on the bases of those a model discrimination was assessed using area under the receiver operation curves (AUROC).This is a retrospective, single-centre cohort study. Patients were included visiting the fever clinic from 4The final analysis included 930 patients, of which 74 (8%) had COVID-19. Anosmia  and ageusia  were strongly associated with COVID-19. High-risk exposure , especially in the same household , was also correlated; the more household members, especially with flu-like symptoms, the higher the risk of COVID-19. Working in an essential workplace was also associated with COVID-19 , whereas smoking was inversely correlated . A model that considered risk factors like anosmia, ageusia, concomitant of symptomatic household members and smoking well discriminated COVID-19 patients from other patients with flu-like symptoms (AUROC 0.84).We report a set of four readily available clinical parameters that allow the identification of high-risk individuals of COVID-19. Our study will not replace molecular testing but will help guide containment efforts while waiting for test results. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the virus that causes coronavirus disease 2019 (COVID-19), rapidly spread all over the globe. The diagnosis primarily relies on molecular techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR) or antigen detection from nasopharyngeal swabs. Especially RT-PCR test results, considered as the gold standard, are often delayed due to transport, slow turnaround times and the requirement of a centralized laboratory .Several studies on prediction models have been performed, but most of them focused on predictors for patients in a hospital setting –6. HowevHere we performed a study in the local fever clinic in Stuttgart, Germany, focusing on mild to moderate outpatient cases of COVID-19. Our study aimed to identify parameters that are readily available in an outpatient setting and help to identify patients with a high risk of SARS-CoV-2 infection.Stuttgart is the capital and largest city of the German state of Baden-Wuerttemberg with a population of around 635,000 people. Unlike other fever clinics which are associated with local hospitals, this fever clinic of Stuttgart was not part of a hospital but rather established by the local health authorities.th of April 2020 to 15th of May 2020. Patients aged 18 or older were either self-referred or sent by their general practitioner in case they had common respiratory symptoms compatible with COVID-19 and/or a confirmed contact to a COVID-19 case.This single-center retrospective cohort study included all patients visiting this fever clinic from 4Data on the type of symptoms, symptom onset, demographics, medical comorbidities, medication, predisposing risk factors, exposure and self-rated health were recorded via a standardized questionnaire used by the treating physician. The questionnaire was introduced in the fever clinic independently of this study to harmonize the workflow. It is shown in Self-rated health was recorded on a scale from one (excellent) to five (poor). Missing data was stated as such. The missing data analysis is shown in Smoking was recorded in pack years (PY). If PY were missing, the number of cigarettes per day was documented. We created two categories for mild to moderate (≤5cig./day or ≤15PY) and heavy (>5cig./day or >15PY) smoking.Essential workers (EW) were defined as people working in critical infrastructure such as retail, health care or police. The definition of contact persons followed the definitions by the Robert Koch Institute responsible for disease control and prevention. High-risk exposure is defined as contact to a COVID-19 case for more than 15 minutes and within distances of 1.5 meters or less without personal protective equipment (PPE). Low-risk exposure was defined as contact in a closed environment with a COVID-19 case for less than 15 minutes or at a distance more than 1.5 meters also without PPE. Exposure as a health care worker (HCW) providing care to a COVID-19 case wearing recommended PPE was looked at independently.SARS-CoV-2 testing was performed on nasopharyngeal swabs via RT-PCR on the Cobas 6800 system . As obliged by the German Infection Protection Act (Infektionsschutzgesetz), patients with suspected or confirmed COVID-19 were reported to the local health department.The study was approved by the ethics committee of the Landesärztekammer Baden-Württemberg, Stuttgart, Germany (vote F-2020-067). Because of the retrospective and anonymized approach, the need for informed consent was waived by the local ethics committee.Continuous data was expressed as mean, median and interquartile range (IQR) while categorical variables were reported as number (n) and percentage (%). Statistical differences across positive/negative SARS-CoV-2 patients were determined using Student’s t-test for continuous variables. Chi-squared test or Fisher’s exact test were performed for categorical variables. Chi-Squared test was used when all cells had at least ten observations. Otherwise, the Fisher’s exact test was applied. Wilcoxon rank-sum test was used to compare BMI between groups. SARS-CoV-2 test result (positive / negative) was used as the dependent variable in logistic regression. Candidate risk factors were entered first individually to assess their relationship with positive SARS-CoV-2 tests. Multivariate modelling was informed both by prior knowledge of the risk factors as well as the results of bivariate models. Logistic regression results are reported as odds ratios (OR) with 95% confidence intervals (95% CI). Reported p-values are 2-tailed, with P≤0.05 being considered statistically significant. Lastly, area under the receiver operating characteristics (AUROC) was used to examine the value of the models discriminating between patients with positive and negative SARS-CoV-2 tests.The flow diagram was created using LibreOffice Draw . Visualization of data was performed using GraphPad Prism . Regressions were modelled using STATA 13 .A flow diagram showing the study population selection is shown in 2; p-value = 0.0165). At least one fifth of the patients in both groups had one comorbidity or more. However, no difference was seen for comorbidities or medication intake.Patient characteristics and demographics are summarized in While most symptoms did not allow any discrimination between SARS-CoV-2 positive and negative patients, some were identified as predictors for COVID-19 after adjustment for age and gender . These wPatients with COVID-19 had more often subfebrile temperatures (37.5–37.9°C) than patients without COVID-19. This was valid for the measured temperature at home  as well as the temperature measurement at the fever clinic . No difference between groups was found for oxygen saturation , systolic  or diastolic blood pressure  nor heart rate .The results are shown in Anosmia and ageusia  had suboptimal performance as sole predictors of COVID-19 (AUROC 0.67–0.74). The same is true for high-risk exposure (AUROC 0.74). The best multivariable model was achieved when age, gender, anosmia, ageusia, smoking status and contact to symptomatic household members were included as parameters. This model achieved an excellent performance with an AUROC of 0.84 . The modThere is an expanding access to fast and reliable laboratory tests for the diagnosis of COVID-19. However, it is still a challenge in emergency departments or in other outpatient settings to decide which patient with flu-like symptoms requires instant isolation and contact tracing.In our study, we identified several predictors for COVID-19 in outpatient setting. We also developed a model that solely depends on information that can be gathered without invasive procedures.Sun et al. published prediction models based on cases at a tertiary hospital in Singapore . Their mInterestingly, smokers are at a reduced risk of SARS-CoV-2 infection in our study, an observation that was also confirmed by other groups . The reaBMI was also identified as a predictor for COVID-19, which falls in line with the published literature, linking obesity to respiratory infections as a general and to COVID-19 , 18. LikUnsurprisingly, the more intense the exposure to COVID-19 cases, the higher the risk for infection. We show that the definition of contact persons of the European Centre for Disease Prevention and Control (ECDC), which was adopted by the German Robert-Koch-Institute, is helpful in determining the associated risk of infection . High-riWe show that symptoms and history of exposure are the strongest predictors for COVID-19. Still, after adjusting for these factors, it was possible for the treating physician to identify individuals with COVID-19 more often as would have been possible by chance. Therefore, as good as a prediction model might theoretically be, it will never replace molecular testing and the practical knowledge of an experienced physician.The strength of our study is the large cohort and the agreement of our data with published studies, proofing to be a reliable data set despite the retrospective approach. Our predictors and model depend solely on non-invasive parameters, allowing for quick identification of individuals at risk.The retrospective approach is a limitation to our findings. The accuracy of the data depends on meticulous documentation in times with a high level of stress during the beginning of the pandemic. Reporting bias is another limiting factor since some patients may have exaggerated their symptoms to receive a test. Furthermore, in some subgroups there were only a limited number of events  and multiple subgroup analysis increases the likelihood of a statistically significant false positive result.In summary we identified that anosmia, ageusia, history of exposure, as well as smoking and BMI are strong predictors of COVID-19 in an outpatient setting. Especially the strong inverse correlation of smoking with COVID-19 warrants further research. Identifying the underlying mechanism might help to find preventive mechanisms.In times of scarce hospital beds and over-crowded emergency departments and family practices, the identified predictors of COVID-19 provide help in the decision process for whom isolation and contact tracing is warranted.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


This study aimed to assess the baseline characteristics and clinical outcomes of coronavirus disease 2019 (COVID-19) in pediatric patients with rheumatic and musculoskeletal diseases (RMD) and identify the risk factors associated with symptomatic or severe disease defined as hospital admission, intensive care admission or death.An observational longitudinal study was conducted during the first year of the SARS-CoV-2 pandemic (March 2020–March 2021). All pediatric patients attended at the rheumatology outpatient clinics of six tertiary referral hospitals in Madrid, Spain, with a diagnosis of RMD and COVID-19 were included. Main outcomes were symptomatic disease and hospital admission. The covariates were sociodemographic and clinical characteristics and treatment regimens. We ran a multivariable logistic regression model to assess associated factors for outcomes.p = 0.00). No statistically significant risk factors for symptomatic COVID-19 were found in the final model.The study population included 77 pediatric patients. Mean age was 11.88 (4.04) years Of these, 30 patients (38.96%) were asymptomatic, 41 (53.25%) had a mild-moderate COVID-19 and 6 patients (7.79%) required hospital admission. The median length of hospital admission was 5 (2–20) days, one patient required intensive care and there were no deaths. Previous comorbidities increased the risk for symptomatic disease and hospital admission. Compared with outpatients, the factor independently associated with hospital admission was previous use of glucocorticoids (OR 3.51; No differences in COVID-19 outcomes according to childhood-onset rheumatic disease types were found. Results suggest that associated comorbidities and treatment with glucocorticoids increase the risk of hospital admission. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection causes COVID-19 disease, which may present with a myriad of clinical symptoms and signs  vs 12 [9.5–15]) than asymptomatic ones, without significant differences. No patients were diagnosed with the pediatric multisystemic inflammatory syndrome. Overall, 81.52% of patients maintained all their immunosuppressive treatment without changes. Around 20% of patients went to the emergency room due to their symptoms.Patients with multisystemic autoimmune disorders were more frequently symptomatic due to COVID-19 (71.4% with symptoms), followed by those with autoinflammatory diseases (66.6%). However, there were no significant differences between RMD groups regarding symptomatic disease.As shown in Table The only patient who required admission to a Pediatric Intensive Care Unit had a recent diagnosis of systemic JIA, was on treatment with Canakinumab and glucocorticoids (> 10 mg prednisone/day), and had associated comorbidities. This patient developed pneumonia and a pleural effusion and was treated with antibiotic therapy and lopinavir/ritonavir. A catheter-associated right iliac vein appeared as a probably multifactorial complication. A few days later The patient’s condition improved after a few days and the recovery was complete at discharge.No laboratory or imaging tests were performed for asymptomatic and mildly symptomatic patients and none received specific treatment for COVID-19.p = 0.95) or synthetic ]), and exposure to glucocorticoids ), did not achieve statistical significance and was not analysed in the final model. Although, long-term treatment with antimalarials seemed to be protective, significance was not demonstrated in the final model .  or synthetic -, did not reach statistical significance in the final model. Comorbidities were not analysed in the final model .  of pediatric patients with RMD and COVID-19 had symptomatic infections. Fever, headache and cough were the predominant clinical features at presentation, with similar rates to those observed in a recent international, multicenter study of pediatric COVID-19 .Presence of comorbidities was associated with an increased likelihood of symptomatic COVID-19 in patients with RMD, although under 8% of the patients required hospital admission.Data from this study also reflect the uncertainties regarding treatment options for COVID-19 with different protocols emerging as knowledge of the disease progressed. In general, admitted patients received few experimental treatments for COVID-19 and in almost all cases symptoms resolved without serious or chronic complications. Patients exposed to disease-modifying agents did not appear to be at higher risk of severe disease or hospital admission due to COVID-19. However, results suggest that pediatric patients with RMD and chronic prior exposure to glucocorticoids have a greater risk of hospital admission, regardless of other factors. Although the number of patients in this study was limited, results are similar to data reported in adult patients with RMD and COVID-19 , 11. IntThe possibility of a less favorable outcome of COVID-19 in patients taking rituximab has recently been discussed . In thisAlthough available data have shown that COVID-19 in children with RMD undergoing immunomodulatory treatment is not usually associated with severe disease, conventional clinical practice includes temporary interruption of immunomodulatory treatment when there is a concomitant infection . In thisThe type of RMD seems to play an important role in the probability of hospital admission in adult patients and COVID-19, and patients with systemic autoimmune conditions seem to have the highest risk compared to patients with chronic inflammatory arthritis , 18. HowNo patients who required admission developed associated complications, except for an adolescent female with systemic lupus erythematosus and secondary antiphospholipid syndrome who had an episode of deep vein thrombosis of a lower limb. Thrombotic complications seem very uncommon in children with SARS-CoV-2, however, in adolescents with previous thrombotic risk factors thromboprophylaxis must be considered .A large majority of children with asymptomatic disease were in remission. This reflects the importance of keeping the disease under control to avoid symptomatic COVID-19. Patients with high or moderate disease activity may be at greater risk for infections, including SARS-CoV-2.The strengths of the study include the comprehensive analysis of pediatric patients with RMD and COVID-19 during the peak of the pandemic. All data were entered by the patients´ own rheumatology healthcare providers, the registry collects information on specific RMD diagnoses and includes cases from the major pediatric rheumatology units in Madrid suggesting that these findings may be more representative than single-centre studies..However, the COVID-SORCOM registry is voluntary and does not capture all cases of COVID-19 in pediatric patients with RMD. This approach to data collection places limitations on causal conclusions and temporal relationships and therefore only limited inferences may be made based on these results. Due to the database design and inherent reporting bias the data may not be used to analyse the incidence of COVID-19 in this patient population.Although the limited sample size makes this a descriptive study with exploratory analyses, detailed information on this population may be obtained. Preliminary results are in line with data published in the adult population with RMD, and may assist with the decision-making process and patient monitoring. Although further studies are required, children with RMD and/or receiving immunomodulatory therapies do not appear to have a significantly increased risk for severe COVID-19 and preventive measures should be similar to those recommended for the general population , 25.In conclusion, overall outcomes in children and adolescents with RMD appear to be generally favorable, and most patients have non-severe infections. Rarely, COVID-19 may be severe in children and comorbidities and the use of glucocorticoids could be considered risk factors in the RMD pediatric population as well as in adults.Overall, no differences in COVID-19 outcomes in pediatric patients according to type of childhood-onset rheumatic disease were found. Some factors may increase the likelihood of complicated symptomatic infection and hospital admission. In this clinical registry, results were in concordance with data reported elsewhere in adult patients with RMD and COVID-19, and special caution should be exerted in patients with thrombotic risk, with comorbidities or with previous exposure to corticosteroids. This study provides novel results in childhood-onset rheumatic disease patients regarding susceptibility to moderate-severe COVID-19.


Klebsiella pneumoniae (pdd-CRKP) infections on KT recipients.Infections remain a major cause of morbidity and mortality in kidney transplant (KT) recipients. This study aimed to investigate the preservation fluid (PF) samples from deceased donors and report the impacts of possible donor-derived carbapenem-resistant p values was set at 0.05.A retrospective study was performed that included all recipients who received kidney transplantation from deceased donors in our hospital between December 2018 and December 2020. A total of 212 patients received kidney transplantation from deceased donors, a total of 206 PF samples were collected, and 20 recipients had a CRKP-positive culture. Both donors and recipients with CRKP-positive PF cultures were divided into two groups, and continuous variables between the two groups were compared using independent-sample t tests and Mann-Whitney tests. Categorical variables were compared using the chi-square test or Fisher’s exact test. The significance level of P < 0.05). Fifteen recipients were infected with pdd-CRKP, and the incidence of pdd-CRKP infection was 7.3% (15/206). The use of antibiotics, including ceftazidime-avibactam (CAZ-AVI), was a potential protective factor against death and graft loss in recipients with a CRKP-positive PF culture (P < 0.05).A total of 337 recipients underwent kidney transplantation, including 212 recipients of organs from deceased donors and 110 corresponding deceased donors. A total of 206 PF samples were collected, and 20 recipients had CRKP-positive PF cultures. The donors’ length of ICU stay was a potential risk factor for CRKP positivity in the PF culture (This study shows that the incidence of pdd-CRKP is high in our centre, recipients with pdd-CRKP infection can still achieve a good prognosis with the use of antimicrobial agents including CAZ-AVI.The online version contains supplementary material available at 10.1186/s12882-022-02733-7. Klebsiella pneumoniae (CRKP) . Continuous variables with a normal distribution are described as the means and standard deviations; otherwise, they are represented as the medians and interquartile ranges (IQRs). The Kolmogorov-Smirnov test was used to assess variable distributions. Independent-sample t tests were used to compare quantitative variables with a normal distribution between groups. When quantitative variables did not follow a normal distribution, the Mann-Whitney test for nonparametric variables was used for comparisons between groups. Categorical variables are presented as frequencies and percentages. The chi-square test or Fisher’s exact test was used for the comparison of categorical variables between groups, as appropriate. For all tests, A total of 337 recipient underwent kidney transplantation between December 2018 and December 2020, including 212 recipients of organs from deceased donors and 110 corresponding deceased donors. A total of 206 PF samples were collected, and 20 recipients had CRKP-positive PF cultures. The incidence of death or graft loss in recipients with CRKP-positive PF cultures was 6/20(30.0%), and the incidence of death or graft loss in recipients with CRKP- negative PF cultures was 15/186(8.1%).P < 0.05), but there were no significant differences in sex, age, cause of death, China classification of donation or warm ischaemia time. In both groups of donors, brain trauma was the most common cause of death, followed by cerebrovascular accidents. The vast majority of donors were classified as China Category III.During the study, twenty CRKP-positive recipient kidneys derived from 11 deceased donors were cultured in PF. The characteristics of the donors are shown in Table P < 0.05), suggesting that the use of antibiotics, including CAZ-AVI, was a potential protective factor against graft loss or death in recipients. Due to the limited sample size, univariate and multivariate analyses could not be performed. The characteristics of the recipients are shown in Table A total of 20 recipients were included in the study, with an average age of 44.3 years, 15 of whom were male. The incidence of pdd-CRKP infection was 7.3% (15/206). The most common aetiology of kidney failure was glomerulonephritis, and 15 recipients developed pdd-CRKP infection. The drug sensitivity profiles of CRKP cultured in the recipients were the same as those in the PF, and the infections occurred shortly after the operation, with an average time of 7.7 days. The difference between the two groups in the use of antibiotics, including ceftazidime-avibactam (CAZ-AVI), was statistically significant  ICU stays, the use of vasopressors, and the need for cardiopulmonary resuscitation were reported to be independent risk factors for predicting potential infections in donors [In our study, a 7.3% (15/206) incidence of pdd-CRKP infection was associated with PF, and the risk of total infection events due to PF contamination ranged from 0.83 to 14.3% in previous reports , 33. Hown donors . Studiesn donors . In our n donors . MicrobiKlebsiella pneumoniae, MDR Acinetobacter baumannii and other bacteria are more likely to cause DDIs associated with PF than other sensitive Cocci species [Whether microbial contamination of PF in SOT is related to DDI is controversial. Bertrand et al. reported a negligible incidence of clinical complications due to PF contamination . However species . Cai et  species , which wMost of the DDIs associated with PF were surgical site infections, including local infection of the graft site, infectious renal graft artery rupture and wound infection , which wDue to the lack of evidence, there is no broad consensus regarding the treatment recipients with of CRKP-positive cultures in PF. Some authors have suggested that when MDR is detected in donors and PF, targeted antibacterial treatment should be performed after transplantation, which could prevent the transmission of MDR gram-negative bacteria. DDIs have been reported despite antibiotic prophylaxis , 42. TheTest protocols for the rapid detection of pathogens carried by donors await further study. The time required for routine microbial culture in PF from specimen collection to final results (including drug susceptibility results) may range from 48 h to 120 h, depending on the testing time and the need for additional resistance testing. This delay affects the time to start appropriate antimicrobial therapy. Although the polymerase chain reaction method can quickly obtain test results, it is expensive, has limited specificity and cannot provide information about the sensitivity of the drug. Delay in communication is another important factor in the transmission of infection . Rapid cThe study has several limitations. First, the lack of routine microbiological screening of donors made it difficult to determine the source of CRKP and the relationship between donor infection and donor-derived CRKP infection after transplantation. Second, there were no data on the use of antibiotics given to donors. Third, regarding the transmission of DDI, genotyping technology provides necessary objective evidence for this event. Unfortunately, this technology is not widely used in our hospital. Therefore, our detection algorithm may overestimate transmission events. Finally, our sample size was small, mainly because of the low incidence of donor-derived CRKP infection, and our results may not be applicable to other transplant centres.In summary, this study showed that the incidence of pdd-CRKP is high in our centre. Recipients with pdd-CRKP infection can still achieve a good prognosis with the use of antimicrobial agents including CAZ-AVI and donor screening for CRKP should be included in measures to contain the incidence of DDI.Additional file 1.Additional file 2.


Clostridioides difficile infection (HCA-CDI). The validated Davis risk score (DRS) indicates that patients with a DRS ≥ 6 are at an increased risk of 30-day HCA-CDI. In the phase 3 OPTIC CABP study, 14% of CABP patients with DRS ≥ 6 who received moxifloxacin developed CDI vs. 0% for omadacycline. This study assessed the potential economic impact of substituting current guideline-concordant CABP inpatient treatments with omadacycline in hospitalized CABP patients with a DRS ≥ 6 across US hospitals. Methods: A deterministic healthcare-decision analytic model was developed. The model population was hospitalized adult CABP patients with a DRS ≥ 6 across US hospitals . In the guideline-concordant arm, 14% of CABP patients with DRS ≥ 6 were assumed to develop an HCA-CDI, each costing USD 20,100. In the omadacycline arm, 5 days of therapy was calculated for the entire model population. Results: The use of omadacycline in place of guideline-concordant CABP inpatient treatments for CABP patients with DRS ≥ 6 was estimated to result in cost savings of USD 55.4 million annually across US hospitals. Conclusion: The findings of this simulated model suggest that prioritizing the use of omadacycline over current CABP treatments in hospitalized CABP with a DRS ≥ 6 may potentially reduce attributable HCA-CDI costs. The findings are not unique to omadacycline and could be applied to any antibiotic that confers a lower risk of HCA-CDI relative to current CABP inpatient treatments.Introduction: Approximately 3% of hospitalized patients with community-acquired bacterial pneumonia (CABP) develop healthcare-associated  Clostridioides difficile (CDI), a common adverse effect associated with use of board-spectrum antibiotic therapy [Infections due to  therapy ,4,5,6,7, therapy . The occ therapy . It is a therapy ,11. Due  therapy  and is p therapy . In rece therapy ,15. Howe therapy ,3,4,5,6, therapy , such asCommunity-acquired bacterial pneumonia (CABP) is a common infection type where there is a potential opportunity to risk stratify patients based on CDI risk and prioritize use of lower CDI risk antibiotics in high CDI risk patients. Data suggest that there are approximately 1 million CABP admissions per year in the United States of America  and apprOne potential alternative to the current guideline-concordant inpatient CABP treatments that has a lower CDI-risk potential is omadacycline. Omadacycline is approved by the FDA for the treatment of adult patients with CABP and has demonstrated a low propensity to induce CDI in preclinical and clinical studies relative to current guideline-concordant inpatient CABP agents ,23,24,25A deterministic framework from the US hospital perspective was used to develop the conceptual healthcare-decision analytic model. The model population was hospitalized adult CABP patients with a DRS ≥ 6 . In the For the current guideline-concordant CABP treatment scenario, it is estimated that 14% of patients with a DRS ≥ 6 would develop an HCA-CDI. The 14% rate of CDI among patients with a DRS ≥ 6 in the guideline-concordant CABP treatment scenario was based on the observed CDI incidence among patients who received moxifloxacin in phase 3 OPTIC CABP study ,25. For Only excess  hospital-related costs for the first HCA-CDI episode in CABP guideline-concordant group and drug acquisition costs in the omadacycline group were considered in the model. HCA-CDI was the only adverse event included in the model as the incidence of other adverse events was found to be similar between omadacycline and moxifloxacin in the OPTIC study. Initial hospital costs were assumed to be equal between treatment scenarios, regardless of treatment assignment. The treatment duration for both groups was assumed to be 5 days . The druTotal attributable costs associated for each treatment scenario and incremental cost differences between treatment scenarios were determined for the base-case analysis. In the CABP guideline-concordant inpatient treatment arm, the attributable HCA-CDI costs for the 14,000 predicted CABP patients with CDI were calculated. In the omadacycline arm, the cost of 5 days of treatment for the estimated 100,000 CABP patients with DRS ≥ 6 was determined.Second-order, probabilistic, parameter sample sensitivity analyses were performed to assess the effect of varying the attributable costs associated with an HCA-CDI. These variables were selected because they were the most influential variables within the structural model. A triangular distribution was applied to capture the variance surrounding this variable. The minimum and maximum values for the hospital costs associated with development of an HCA-CDI were set at USD 10,900 and USD 29,300, respectively [One-way sensitivity analyses were performed to determine the absolute difference in HCA-CDI rates per 100 treated CABP patients with a DRS ≥ 6 that still resulted in cost saving with omadacycline relative to the guideline-concordant standard of care agents (dominance threshold). Like the primary analysis, the only costs included in the guideline-concordant standard of care scenario were the attributable HCA-CDI costs among those who experienced HCA-CDI. The only costs included in the omadacycline scenario were the cost of 5 days of IV omadacycline for the 100 treated CABP patients with a DRS ≥ 6. The attributable HCA-CDI cost from the AHRQ meta-analysis of USD 20,100  was usedTo capture the range of attributable costs associated with an HCA-CDI, the range of values identified in the literature were also evaluated. For the lower end, USD 10,860 (inflated to 2021 US dollars) was used as it represented the lower end of attributable costs for HCA-CDI by Gabriel and colleagues . This loThe total attributable costs associated for each treatment scenario and incremental cost differences between treatment scenarios that were determined in base-case analysis are shown in Results of the one-way sensitivity analyses to determine the absolute difference in attributable CDI rates between groups per 100-treated CABP patients with a DRS ≥ 6 that still resulted in cost saving with omadacycline is shown in There are 1) well-documented concerns with HCA-CDIs ,10,11,  well-doc. Based oSeveral items should be noted when interpreting these findings. Findings are not unique to omadacycline and could be applied to any antibiotic that confers a lower risk of CDI relative to current CABP inpatient treatments ,40. We fThe major drivers of cost in the model were the attributable HCA-CDI hospital costs  and currC. difficile antibiotics, advanced age, and use of antacid medications places patients at an increased risk for recurrence [The reported value of omadacycline relative to guideline-concordant inpatient CABP therapies in CABP patients with DRS ≥ 6 were likely highly conservative and did not capture the full range of costs associated with the acquisition of an HCA-CDI. The model was limited to the attributable HCA-CDI hospital costs associated with the first CDI episode. Data suggest that the attributable healthcare may extend the first HCA-CDI hospitalization ,42,43,44currence  and thescurrence ,21. Attrcurrence ,38,41,44currence ,10,11,38currence . There are also other aspects of these analyses that suggest the observed study results were highly conservative. The model was constructed from the hospital perspective and mortality was not incorporated into the model . Each CDIn conclusion, the hospital costs attributable to primary HCA-CDI and recurrent CDI are substantial ,29,37,44


Developing positive learning experiences in team-based geriatric care is challenging. This presentation will highlight an inter-University geriatric case competition for developing interprofessional competencies in health professional students sponsored by the Geriatric Workforce Enhancement Programs at Saint Louis University and the University of Minnesota. The virtual competition involved teams of 4-5 undergraduate and graduate students from multiple health professions who designed a comprehensive care plan using a simulated complex geriatric patient case. Students were assigned to an interprofessional team with a faculty or community expert coach, attended an orientation, and developed a 20-minute recorded presentation. A panel of judges rated team presentations using a scoring rubric based on the Core Competencies for Interprofessional Collaborative Practice. Local competitions included a first and semi-final round, with the winning teams presenting at the inter-university competition held via live videoconferencing that involved a question-and-answer session. Prizes were given to the top teams. Students, coaches, and judges completed evaluation surveys focused on satisfaction with the competition format/procedures and achievement of interprofessional competencies. Twenty-one teams and 117 students from 12 disciplines participated. Team scores ranged from 2.2 to 4.3  on a 1-5 scale. Judge, coach, and student evaluations were positive, indicating students learned valuable lessons in group dynamics, team-based care, and geriatric care. Most students (82%) preferred the virtual competition format or had no preference on format. The virtual case competition provided a positive, engaging experience to introduce health professional students to geriatric team-based care and develop their readiness for collaborative practice.


Anal cancer, despite being a rare malignancy, is increasing in incidence, accounting for 0.5% of all new cancer cases in the United States, with rate of new cases being 1.9 per 100,000 men and women. It is common in immunocompromised individuals, especially those with malignancy, human immunodeficiency virus (HIV) and human papillomavirus (HPV) infection. Despite similar treatment of anal cancer in both HIV-positive and negative patients, guidelines for prevention and treatment of therapy-related side effects are rarely studied. While these patients have a better prognosis on HAART, limited guidelines exist regarding appropriate therapy. There is a common link between HPV and HIV and the transmission of one is associated with increased risk of transmission of the other. HPV vaccine which is known to prevent high-grade cervical intraepithelial neoplasia is thought to also decrease the incidence of anal intraepithelial neoplasia. The association of HPV vaccine in the prevention of anal cancer in high-risk groups with HIV is a scarcely studied subject that requires further research. Anal cancer is a rare malignancy, accounting for 0.5% of all new cancer cases in the United States, with rate of new cases being 1.9 per 100,000 men and women [The incidence of anal cancer has been steadily rising over the years, between 2001 and 2015 anal cancer cases rose by 2.7% each year [Major risk factors associated with an increased incidence of anal cancer include female gender, human papillomavirus infection (HPV), human immunodeficiency virus infection (HIV), multiple sexual partners, receptive anal intercourse, history of cervical, vulvar or vaginal cancer, immunosuppression associated with solid organ transplantation, hematological malignancy, autoimmune disease and smoking -8.With the rise of HIV, HPV infections, and anal cancer, in this article, we aim to provide a comprehensive review of their associations, current treatment modalities and highlight areas for future research.PathologyThe anal canal ranges from the perianal skin, or anal verge, to the rectal mucosa. Squamous mucosa lines the anal canal from the distal-most anal verge up to the dentate or the pectinate line, following which is a transition zone consisting of both squamous and nonsquamous  mucosa . Tumors Associations between HPV, HIV and anal cancerAnal cancer incidence rates are 30 times higher in HIV-infected individuals compared to the general population . In a laThe correlation between HIV and anal cancer is difficult to understand, due to the confounding factors. A systematic review of 95 studies was done by Lin et al., which included studies published from January 1986 to July 2017 in MEDLINE, Embase, and the Cochrane Library on anal HPV infection . It showTreatmentHistorically, anal cancer was treated by abdominoperineal resection (APR) with permanent colostomy. This changed when a landmark trial by Nigro et al. proved the success of radiotherapy (RT) with chemotherapy alone in complete pathological cure . The medThe widespread use of antiretrovirals started around 1999-2000. These drugs control HIV proliferation and amplification. Along with a high CD4 count, being on HAART leads to favorable outcomes in HIV patients. Stadler et al. compared pre-HAART group of HIV-positive individuals receiving therapy for anal cancer with those receiving HAART and reported a marked difference in their 24-month survival- 17% for pre-HAART group versus 67% for those on HAART. The mean CD counts were 190 and 255, respectively, in the pre-HAART and HAART treatment groups. Of note, all six patients in the pre-HAART group passed away with SCC versus only two in the HAART group. Four out of six remaining in the HAART group were disease-free on all follow-up visits . Other sOf note, an appropriate choice of antiretrovirals is also imperative. Recommended initial regimens include two nucleoside reverse transcriptase inhibitor  and a third single/boosted drug which is either an integrase strand transfer inhibitor , a non-nucleoside reverse transcriptase inhibitor  or a boosted protease inhibitor . As per the 2014 recommendations of the International Antiviral Society-USA panel, dolutegravir- or raltegravir-based regimens are to be used in the setting of anticancer and immunosuppressive drug use . ZidovudTable Management of adverse drug reactionsMajor toxicities resulting from the treatment of anal cancer are diarrhea, skin desquamation and immunosuppression. HIV-positive patients receiving treatment for anal cancer experience higher toxicities and require more frequent and longer treatment breaks than their HIV-negative counterparts. This effect is further exacerbated by lower CD counts and untreated HIV . Dermal HPV vaccine in anal cancerThe causal relationship between HPV infection and anal cancer is well established . The quaAnal cancer is common in HIV-positive patients. Treatment in HIV-positive patients is similar to HIV-negative patients but is associated with increased toxicity and needs careful monitoring. Patients on HAART have a better prognosis compared to those not on HAART. Effect of HPV vaccine on incidence of anal cancer and interaction with HIV needs further research.


Escherichia coli commonly evolved minority mucoid phenotypes in response to predation by the bacterial predator Myxococcus xanthus by one time point of a coevolution experiment now named MyxoEE-6. Here we examine mucoid frequencies across several MyxoEE-6 timepoints to discriminate between the hypotheses that mucoids were increasing to fixation, stabilizing around equilibrium frequencies, or heading to loss toward the end of MyxoEE-6. In four focal coevolved prey populations, mucoids rose rapidly early in the experiment and then fluctuated within detectable minority frequency ranges through the end of MyxoEE-6, generating frequency dynamics suggestive of negative frequency-dependent selection. However, a competition experiment between mucoid and non-mucoid clones found a predation-specific advantage of the mucoid clone that was insensitive to frequency over the examined range, leaving the mechanism that maintains minority mucoidy unresolved. The advantage of mucoidy under predation was found to be associated with reduced population size after growth (productivity) in the absence of predators, suggesting a tradeoff between productivity and resistance to predation that we hypothesize may reverse mucoid vs non-mucoid fitness ranks within each MyxoEE-6 cycle. We also found that mucoidy was associated with diverse colony phenotypes and diverse candidate mutations primarily localized in the exopolysaccharide operon yjbEFGH. Collectively, our results show that selection from predatory bacteria can generate apparently stable sympatric phenotypic polymorphisms within coevolving prey populations and also allopatric diversity across populations by selecting for diverse mutations and colony phenotypes associated with mucoidy. More broadly, our results suggest that myxobacterial predation increases long-term diversity within natural microbial communities.Predator impacts on prey diversity are often studied among higher organisms over short periods, but microbial predator-prey systems allow examination of prey-diversity dynamics over evolutionary timescales. We previously showed that  Predation is one of the most common forms of inter-specific antagonism . Under pBacteria fall prey to a wide variety of predators, including unicellular eukaryotes , amoebaeLong-term coevolution studies have examined the emergence and subsequent evolution of adaptive defensive traits of bacteria under attack by phage ,16,17, bMyxobacteria are soil- and sediment-dwelling bacteria that kill and consume diverse other microbes, including both Gram+ and Gram– bacteria and fungi, by mechanisms that remain poorly understood ,19,20. BEscherichia coli as prey and the myxobacterium Myxococcus xanthus as predator recently named MyxoEE-6  to mimic a scenario in which the actual colony count N for the plate was increased by one and the additional hypothetical colony was mucoid. The four populations with the highest mucoid frequencies at cycle 18  were selected for examination at more time points to allow the greatest opportunity to accurately resolve frequency dynamics. The colonies scored to assess mucoid frequencies were not subsequently cultured or stored frozen.Isolation of cycle 18 mucoid and non-mucoid clones. One mucoid and one non-mucoid clone each were isolated from cycle-18 co-evolved populations in which mucoids were detected and subsequently stored frozen. The clone pair from ME4 was used for the direct competition experiment measuring mucoid vs non-mucoid relative fitness and all nine clone pairs were used in the survival-under-predation and productivity assays and for colony-phenotype imaging. Single colonies of each phenotype were picked and streaked onto fresh LB agar plates to isolate a sub-colony and thereby purge any genetic variation that may have been present in the colony from the original plating. Isolated colonies of each phenotype were picked, grown to high density in LB liquid and samples of the resulting cultures were stored as frozen stocks (20% glycerol) for further experiments. The same mucoid and non-mucoid clone from each co-evolved population were used in the productivity and predation assays described below and were used for imaging colony phenotypes. For colony-phenotype imaging, aliquots of frozen stocks from mucoid and non-mucoid clones from nine different lineages were thawed in 100 E. coli mucoid vs. non-mucoid relative fitness in the presence and absence of M. xanthus, survival in the presence of M. xanthus and population size after 25 h of incubation (productivity) were performed under the same abiotic conditions as MyxoEE-6 )In our first study of MyxoEE-6, we reported that fewer cells of a mucoid clone isolated from the coevolving ME4 prey population at cycle 18 are killed by the ancestral predator compared to a contemporary non-mucoid isolate , but dirM. xanthus also isolated from the ME4 community after cycle 18) was either absent or added at a population size of ∼5 × 10E. coli populations generally increased more than 100-fold during the competition period. As expected, the mucoid genotype had higher fitness than the non-mucoid clone in the presence of M. xanthus but not in its absence (p = 5.49 × 10p = 0.558). These results thus do not support the hypothesis that mucoids fail to reach fixation due to cheating-mediated NFD. However, we note that this outcome does not generally exclude the NFD hypothesis for other experimental conditions or for other mucoid and non-mucoid competitors.The mucoid and non-mucoid prey clones were mixed at initial starting mucoid frequencies of 0.1, 0.25 and 0.5 and allowed to compete while growing under the same the experimental conditions as MyxoEE-6, except that the predator (a clone of Adaptations of prey that decrease susceptibility to predation often come at a cost to other components of fitness. We previously found no cost of mucoidy to growth rate from 5–20 h after inoculation into the MyxoEE-6 selective regime for a pair of ME4 cycle 18 clones in the absence of predators . HoweverM. xanthus than the corresponding non-mucoid clones from the same population (t-test: tp = 0.011), as was expected from previous results with the clone pair from population ME4 [t-test: tp = 0.0015). Thus, mucoidy protects against predation, but at a cost to total productivity in the absence of predation. This productivity cost of mucoidy led us to speculate that it may mediate a form of NFD in which mucoid frequency impacts total E. coli productivity, which, along with and decreased susceptibility to predation by mucoids, impacts predator productivity at the end of a growth cycle, which in turn impacts mucoid vs non-mucoid relative fitness in the subsequent growth cycle. We elaborate this hypothesis further in the Discussion.We first confirmed that the mucoid clones are on average less susceptible to killing by tion ME4 . To examM. xanthus than non-mucoids (p = 0.007). Additionally, while mucoids collectively have lower productivity than non-mucoids in the absence of predation (p = 0.004). Thus, while mucoidy in general is clearly selected by bacterial predation, the broader categorical phenotype can be associated with a diversity of detailed colony phenotypes and predation-related parameter values.We observed visually that colonies of the nine mucoid clones isolated from different lineages at the end of cycle 18 were phenotypically diverse, varying with regard to colony size, opacity, color and degree of phenotypic differentiation from non-mucoid colonies from their respective populations . We ther-mucoids , the degredation , the isoredation c, one-wayjbEFGH operon, with only one gene (yjbH) mutated in more than one population  and other evolved mutations in the same genetic background. To generate hypotheses regarding genetic causation of mucoidy in MyxoEE-6, we compared whole-genome sequences of one mucoid and two non-mucoid clones each from the four focal coevolved prey populations examined here for mucoidy dynamics across MyxoEE-6  . Mucoid pulation . In conton  resulting from immediate effects of prey-type frequency on prey-type fitness that would be observable over the course of a single cycle of MyxoEE-6 community growth. Such single-cycle NFD could result, for example, if non-mucoids can cheat on mucoids with respect to EPS production , not incE. coli grows much faster than M. xanthus and M. xanthus is completely dependent on E. coli for growth. For these reasons, we hypothesize that E. coli populations generally reached carrying capacity within each growth cycle before M. xanthus predator populations increased sufficiently to impose strong predation pressure that would selectively favor mucoids. Due to the productivity cost of mucoidy, non-mucoids may have generally outcompeted mucoids upon completion of prey growth earlier in each cycle, while mucoids may have often outcompeted non-mucoids later in each cycle, after predator density had increased sufficiently to impose strong predation pressure and thus favor mucoidy. In this scenario, sufficiently high late-cycle predation pressure promotes the net increase of mucoids over the entire cycle, despite a disadvantage of mucoids upon completion of prey growth before predators increase to large population sizes. However, mucoid frequency in one MyxoEE-6 cycle might impact the degree of late-cycle predation pressure in the next cycle if predators exhibit lower productivity on mucoid cells than non-mucoids.We speculate that the productivity cost and predation-resistance of mucoidy have the potential to collectively mediate NFD of mucoid fitness by reducing predator numbers when mucoids are sufficiently frequent in one growth cycle, which may in turn impact mucoid vs. non-mucoid fitness in the subsequent growth cycle. Under MyxoEE-6 conditions, This latter scenario would require first that predators exhibit lower productivity on mucoids than on non-mucoids, a plausible scenario given that mucoid prey have lower productivity and are less susceptible to predatory death. It would also require that lower predator productivity caused by relatively high mucoid frequency in one cycle decreases total predation pressure over the next cycle sufficiently to give non-mucoids a net whole-cycle advantage over mucoids in that subsequent cycle. This hypothetical scenario involves a negative feedback loop analogous to such loops proposed or demonstrated to maintain diversity in other predator-prey systems . A test E. coli are associated with increased production of exopolysaccharides, especially colanic acid (CA-EPS) [rcs, yjb and yrf genes [E. coli clones carried mutations in the yjbEFGH operon, while none of the non-mucoid clones had such a mutation, it appears that mucoidy in MyxoEE-6 was achieved predominantly by mutation of this operon, which is involved in the synthesis of an uncharacterised exopolysaccharide [rpoS background has been shown to result in CA-EPS synthesis and mucoidy [yjbEFGH, the mucoid strain from lineage ME4 had an additional mutation upstream of rcsA, which is a known positive regulator for colanic acid synthesis [yjbEFGH and rcsA are likely to mediate the apparent tradeoff between resistance to M. xanthus predation and productivity, although additional genetic experiments would be required to demonstrate this directly. Collectively, mucoidy seems to have been achieved largely through mutating the same operon, but in different genes and sites. These distinct mutations may have themselves generated the phenotypic variation among mucoid genotypes documented here, or they may have interacted with other mutations to generate such diversity.Mucoid phenotypes in (CA-EPS) ,49 and hrf genes ,48. Giveccharide . Interesynthesis ,50. ThusNatural microbial communities contain myriad species of prey and predators co-existing in complex food chains and webs ,52,53. TAt the inter-specific level, it is now well understood that variable levels and forms of intraspecific diversity impact community evolution e.g., ,55,56). ,56. 55,5


Although blood–heart-barrier (BHB) leakage is the hallmark of congestive (cardio-pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus variants such as the severe acute respiratory syndrome novel corona virus 2 (SARS-CoV-2) known as COVID-19, the mechanism is unclear. The goal of this project is to determine the mechanism of the BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase-9 (MMP-9) during CHF. MMP-9 degrades the connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP-9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP-9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial-myocyte coupling. To determine this, CHF was created by aorta-vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling-induced mitochondrial-myocyte, and endothelial-myocyte contractions were measured. Microvascular leakage was measured using FITC-albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP-9 activation, endocardial endothelial leakage, endothelial-myocyte (E-M) uncoupling, dyssynchronous mitochondrial fusion-fission (Mfn2/Drp1 ratio), and mito-myocyte uncoupling in the AVF heart failure were found to be rampant; however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID-19 in human subjects. Numerous studies have focused on CHF, but a very few have determined the BHB function . The endAlthough well-done, randomized clinical trials are needed urgently to test potential therapies including HCQ once the pharmacokinetics and pharmacodynamics (PK/PD) of HCQ are fully understood for the identification of an effective and safe therapeutic regimen for treatment of patients, we decided to study the potential mechanism(s) in an animal model, in anticipation of any benefits, if at all, regarding repurposing of HCQ in treating viral infections such as COVID-19 ,34,35,36We know that CQ is a weak base and that is trapped upon protonation in the acidic compartments such as late endosomes and lysosomes, wherein it alkalizes these compartments thus affecting the fusion events and disrupting the endosomal and autophagic cargo degradation. In this way, it blocks lysosomal function. By rendering these functions, CQ normalizes vessel structure, their function and helps increase the perfusion. CQ’s vessel normalization activity relies on alterations of endosomal Notch1 trafficking and signaling in the endothelial cells ,50,51,52Heart weight/body weight was increased in AVF mice hearts suggesting cardiac hypertrophy in AVF as compared to control mice. The treatment with HCQ mitigated this cardiac hypertrophy. Similarly, the lung weight/body weight was also increased in AVF mice indicating congestive (cardio-pulmonary) dysfunction in AVF mice; however, the treatment with HCQ attenuated this congestive heart failure phenotype .The serum levels of in gel-muscle injury markers were robust in AVF mice but the treatment with HCQ slowed the muscular  injury in chronic volume overload-induced heart failure . Tissue Because echocardiography presents whole heart function, we decided to separate LV from RV and from these ventricles we prepared the cardiac LV rings that were separate from RV and the atrium. By doing so, we were able measure the role of cardiac endothelial cells in myocyte contraction from the cardiac LV ring preparations. The results suggested that endothelial was impaired in AVF hearts and the same was mitigated by the treatment with HCQ . In vivoThe total vascular density in the heart was measured by barium sulfate contrast X-ray fluorography. The results revealed that there was a decrease in vascular density in the AVF hearts of the mice but treatment with HCQ seemed to normalize the vascular density post-AVF . FurtherHeart lung weights were increased in AVF-CHF, suggesting congestive heart failure post AVF in mice. The treatment with HCQ mitigated this increase in weights. Since AVF creates heart, lung, and vascular overload, we measured multiorgan damage by measuring the CKs activities. The findings suggested a robust increase in CK MM muscle activity indicating the multiorgan damage by chronic volume overload. Interestingly, the HCQ mitigated the multiorgan damage post treatment. Previously, we have shown that MMP-9 mediated the leakage of blood brain barrier (BBB) , therefore, in this study MMP-9 mediated breakdown of BHB was measured. It is known that MMP-9 degrades EE connections and make EE leaky. Since CHF and AVF involve RV, lung and then LV dysfunction, it is thus important to measure the RV, LV and pulmonary diaphragm function and MMP-related muscle remodeling.Several drugs are being tried for mitigating the COVID-19 symptoms including dapagliflozin (SGLT2-antidiabetic) lopinavir/ritonavir, darunavir/umifenovir (anti-HIV), remdesivir (anti-Ebola), favipiravir and dipyridamole (anti-hypertensive). We consider HCQ as a unique one in the sense that it mitigates unhygienic conditions related viral induced illnesses in the developing countries. Although we employed HCQ to test its efficacy whether it could help mitigate CHF in the mouse model, our work has relevance to the heart failure (HF) phenotype that is also the sequel of many viral myocarditis including the COVID-19 infections affecting the cardiovascular function in humans. Therefore, in future it would be important to further determine, in detail, whether the MMP-9 activation induces EMMPRIN (CD147), decreases connexin-37 and causes endocardial endothelial leakage and that HCQ mitigates MMP-9 activation, EE leakage and pulmonary diaphragm muscle dysfunction.Although, MMP9 degrades collagen/elastin and connexins, the turnover of collagen is rapid as compared to elastin or connexins, that in turn, is replaced by stiffer collagen . In this2O) as an intra-peritoneal (IP) injection @100 µL/mouse, twice a week for a total period of 6 weeks. Control/sham mice were given only the sterile water. AVF was created in anesthetized mice between the aorta and caudal vena cava, ~0.5 cm below the kidneys, using a 30-gauge needle. A minimum of 5–8 mice were used in each group. Most of the animals survived the surgical procedure. On completion of the experiments, heart, and lung weights of AVF and sham mice treated with and without the HCQ were recorded and the weights were normalized with weight of the tissue. An infrarenal AVF creates an unambiguous model of chronic volume overload. In this model, arterial blood below the kidneys rapidly enters venous circulation and overloads the ventricles without contribution from the stimulation of circulating factors [Because aging and viral myocarditis induce CHF primarily due to chronic increase in preload, therefore, we created AVF below the kidney in adult 12–15 weeks age old male wild type  mice. The animals were allowed to recover from the anesthesia, and then starting next day they were treated with or without HCQ . The gels are typically run at 400 V for 4:15 min. The standard (ST) amounts of CK isoforms (that are supplied by the kit manufacturer as loading controls) were also loaded in parallel to the samples for comparison [Multi-tissue specific injury was determined by measuring creatine phosphokinase (CK) isoforms in the plasma as described earlier . Brieflymparison ,91.The in situ FITC-gelatinase activity and protein-array for MMPs and TIMPs levels in sham and AVF groups along with the matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) activities were determined in whole heart and the tissue samples respectively prepared from the ventricle (RV), left ventricle (LV) and diaphragm. These tissues were collected from AVF and sham mice that were treated with and without HCQ and loaded in the gels and visualized as reported previously . Type I TM Acquisition Software version 9 [The single myocytes were isolated by collagenase digestion and contraction of single myocyte was measured using a modified protocol via the by IonOptix  as previously described ,95,96,97ersion 9 . RandomlThe determination of endothelial function in an isolated papillary muscle preparation does not demonstrate what happens in the entire trans-myocardial wall . TherefoThe endothelial permeability was measured in the coronary that were perfused with FITC. The measurement of florescence outside the coronary was recorded . SimilarTo determine vascular density barium sulfate contrast, X-ray angiography was performed as per the method described in our previous work . In shorA mixture of FITC (300 µg/mL) and BSA-647 (3.3 mg/mL) in PBS ~100 µL solution was infused through the tail vein injection in the anesthetized mice and FITC-BSA and allowed to circulate for 30–40 min. The in-situ leakage of the coronary was observed by exposing the hearts after performing thoracotomy under the microscope. The microscopic images were acquired. The results were averaged for each experimental group as per the protocol described ,108,109.To determine overall myocardial function, Echo was performed as previously described . Brieflyn = 5–8 animals/group), * p < 0.05, ** p < 0.01.Data from sham, AVF, sham + HCQ and AVF + HCQ were collected and statistically analyzed using the one-way ANOVA to analyze the difference between the groups, including a Tukey’s post hoc analysis for groups’ comparison, comparing AVF with sham and AVF + HCQ with AVF groups. Data are Mean ± SEM  of the University of Louisville School of Medicine, Louisville, KY, USA. Further, the animal care and guidelines of the National Institutes of Health  were also adhered to during all the experimental procedures.The primary cause of CHF leading to death during old age and viral myocarditis is not fully understood. The goal of this project was to determine the mechanism of BHB during CHF. Using an animal model of CHF by creating AVF, the control as well as AVF mice were treated with HCQ. Both structural, as well as functional parameters were measured. Our findings reveal that metalloproteinase activation, EE leakage, E-M uncoupling, altered Mfn2/Drp1 ratios, and MM uncoupling during AVF heart failure were the abundant features but treatment with HCQ could successfully mitigate deleterious CHF phenotype highlighting the potential application of CHQ as a possible therapeutic option for the clinical consideration ,77,78,79


Background: A subgroup of patients recovering from COVID-19 experience persistent symptoms, decreased quality of life, increased dependency on others for personal care and impaired performance of activities of daily living. However, the long-term effects of COVID-19 on physical activity (PA) in this subgroup of patients with persistent symptoms remain unclear. Methods: Demographics, self-reported average time spent walking per week, as well as participation in activities pre-COVID-19 and after three and six months of follow-up were assessed in members of online long-COVID-19 peer support groups. Results: Two hundred thirty-nine patients with a confirmed COVID-19 diagnosis were included  age: 50 (39–56) years). Patients reported a significantly decreased weekly walking time after three months of follow-up (three months: 60 (15–120) min. vs. pre-COVID-19: 120 (60–240) min./week; p < 0.05). Six months after the onset of symptoms walking time was still significantly lower compared to pre-COVID-19 but significantly increased compared to three months of follow-up (three months: 60 (15–120) min. vs. six months: 90 (30–150) min.; p < 0.05). Conclusions: Patients who experience persistent symptoms after COVID-19 may still demonstrate a significantly decreased walking time six months after the onset of symptoms. More research is needed to investigate long-term consequences and possible treatment options to guide patients during the recovery fromCOVID-19. At the time of writing, the number of people with a confirmed diagnosis of coronavirus disease 2019 (COVID-19) has risen to 167 million globally, with over 1.6 million confirmed cases in the Netherlands and over 1 million cases in Belgium [It has already been demonstrated that hospitalized patients with COVID-19 present with low physical functioning and impaired performance in activities of daily living (ADLs) immediately after discharge . SeveralTherefore, the aim of this study was to assess the impact of COVID-19 on the level of self-reported PA  in patients with post-COVID-19 syndrome . We hypothesized a significant decrease in PA approximately three months after the infection, and at least a partial recovery of PA six months after the infection.www.coronalongplein.nl) (accessed on 4 June 2020). In total, 1556 participants who agreed to take part in a follow-up of this study were asked to complete a second survey between August 31 and September 8 of 2020 (T2). The medical ethics committee of the Maastricht University stated that the Medical Research Involving Human Subjects Act (WMO) did not apply to this study and that an official approval of this study by the committee was not required . The medical ethics committee of Hasselt University formally judged and approved the study (MEC2020/041). All adult respondents (aged 18 years or older) gave digital informed consent at the start of the questionnaires. Data about symptoms, care dependency, information needs, functional status, work productivity and quality of life have been published before [As part of a large longitudinal study conducted in the Netherlands and Flanders (Belgium), an online questionnaire was made available between June 4 and June 11 of 2020 (T1) to all members of two long COVID Facebook groups  and to ad before ,24,25,262), marital status (married or living with partner: yes/no), education level (low/medium/high)), self-reported pre-existing comorbidities, COVID-19 diagnosis (based on reverse transcription polymerase chain reaction (RT-PCR) test and/or computed tomography (CT) scan of the thorax; symptom-based medical diagnosis; no test/medical diagnosis), received care (no care needed/physiotherapy/rehabilitation), symptoms and admission to hospital.The survey contained questions regarding demographics , age (years), body mass index (BMI)  and at the time of completing the two questionnaires  (A summary of the questionnaire is added in the online supplement). The World Health Organization (WHO) recommends a minimum of 150 min per week of moderate-intensity aerobic physical activity . Therefon = 766; data presented in the online supplement) were excluded from the primary analyses.Initial analyses were performed in participants with an RT-PCR or CT-confirmed diagnosis. Participants with a presumed COVID-19 diagnosis (http://sankeymatic.com/build/) (accessed on 4 June 2020) and GraphPad Prism 8.3.5. . Data were presented as mean and standard deviation (SD), median and interquartile range (IQR) or frequency and proportion, as appropriate. Data were tested for normality with a Kolmogorov–Smirnov test. Within-group comparisons were performed using the Friedman test, McNemar’s test or standard Cochran’s Q test (with Bonferroni corrected post-hoc test). Between-group comparisons were performed using a Mann–Whitney U test or Fisher’s exact test. A priori, the level of significance was set at p < 0.05.Statistical analyses and visualizations were performed using SPSS v25.0 , SankeyMATIC . Although there was a recovery in walking time between three months and six months of follow-up (from 60 (15–120) min. to 90 (30–150) min.; p < 0.05), walking time was still significantly lower compared to pre-COVID-19.Results regarding self-reported time spent walking per week at the three time points (pre-COVID-19 (T0) and three (T1) and six months (T2) of follow-up) are presented in p = 0.04). Additional analyses of walking time stratified for sex and number of symptoms are reported in the online supplements , outdoor cycling  and (physio)fitness/exercise groups  as the three most reported activities .p < 0.05) and the proportion of participants reporting walking, cycling outdoors/indoors, participating in (physio)fitness/exercise groups and running significantly increased. The proportion of participants that reported walking and cycling indoors at six months was significantly higher compared to pre-COVID-19.At three months of follow-up, participants reported performing fewer activities compared to pre-COVID-19 and almost 44% of the participants were not able to be physically active or perform sports or activities due to COVID-19. From three months to six months of follow-up the proportion of participants unable to be physically active significantly decreased  in patients with post-COVID-19 syndrome . As hypothesized, a significant decrease in walking time approximately three months after the infection and a partial recovery of walking time six months after the infection was found.These results indicate a possible recovery pattern similar to that which has been established in influenza A, acute respiratory distress syndrome survivors and severe acute respiratory syndrome, where patients experience impaired health-related quality of life, functional disability, psychological problems and impaired exercise capacity after up to two years of follow-up ,29,30,31Since the Dutch healthcare system was completely overwhelmed by the COVID-19 pandemic, the primary focus was the treatment of patients with the most life-threatening symptoms. People without the need for ICU admission, were understandably not prioritized during these hectic times, and therefore not intensively monitored or guided in their recovery. Complications like myocarditis or thromboembolic problems might have been missed and therefore might have hindered the recovery of PA in this subgroup of patients ,33.Furthermore, since the effects of COVID-19 on the human body are still not completely clear, it is important to consider that patients might recover along different trajectories. Indeed, Gandotra et al. have investigated recovery in patients with respiratory failure due to several causes and identified different recovery trajectories and characteristics . It is fAlthough encouraging patients to return to performing daily activities and to start low/moderate-intensity exercise at home is currently recommended for patients recovering from COVID-19 ,37, HumpWhile interpreting the abovementioned results, some strengths and limitations of this study need to be considered.One of the strengths of this study is the fact that this is the first longitudinal study investigating PA in patients with post-COVID-19 syndrome. We were able to follow up with a significant number of participants during the course of six months. A team of scientists, methodologists and patients worked together during the preparation of this study in order to create a study that provided a complete overview of relevant parameters in this specific population.p = 0.046) while walking time before COVID-19 was similar. This indicates that non-responders showed even less recovery of PA compared to responders.However, since participants were recruited through platforms that targeted patients with persistent symptoms, there is the possibility of selection bias and the external validity of these results might be limited. It is possible that participants that recovered after three months did not feel the need to fill in the questionnaire at six months and therefore are underrepresented. However, the additional analyses demonstrate that non-responders had a significantly lower walking time three months after the onset of symptoms, compared to the responders (median (IQR) 40 (10–95) vs. 59 (15–105) min. respectively; Additionally, females are overrepresented in this sample, possibly due to the higher proportion of women that are part of online long COVID support groups . This isDue to the national regulations that were in place during the first wave of COVID-19 infections, the possibilities for sports and activity were limited. In both the Netherlands and Belgium, a lockdown was proclaimed, people were asked to stay at home as much as possible and sport facilities were closed. Therefore, the decrease in activities that were not possible during a large part of the three months after symptom onset  is probably caused by the regulations that were in place at that time. However, at the same time, many sports clubs initiated alternative options like online dance and exercise classes to provide the possibility to exercise at home or outdoors. The regulations that were in place at the time also do not explain the difference in walking time. However, the increase in the proportion of participants who reported walking as an activity might be related to the regulation.Since the pandemic had a huge impact on healthcare resources, medical treatments were focused on the most severe cases of COVID-19 and patients with “milder” symptoms were often not tested. As a result, many patients who presented with milder symptoms were not admitted to the hospital or even officially diagnosed with COVID-19 and remained under the medical radar. The NICE guidelines have indicated that having a positive RT-PCR test or hospitalization is not a requirement for a COVID-19 diagnosis and healthcare should also focus on patients with suspected COVID-19 . In lineParticipants were asked to retrospectively assess their physical activity by filling in questionnaires. Therefore, the effect of recall bias cannot be excluded. However, since participants were asked questions that referred to the previous three months or the previous seven days, we expect that the influence of recall bias is limited. Besides recall bias in general, Dyrstad et al. have shown that a self-reported assessment of PA tends to overestimate vigorous activity and underestimate sedentary time . This efParticipants with persistent symptoms while recovering from COVID-19 who were all members of online long COVID support groups still demonstrated a significantly decreased self-reported walking time six months after the onset of symptoms. In contrast, the proportion of participants that reported walking or cycling indoors increased over the course of six months after the onset of symptoms. More research is needed to investigate the long-term consequences and possible treatment options to guide patients during the recovery from COVID-19.