Standardized reading schemes, the use of indicators derived from medical images and the use of deep learning-based algorithms become very popular in medical imaging. In this retrospective study, we evaluated the performance of an automatic deep-learning-based algorithm for computer-assisted diagnosis in the field of oncological whole-body bone imaging in nuclear medicine. In addition to prostate cancer, representing a tumor entity evaluated thoroughly using the examined methodology (Bone Scan Imaging (BSI) methodology), a modification of the BSI based standard rating scheme facilitate the use of the methodology for other tumor entities . Diagnostics in clinical routine can benefit from the examined methodology, mainly due to its sensitivity and the high negative predictive value. Non-pathological bone scans may be easily identified. This may lead to a reduced working load in nuclear medicine departments and may result in an improved and more standardized workflow.The bone scan index (BSI), initially introduced for metastatic prostate cancer, quantifies the osseous tumor load from planar bone scans. Following the basic idea of radiomics, this method incorporates specific deep-learning techniques  in its development to provide automatic calculation, feature extraction, and diagnostic support. As its performance in tumor entities, not including prostate cancer, remains unclear, our aim was to obtain more data about this aspect. The results of BSI evaluation of bone scans from 951 consecutive patients with different tumors were retrospectively compared to clinical reports . Statistical analysis included entity-specific receiver operating characteristics to determine optimized BSI cut-off values. In addition to prostate cancer  = 87%, specificity (SP) = 99%), the algorithm used provided comparable results for breast cancer  and colorectal cancer . Worse performance was observed for lung cancer  and renal cell carcinoma . The algorithm did not perform satisfactorily in melanoma (SN = 60%). For most entities, a high negative predictive value  was determined, whereas positive predictive value (PPV) was clinically not applicable. Automatically determined BSI showed good sensitivity and specificity in prostate cancer and various other entities. Particularly, the high NPV encourages applying BSI as a tool for computer-aided diagnostic in various tumor entities. Planar entities . SPECT, entities ,6,8. PlaThe evaluation of bone scans is observer-dependent and shows significant inter-observer variability . Two decThe aim of this study was to validate the performance of BSI, automatically calculated by an ANN trained with bone scans from metastatic prostate cancer patients, in the detection of osseous metastatic disease from bone scans in different tumor entities. Additionally, we examined the potential for improving BSI methodology in clinical application by using entity-specific cut-off values to distinguish between the absence and presence of bone metastases.n = 1067), e.g., loosening of joint prosthesis or rheumatic affection, were excluded from further analysis. Only the first whole-body scan of each patient during the observed period was included in the analysis (n = 2740). Some data were excluded due to methodological constraints, e.g., an incomplete whole-body scan or signs of extravasation (n = 24). Final count statistics had to exceed 1.0 million counts in the geometric mean image, calculated from anterior and posterior projections (n = 733 examinations excluded), to ensure the good reliability of the BSI [n = 1032) for localization of suspect tracer accumulation. Corresponding clinical reports presented the condensed information from planar imaging and SPECT/(CT). As a result, for these patients, the classification could have been biased by findings from SPECT/(CT). Patients with SPECT/(CT) data were excluded from primary evaluation because BSI evaluation is based on planar images only (effect from SPECT/(CT); The data of 4702 patients referred to our department  with a clinical indication for a bone scan between January 2009 and December 2017 were retrospectively screened for analysis. Standardized eligibility criteria were defined as follows to guarantee a comparable data quality for the validation procedure. Patients with suspicion of benign disorders ( the BSI . SPECT/(n = 11), the current entity triggering the imaging procedure was used for grouping.The remaining scans were classified according to the primary tumor entity . In caseAll data analyzed were collected as part of routine diagnosis. The retrospective study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethics Committee of the Otto-von-Guericke-University , Registration Number: R05/20 (15 July 2020).99mTc-2,3-dicarboxypropane-1,1-diphosphonate . The administrated activity was 646 ± 77 MBq. In adipose patients, the injected dose was increased to 11–13 MBq/kg body weight in accordance with the corresponding guidelines [Bone scintigraphy was performed by using idelines .Whole-body planar imaging was performed using one of three scintillation gamma camera systems: (1) two dual-head SPECT gamma cameras of identical design  and (2) a dual-head SPECT/(CT) . Each camera was equipped with a manufacturer-specific, low-energy, high-resolution (LEHR) collimator. All gamma cameras were monitored by dedicated image quality management procedures. Imaging was performed with a matrix size of 1024 × 256  and 2.21 × 2.21 mm (NM/CT 670)) and a scan speed of 16 cm/min. The energy window was set to 140.5 keV ± 10%. Whole-body imaging was performed using the automatic body contouring system of the gamma camera systems to minimize the detector to patient distance.In the cases of a two-/three-phase protocol applied for different clinical indications, whole-body images from the late phase  were used for BSI analysis. Images were acquired in concordance to clinical standard 2.5 to 4 h after injection.BSI was calculated automatically by dedicated software . The software automatically segments the anterior and posterior projection of the bone scan by dividing the skeleton into groups . The bladder was segmented automatically, and counts inside the bladder were excluded from further analysis. Paired bones were distinguished in each planar image into right and left sides . Areas of increased tracer accumulation, s pixels . The thrs pixels . The intotection .(1)BSI ion (1)) . ClinicaThe R software package  was used for all statistical evaluation . Descripp-value < 0.05 for two-sided testing.Differences in the BSI values for patients with and without bone metastases were tested for significance by an independent two-samples Wilcoxon’s rank sum test. Receiver operating characteristic (ROC) curves  were useFor nearly all examined cancer types, BSI was significantly higher in patients with expert-confirmed M1 state compared to the corresponding M0 cohort . BSI wasp = 0.016; breast cancer, p = 0.048). The BSI cut-off estimated for scanners #1 and #2 is higher in both tumor entities compared to the cut-off values estimated for scanner #3 .The data for prostate and breast cancer were used to analyze the impact of the gamma camera model. A scanner-specific ROC analysis was conducted for these specific sub-cohorts. The respective values of the AUC were calculated as 0.923 (scanners #1 and #2) and 0.984 (scanner #3) in patients with prostate cancer, and 0.877 (scanners #1 and #2) and 0.968 (scanner #3) in breast cancer patients. Given these values, a significant effect of the gamma camera model on BSI was detected  to estimate the test performance using the standard setup of the algorithm. We improved the test performance in detecting metastatic disease by modifying the BSI methodology using entity-specific cut-off values for discriminating M0/M1 states. In parallel, we evaluated the effect of different gamma camera systems on the optimal BSI cut-off value. Data from the two largest sub-cohorts (patients with prostate and breast cancer) were used for this methodological analysis.The high sensitivity and specificity in detection of bone lesions in patients with prostate cancer, known from the literature, were reproduced ,19. UsinA positive BSI in the M0 sub-cohorts is the result of a false-positive classification of benign areas of increased uptake  as malignant even though the algorithm is supposed to reject them. Many scans therefore had a positive definite BSI due to a solitary area of increased uptake, even though there was no obviously suspicious uptake according to the physician’s statement. This effect was already analyzed by Petersen et al. , who useSadik et al.  reportedThe potential bias from the different gamma camera models was assessed by analyzing data from breast and prostate cancer, representing the most frequent cancer entities in our cohort. Comparing ROC curves, we observed significant differences between AUCs and cut-off values. For further examined tumor entities, the evaluation was not applicable due to the limited number of patients in the sub-cohorts. We hypothesize that scanner-individualized (model-specific) training of the ANN and cut-off values can further improve the diagnostic performance of the BSI. A deeper investigation is necessary to substantiate this statement.n = 1211) with prostate cancer using 99mTc-methylene diphosphonate (99mTc-MDP). In contrast, in our study, imaging was performed with another similar radiopharmaceutical 99mTc-2,3-dicarboxypropane-1,1-diphosphonate. We did not expect this difference to have any significant effect due to comparable pharmacokinetics and accumulation patterns reported by studies conducted during the approval process of the corresponding pharmaceuticals [The software’s algorithm of the examined tool was trained for a database of bone scans performed in patients . For that reason, an information bias  has to be discussed. The effect was always considered when examining the influence of an additional SPECT or SPECT/(CT) on the clinical decision . This waFinally, the BSI is correlated with prognosis and likelihood of response to various therapies in patients with metastatic prostate cancer ,41,42,43In this retrospective study, we evaluated the performance of an automatic deep-learning-based algorithm for computer-assisted diagnostics in the field of oncological whole-body bone imaging. In addition to prostate carcinoma, representing a tumor entity evaluated thoroughly using the BSI methodology, entity-specific BSI cut-off values facilitate the use of the BSI methodology for other tumor entities . Diagnostics in clinical routine can benefit from BSI methodology, mainly due to its sensitivity and the high NPV supporting the identification of non-pathological bone scan.


To assess the association between routinely prescribed non-steroidal anti-inflammatory drugs (NSAIDs) and deaths from COVID-19 using OpenSAFELY, a secure analytical platform.We conducted two cohort studies from 1 March to 14 June 2020. Working on behalf of National Health Service England, we used routine clinical data in England linked to death data. In study 1, we identified people with an NSAID prescription in the last 3 years from the general population. In study 2, we identified people with rheumatoid arthritis/osteoarthritis. We defined exposure as current NSAID prescription within the 4 months before 1 March 2020. We used Cox regression to estimate HRs for COVID-19 related death in people currently prescribed NSAIDs, compared with those not currently prescribed NSAIDs, accounting for age, sex, comorbidities, other medications and geographical region.In study 1, we included 536 423 current NSAID users and 1 927 284 non-users in the general population. We observed no evidence of difference in risk of COVID-19 related death associated with current use  in the multivariable-adjusted model. In study 2, we included 1 708 781 people with rheumatoid arthritis/osteoarthritis, of whom 175 495 (10%) were current NSAID users. In the multivariable-adjusted model, we observed a lower risk of COVID-19 related death  associated with current use of NSAID versus non-use.We found no evidence of a harmful effect of routinely prescribed NSAIDs on COVID-19 related deaths. Risks of COVID-19 do not need to influence decisions about the routine therapeutic use of NSAIDs. There have been concerns that non-steroidal anti-inflammatory drugs (NSAIDs) may increase the risk of COVID-19 disease. Recent observational studies reported no evidence of a harmful effect of NSAID use on COVID-19 severity among patients with COVID-19.However, most studies were of much smaller sample size, not general population based or did not specifically investigate individual NSAIDs .In addition, limited clinical data are available to advise patients using long-term NSAID treatment (including people with rheumatoid arthritis and osteoarthritis) whether the treatment should be continued or stopped in the context of COVID-19 pandemic.We identified two study populations  in England using OpenSAFELY platform. We then grouped them into current users and non-users, respectively, in each study population.In both populations, no association between NSAIDs and COVID-19 related death was found.This study does not support the hypothesis of any harmful effect of NSAIDs on COVID-19 related deaths among regular NSAID users.Treatment decisions about the routine use of NSAIDs do not need to be influenced by fears of an effect on COVID-19 outcomes.COVID-19, caused by the SARS-CoV-2, has been diagnosed in approximately 18 million patients with >690 000 deaths in >200 countries as of 5 August 2020.Non-steroidal anti-inflammatory drugs (NSAIDs) are widely prescribed for relief of pain and inflammation with nearly 11 million NSAID prescriptions dispensed in primary care in England in the last 12 months.There is now a debate over whether NSAIDs may worsen the prognosis of COVID-19. On 14 March, it was recommended in France that patients should avoid NSAID use due to an apparent worsening of COVID-19 in those taking ibuprofen, based on unpublished reports.We therefore investigated the association between NSAID use and deaths from COVID-19 using linked data from >17 million patients in England. We further examined whether the association varied by types of NSAID.We conducted two cohort studies using primary care electronic health record data linked to death data from the Office for National Statistics between 1 March 2020 and 14 June 2020.Primary care records managed by the software provider The Phoenix Partnership (TPP) were linked to Office for National Statistics death data through OpenSAFELY, a data analytics platform created by our team on behalf of NHS England.We identified two cohorts, anticipating that underlying factors influencing NSAID use and therefore potential biases would differ between them. The first cohort was all people with ≥1 oral NSAID prescription within the 3 years before study start (1 March 2020), identified from the general population. It was chosen to minimise confounding by restricting to people who were currently prescribed NSAIDs and those who recently stopped NSAIDs as their characteristics were likely to be more comparable than never-users. The second cohort was all people with a diagnosis of rheumatoid arthritis (RA)/osteoarthritis (OA) before study start. It was chosen because they were potential NSAID users with similar underlying diseases to reduce confounding by indication. A patient could be included in both cohorts.In both cohorts, people with missing data for gender, index of multiple deprivation, <1 year of primary care records or aged <18 or >110 years were excluded. Aspirin is used at lower doses as an antiplatelet to prevent cardiovascular disease,In the main analysis, we defined current NSAID users as those ever prescribed NSAID in the 4 months prior to study start, and non-users are those with no record of NSAID prescription in the same time period.We examined whether the association varied by types of NSAID, specifically: (1) naproxen dose , low-dose naproxen (250 mg) and other NSAIDs based on the strength of the formulation), (2) COX-2 specific NSAIDs  and non-specific NSAIDs) and (3) ibuprofen .Follow-up for each cohort began on the 1 March 2020 and ended either on date of death or study end date (14 June 2020). If people in the non-user group received a NSAID prescription after 1 March 2020, they were censored at the date of this prescription .10.1136/annrheumdis-2020-219517.supp1Supplementary dataThe outcome was COVID-19 related death as registered in Office for National Statistics data using International Classification of Diseases (ICD)-10 codes U07.1  and U07.2  listed either as the underlying or any contributing cause of death. The latter ICD-10 code is used when laboratory testing is inconclusive or unavailable.https://codelists.opensafely.org/.Baseline characteristics in each cohort were summarised using descriptive statistics, stratified by exposure status. Time to COVID-19 related death was displayed in Kaplan-Meier plots. We present adjusted cumulative mortality curves and the difference between curves using the Royston-Parmar model. We estimated HRs with 95% CIs for the association between current NSAID use and COVID-19 related death using Cox regression with time since cohort entry as the underlying timescale. We accounted for competing risk by modelling the cause-specific hazard . We used graphical methods and tests based on Schoenfeld residuals to explore violations of the proportional hazards assumption.Unadjusted models, models adjusted for age (using restricted cubic splines) and sex and multivariable-adjusted models including covariates listed in We used e-value formulae to calculate the minimum necessary strengths of association between an unmeasured confounder and exposure or outcome, conditional on measured covariates, to fully explain observed non-null adjusted associations .https://github.com/opensafely/nsaids-covid-research.Data management was performed using Python V.3.8 and SQL, with analysis carried out using Stata V.16.1. All study analyses were preplanned unless otherwise stated. All code for data management and analyses in addition to the prespecified protocol are archived at: https://opensafely.org/ through which we invite any patient or member of the public to contact us regarding this study.Patients were not formally involved in developing this specific study design that was developed rapidly in the context of a global health emergency. We have developed a publicly available website We included 536 423 current NSAID users and 1 927 284 non-users . Median Current users were more likely to be obese, former smokers and have a medical history of hypertension, diabetes, other respiratory diseases, cancer, chronic kidney disease, OA and RA than non-users. Current users were also more likely to have a prescription for statins, proton pump inhibitors and disease-modifying antirheumatic drugs and to have had more primary care consultations and vaccinations than non-users.We included 175 495 current NSAID users and 1 533 286 non-users . A higheCurrent users were more likely to be obese, more deprived, former/current smokers and to have had more primary care consultations and a prescription for proton-pump inhibitors and disease-modifying antirheumatic drugs than non-users. However, non-users were more likely to have comorbidities than current users.After we excluded people who were ever prescribed aspirin, we observed no difference in risk of COVID-19 related death associated with current use compared with non-use  in RA/OA population . In the To fully explain the multivariable-adjusted HR (0.78) or the upper bound of the 95% CI (0.94) in the RA/OA population, an unmeasured confounder would need to be associated  with either non-use, relative to current use or COVID-19 mortality by at least risk ratio (RR) of 1.88 (effect estimate) or 1.29 (upper bound) and with both non-use and COVID-19 mortality by at least RR of 1.28 (effect estimate) or 1.06 (upper bound) .Based on routinely collected data, our study showed no overall increased risk of COVID-19 related death associated with current NSAID use in adults, compared with non-use. This was consistently seen across all analyses.In this study, we used two different populations to explore the potential impact of confounding. Current users were generally older and had more comorbidities than non-users in the general population cohort. As expected, this was associated with an increased risk of COVID-19 related death in current users compared with non-users in the unadjusted model. In contrast, current NSAID users were younger and had more comorbidities than non-users in the RA/OA population, associated with a decreased risk of COVID-19 related death in the unadjusted model. Notably, both associations were largely removed on adjustment for age. We observed a small decreased risk of COVID-19 related death among current users in the RA/OA population but not in the general population in the multivariable-adjusted models. In a post hoc analysis informed by a DAG that captures the complexity of relationships between variables, this protective effect was somewhat attenuated, suggesting it is not a robust finding and is subject to model variable selection. Moreover, our main analysis in the RA/OA population might also be subject to residual confounding. As demonstrated in quantitative bias analysis, an unmeasured confounder of only moderate strength could potentially fully explain this observed association. As we consistently found no evidence of harmful effect of NSAIDs on COVID-19 related death, using two populations provides a useful context for result interpretation.It was postulated that NSAIDs might delay diagnosis and thus clinical care by masking the symptoms of a worsening infection.In line with our results, five observational studies reported no evidence of a harmful effect of NSAID use on COVID-19 severity among patients with COVID-19Notably, we assessed exposure as NSAID use prior to the outbreak in England to establish who were current users, but we did not evaluate any potential therapeutic role of NSAIDs to treat patients with COVID-19. While our study mainly focused on current NSAID use for routine clinical care, there are some ongoing clinical trials investigating the role of NSAIDs in management of COVID-19. They are due to complete later this year or next year (NCT04325633The greatest strength of this study was the power we had to examine the association between NSAIDs and COVID-19 death, particularly on types of NSAID as our dataset included medical records from 24 million individuals. We also used two different study populations for comparisons to understand the impact of confounding by indication. The breadth of data available in primary care allows us to account for a wide range of potential confounders. We prespecified our analysis plan and have openly shared all analytical code.We recognise possible limitations. First, we do not know whether patients truly took the medications as prescribed. Second, the supply of NSAIDs ‘over the counter’ is not captured. However, ‘over the counter’ purchases are likely to be for ibuprofen, used for acute, irregular conditions and may mean some non-users were in fact taking ibuprofen. This would tend to bias results towards the null. However, this is unlikely to impact the result in the RA/OA population as GPs in England prescribe NSAIDs for long-term conditions such as RA/OA.We found no evidence of a harmful effect of routinely prescribed NSAIDs on COVID-19 related death. People currently prescribed NSAIDs for their long-term conditions should continue their treatment as part of their routine care.NHS England is the data controller; TPP is the data processor; and the key researchers on OpenSAFELY are acting on behalf of NHS England. This implementation of OpenSAFELY is hosted within the TPP environment, which is accredited to the ISO 27001 information security standard and is NHS IG Toolkit compliant


Coronavirus disease 2019 (COVID-19) is now a global public threat. Given the pandemic of COVID-19, the economic impact of COVID-19 is essential to add value to the policy-making process. We retrospectively conducted a cost and affordability analysis to determine the medical costs of COVID-19 patients in China, and also assess the factors affecting their costs.t test and Mann-Whitney test when appropriate. A multiple logistic regression model was built to determine the risk factors associated with high cost.This analysis was retrospectively conducted in Shandong Provincial Chest Hospital between 24 January and 16 March 2020. The total direct medical expenditures were analyzed by cost factors. We also assessed affordability by comparing the simulated out-of-pocket expenditure of COVID-19 cases relative to the per capita disposable income. Differences between groups were tested by student A total of 70 COVID-19 patients were included in the analysis. The overall mean cost was USD 6827 per treated episode. The highest mean cost was observed in drug acquisition, accounting for 45.1% of the overall cost. Total mean cost was significantly higher in patients with pre-existing diseases compared to those without pre-existing diseases. Pre-existing diseases and the advanced disease severity were strongly associated with higher cost. Around USD 0.49 billion were expected for clinical manage of COVID-19 in China. Among rural households, the proportions of health insurance coverage should be increased to 70% for severe cases, and 80% for critically ill cases to avoid catastrophic health expenditure.Our data demonstrate that clinical management of COVID-19 patients incurs a great financial burden to national health insurance. The cost for drug acquisition is the major contributor to the medical cost, whereas the risk factors for higher cost are pre-existing diseases and severity of COVID-19. Improvement of insurance coverage will need to address the barriers of rural patients to avoid the occurrence of catastrophic health expenditure. The costs of medical care were primarily paid by health insurance, and the remaining costs were afforded by local government.Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now a global public health threat . As of 3The spectrum of the COVID-19 severity is broad, ranging from asymptomatic to severe illness that requires mechanical ventilation . In mostShandong Province is located in eastern China, with a population of 100.7 million. As the second most populated province, it has fast economic development in the country. Despite not being close to Hubei Province, as of 3 May 2020, human-to-human transmission has driven its spread with a total of 788 confirmed cases, including 763 non-imported cases and 25 imported cases. To address this public health concern, we retrospectively conducted a cost and affordability analysis to determine the medical costs of COVID-19 patients in China, and also assess the factors affecting their costs.This analysis was retrospectively conducted in Shandong Provincial Chest Hospital. This hospital is the only provincial-level tertiary hospital designated hospital for respiratory diseases in Shandong, including tuberculosis and other newly emerging respiratory illnesses, such as COVID-19. Between 24 January and 16 March 2020, 70 laboratory-confirmed patients consistent with national guidelines, including clinical symptoms, radiological abnormalities and positive molecular testing results for SARS-CoV-2 , were adThe costs of medical care were paid by health insurance and local government. Costs in this analysis were identified for drugs, radiological examinations, laboratory diagnostic tests, clinical measures, drug prescriptions, and hospitalization. Total costs were calculated from the following cost factors comprising the listed single positions: (i) laboratory diagnostic measures included microbiology, hematology, biochemistry, and blood gas analysis; (ii) radiographic measures included X-ray, computed tomography (CT) and B-mode ultrasonography; (iii) therapeutic measures included supplemental oxygen, hemopurification, salvage, and other measures used for treatment of COVID-19 and comorbidities; (iv) drug acquisition included all drugs indicated by the physician to be necessary for treatment of COVID-19 and comorbidities; (v) bed costs included costs of regular wards and intensive care units. Given that the medical cost in China was not separately accounted for staff expense, these expenditures were included in therapeutic measures. The costs of drugs were further divided according to their efficacy. Expenditures were calculated in US dollars (exchange rate: CNY 7.0 = USD 1.0).http://www.stats.gov.cn/), and family size according to the recent data from national population census (http://www.stats.gov.cn/ztjc/zdtjgz/zgrkpc/). The catastrophic expenditure was defined as the families who spend 50% or more of their disposable income [We assessed affordability by comparing the simulated out-of-pocket expenditure of COVID-19 cases relative to the per capita disposable income. The simulated out-of-pocket expenditure was calculated according to the reimbursement ratios by health insurance. In addition, we collected the rural and urban per capita disposable income of China from the National Bureau of Statistics , while the categorical variables were expressed as percentages. A descriptive analysis was undertaken to compare the cost stratified to different patient groups. The comparison for normal variable was conducted using student t test. Mann-Whitney tests were performed for nonparametric data. Variables were introduced into a multiple logistic regression model if they were significantly associated with high cost at P < 0.10. The adjusted odds ratios (ORs) and 95% confidence interval (CI) were calculated according to this model.Demographic and treatment costs data were collected from electronic patient record system. Microsoft Excel 2013  was used for data entry. Statistical calculations were conducted using the statistical software SPSS 20.0 . A total of 70 COVID-19 patients were included in the analysis. All patients were treated in isolation. Demographic and clinical characteristics on hospital admission are shown in Table The mean costs for clinical management of COVID-19 patients are summarized in Fig. P < 0.001). The increasing trend in mean total cost was observed with advanced age, ranging from USD 2752 for 0–34 years group to USD 11668 for ≥ 70 years group (P = 0.002), which majorly contributed to the greater odds for pre-existing diseases in elderly patients. We also found that the mean cost was associated with the disease severity, the value of critically ill cases (USD 16 652) was significantly higher than that of mild cases. In contrast, the sex and residence had no significant effect on the total cost. Notably, the statistically higher expenses in patients with pre-existing diseases were identified in all individual cost factors investigated in this study (P < 0.01) . In addition, the advanced disease severity was also associated with higher cost .A regression model was modeled to identify variables independently associated with high cost (USD 6877). The results of the regression model are presented in Table http://www.nhc.gov.cn/xcs/yqtb/list_gzbd.shtml). The number of cases with varying severity was estimated by the recent report from Chinese Center for Disease Control and Prevention, demonstrating that the proportion of mild, severe and critically ill cases were 81, 14 and 5%, respectively. On the basis of these estimates, the total burdens were USD 0.30 billion (61%) for mild cases, USD 0.13 billion (26%) for severe case, and USD 0.07 billion (13%) for critically ill cases, respectively. In total, around USD 0.49 billion were expected for clinical manage of COVID-19, and financed a significant proportion of the proposed national health care plan  , community-acquired pneumonia (about USD 650) and severe acute respiratory syndrome (about USD 2700) in China –16. The Of note, drug acquisition accounts approximately half of total direct cost in our cohort, of which immunomodulator, Chinese medicine and antibiotic occupy the top three rank positions by mean cost per case. In our opinion, this cost rank is associated with the diversity of unit price of each drug. Intravenous immune globulin therapy is the predominant immunomodulator used in the management of patients with COVID-19 . As the As expected, the patients with pre-existing diseases are strongly associated with higher cost than those without pre-existing diseases. The reasons for this observation are multifactorial. First, there is no doubt that the use of drugs for pre-existing diseases will increase the medical expense. Second, the concurrent diseases at admission imply more severity and complexity. Several studies documented that the comorbidities are recognized as independent factors in increased disease severity , 24, thuAlthough the costs of medical care were paid by health insurance and local government in China, we constructed an estimated model taking the household income and health insurance scheme into consideration to assess the affordability for clinical management of COVID-19. For urban households, the proportion of insurance payment is about 70% of total in-hospital payment . In thisThere were several obvious limitations in this study. First, despite enrolment of all COVID-19 cases in our hospital, the small sample size from the single center may weaken the significance of our conclusion. Second, the present study excluded partial direct costs to patients and indirect costs, such as lost working days. Therefore, the total cost of COVID-19 patients is underestimated. Third, the insurance coverage and health benefits were significantly different across different regions in China. Hence, this study was more likely to be a theoretical cost analysis in view of that fact that Chinese government affords medical expenditures for COVID-19 patients. Fourth, despite exhibiting promising efficacy for treatment of patients infected with COVID-19, the traditional Chinese medicines are composed of several classical herbal materials for exogenous fever. Thus, it is difficult to elucidate the systemic pharmacological mechanisms of action of Chinese medicine against SARS-CoV-2. Finally, drug prices can be expected to change substantially across regions. This diversity may lead to potential bias in estimation of national financial burden. Nevertheless, our study firstly provides important insights in direct medical cost of COVID-19 patients in China.Our data demonstrate that clinical management of COVID-19 patients incurs a great financial burden to national health insurance. The cost for drug acquisition is the major contributor to the direct medical cost, whereas the risk factors for higher cost are pre-existing diseases and severity of COVID-19. Despite government financial support to clinical management of COVID-19 patients, an estimated model indicates that improvement of insurance coverage will need to address the barriers of rural patients to avoid the occurrence of catastrophic health expenditure.


Foot-and-mouth disease (FMD) is an acute viral infection affecting cloven-hoofed animals causing vesicular erosions in the oral cavity and interdigital space. The present study was undertaken to ascertain the time-dependent changes in clinical, hematological, and biochemical profiles in different breeds of cattle following experimental infection.4 50% bovine tongue infectious dose (BTID50) by intradermolingual route. Clinical signs were observed, and blood/serum samples were collected at different time intervals.The animals were inoculated with 1.0×10The white blood cell count declined sharply on days 7-13 and recovered on day 14 post-FMD infection. Biochemical analysis of serum markers for vital organ profile revealed no marked damage. However, a significant increase in blood urea nitrogen (BUN) value indicated pre-renal azotemia. Transient hyperthyroidism was indicated by the rise in T3 and T4 that can be correlated with a decrease in triglyceride and total cholesterol levels. In the cardiac damage assessment study, a distinct breed difference was observed wherein Malnad Gidda calves showed no cardiac damage.Except thyroid profile, BUN, and creatine kinase-myocardial band, all other serum biochemical parameters showed no significant abnormalities, whereas lymphopenia is the only hematological change and it is suggested that effective ameliorative measures should be targeted mainly on the feed/water intake, thyroid gland, and the level of lymphocytes. Foot-and-mouth disease (FMD) is the most economically important viral disease of cattle, caused by FMD virus (FMDV) and is characterized by fever, depression, anorexia, and excessive stingy or foamy salivation with vesicles or blisters appearing on the tongue. Some infected animals remain asymptomatic, but they carry the virus and can transmit it to other animals. The disease is generally not fatal (mature livestock) but increases the risk of abortion  and of mortality (young livestock). FMD leads to reduced productivity and requires increased expenditures on feed, medication, and shelter . The ecoThe initial diagnosis of the disease is usually done on the basis of clinical signs. In endemic regions, the partial signs are generally overlooked due to natural or vaccinal immunity . The levBased on the above-mentioned facts, the present study has been undertaken to elucidate the effect of FMD infection on certain clinical, hematological, and serum biochemical parameters in few breeds of cattle during the course of the disease after experimentally infecting the calves.The animal experimental protocol was approved by the Institutional Animal Ethics Committee of ICAR- Indian Veterinary Research Institute, Bengaluru and carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forests and Climate Change, Government of India.Bull calves of 6-9 months of age  with a history of no previous infection and not vaccinated for FMD, were used in the study. They were dewormed and kept under quarantine and acclimatization for 4 weeks. All the animals were negative for non-structural protein antibodies . They we4 50% bovine tongue infectious dose (BTID50) of virus suspended in 0.2 mL media by intradermolingual route, in containment animal facility. Observations on day 0 (the day of infection) were made before inducing experimental FMDV infection.A bovine-derived FMDV serotype O/IND/R2/75 was used for experimental infection. Four calves per breed were inoculated with 1.0×10g for 15 min and stored at −80°C until assay for serum biochemical parameters. For hematological analysis, 2 mL blood sample was collected in a green vacutainer containing heparin as an anticoagulant .Blood samples of 10 mL were collected by jugular venipuncture in a red vacutainer containing clot activator  and serum was separated by centrifugation at 3000× Complete blood count analysis was carried out using a double chamber hematology analyzer .Calves were observed for the onset, progress, and remission of secondary lesions in the foot at 24 h interval for 30 days post-infection. The foot lesion was scored on a scale of 1-4 based on the number of limbs involved. Rectal temperature was recorded from day 0 to 10 post-infection on a daily basis.Total serum protein, albumin, triglycerides, and total cholesterol were estimated with commercial kits using bench-top semi-automatic biochemistry analyzer . The concentration of globulin and albumin to globulin (A: G) ratio was derived from total protein and albumin. Cardiac damage was assessed by the immunoinhibition method using the creatine kinase-myocardial band (CK-MB) kit . Liver function was evaluated by aspartate transaminase (AST), alanine transaminase (ALT), and gamma-glutamyl transferase (GGT) levels, while azotemia was assessed by blood urea nitrogen (BUN) and creatinine. The concentration of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone was estimated by standard kit.Electrocardiography (ECG) was recorded in bipolar base apex lead  using limb lead I on day 0 (pre-infection), 1-5 days, and 21-day post-infection. ECG was recorded in standing position by placing the rubber mat underneath the animal without any tranquilizer or sedative. Alligator type electrodes were attached to skin after cleaning it with ethanol and applying electrocardiographic jelly. The positive electrode of the lead I was attached to the skin of the fifth intercostal space just caudal to olecranon process and negative electrode on jugular furrow about lower one-third of the left side of the neck. ECG was recorded in a single channel with a paper speed of 25 mm/s and calibration of 10 mm equal to 1 mV.post hoc test was done for viremia, CK-MB, and one-way repeat measure ANOVA was done with orthogonal contrast (pre-infection vis-à-vis post-infection) using Tukey’s post hoc test. Data were analyzed by GraphPad Prism 6.0. The value of p=0.05 or less was considered statistically significant.The sample size for infected samples comprised 12 animals (n=4 per breed) for the parameters observed till day 10 post-infection. However, for later days, it was n=11, as one Malnad Gidda calf died on 11 dpi. Two-way ANOVA with Tukey’s The onset of pyrexia (>103° F) on day 2 and foot lesions on day 3 indicates that the animals were infected with FMDV (O/R2/75). Pyrexia and lesions reached peak values on days 3 and 5 post-infection, whereas remission was observed on day 5 and 21-day post-infection, respectively  and b.< 0.05) increase during days 3-14 post-infection  of Tnfection  and b. Tnfection  and d.Since FMDV is known to induce myocarditis in calves, CK-MB was estimated in serum samples to assess the severity of damage from pre-infection to day 7 in the experimental calves . A signiTotal leukocyte count revealed a significant decrease (p<0.05), which was apparent during days 7-14 post-infection indicating leukopenia . The neuDespite the widespread use of antimicrobials and vaccinations, infectious diseases pose a major threat to livestock health. Therefore, research focused on the influence of host genetic variations in susceptibility to infectious diseases including FMD, is gaining relevance, to aid develop strategies for selective breeding. In addition, the stress posed by the viral infection on the animal result in production loss. An understanding of the changes in the pattern of clinical, serum biochemical, and hematological parameters during the course of FMD infection is important in developing therapeutic approaches for circumventing the stress and thereby reducing the negative effect of the infection in the animal. In addition, as mentioned elsewhere, a few Indian native breeds are said to be relatively tolerant of many infectious diseases including FMD. Therefore, the present study was aimed at studying the changes in some clinical, biochemical, and hematological parameters in different breeds of cattle experimentally infected with FMDV. Although hematological and serum biochemical changes have been reported in different species of animals -12, studTherefore, in this study, we evaluated the clinical, serum biochemical, and hematological responses during the course of experimental FMDV infection in Malnad Gidda, Hallikar (another Indian native breed), and Holstein Friesian crossbred calves.th day in all breeds while the healing of wound was early by 21 days post-infection in Malnad Gidda and Hallikar and 28 days in crossbred HF indicating Indian native breeds have better healing ability from infections.The onset of pyrexia started by day 1 in all the breeds, while peak pyrexia was observed on day 2 in Malnad Gidda and day 3 in Hallikar and crossbred HF. Peak lesions appeared on 53 and T4 on 7 days post-infection indicates the release of the stored hormones due to FMDV induced damage to follicular cells of thyroid rather than altering the negative feedback regulation. Further, a significant reduction in the serum triglyceride and total cholesterol on 7 days post-infection is likely due to the transient elevation of thyroid hormones, as reported earlier [The serum biochemistry showed no significant change in total protein, albumin, and A: G ratio. This is in contrast to a significant decrease in serum total proteins observed after natural infection in the previous studies -13. Furt earlier . Notably earlier .et al. [Interestingly, the alteration in the CK-MB level in Malnad Gidda was insignificant compared to Hallikar and HF crossbred calves. This suggested that Malnad Gidda suffered less myocardial tissue damage during FMDV infection. Hallikar and HF crossbred calves had significantly elevated CK-MB levels, which were corroborated by the typical arrhythmic changes such as preventricular complexes and prolonged ST waves observed in ECG on day 3 post-infection indicating myocardial damage. The ECG recorded on day 3 for Malnad Gidda and day 14 post-infection for all animals showed normal sinus rhythm. However, Aktas et al.  observedWith regard to hematological analysis, there was a significant decrease in total leukocyte, absolute counts of neutrophil, lymphocyte, and monocytes in FMDV infected calves. This is in agreement with previously reported leukopenia in cattle and swine during the virus infection ,19. AlthTo sum up, the FMD infected animals showed significant clinical signs during the course of infection, and the animals completely recovered as early as 21 and 28 days in case of native breeds and HF cross-breed animals, respectively. Except for thyroid profile, BUN, and CK-MB, all other serum biochemical parameters showed no significant abnormalities. Lymphopenia is the only hematological change. Based on these observations, it is suggested that effective ameliorative measures should be targeted mainly on the feed/water intake, thyroid gland, and the level of lymphocytes. As far as the breed difference is concerned, Malnad Gidda calves showed relatively less impact on myocardial changes and leukopenia. However, further studies on immunological and molecular pathways are needed to confirm their relative tolerance for FMD.SHB, AS and VU conceived the problem, designed the work, and finalized the manuscript. SS and MH carried out animal and laboratory experiments, MP and BHMP carried out animal experiments including ECG work, KN and BPS analyzed and interpreted the data. All authors read and approved the final manuscript.


P<0.05 was set as statistical significance. In total, 10 studies were included in this analysis. After pooled estimation, it was demonstrated that SARS-CoV-2 positive patients taking ACEIs/ARBs were not associated with an increased risk of mortality compared to those not taking ACEIs/ARBs . Furthermore, the risk of composite severe clinical manifestations was not significantly different between the positive patients with or without ACEIs/ARBs users . There was no risk difference for SARS-CoV-2 test positivity in patients with or without ACEIs/ARBs users . These findings may augment current professional society guidelines for not discontinuing ACEIs/ARBs in treating COVID-19 patients where it is clinically indicated.The effects of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) in the treatment of COVID-19 are highly debated. This study was aimed to assess aggregated risk by investigating the association of ACEIs/ARBs users against non-users of ACEIs/ARBs with the risk of mortality or severe clinical manifestations or magnitude of SARS-CoV-2 test positivity in COVID-19 patients. Systematic literature search was carried out in different databases for eligible studies. The pooled relative risks (RRs) were measured using RevMan software where  It has already been established that SARS-CoV-2 binds to the angiotensin-converting enzyme 2 (ACE2) receptor of the extracellular domain of the transmembrane to gain entry into host cells2. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) are frontline therapies in treating cardiovascular disorders including hypertension and diabetes; however, they may upregulate ACE2 expression in some animal models4. Data remains limited for human studies showing variations on plasma ACE2 levels, most importantly no study has identified their effect on lung-specific expression of ACE25.Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to substantial morbidity and mortality throughout the world since it was first detected in early December 2019 in Wuhan, China7. This may lead to clinical concerns that patients who are taking ACEIs/ARBs for combating cardiovascular diseases along with hypertension and diabetes are at an increased risk for becoming infected with SARS-CoV-2 and are at the highest risk of worst clinical outcomes8. However, in this clinical debating situation, it has also been postulated that ACE2 upregulation may improve clinical outcomes in SARS-CoV- 2 infected patients by protecting against lung injury9. Furthermore, in certain high-risk SARS-CoV-2 infected patients, the removal of ACEIs or ARBs may be detrimental5. Despite the lack of sufficient robust evidence, several professional societies have recommended continuing the administration of these medications in patients with COVID191.It is alarming that COVID-19 patients with comorbidities especially hypertension, diabetes, and cardiovascular diseases, have been found to be associated with the highest adverse clinical outcomes, e.g. deathsIt is therefore expected that researchers may have explored the associations of taking ACEIs/ ARBs with an increased risk of mortality or severe clinical manifestations (e.g. admitted to the intensive care unit (ICU)/using mechanical ventilation/deaths) or even the magnitude of positivity with the SARS-CoV-2 infection test during the COVID-19 pandemic.10. First, it did not clearly group patients with or without ACEIs/ARBs users for estimating its effects on SARS-CoV-2 infection. Second, when estimating aggregated risk of clinical outcomes, e.g. mortality or severity, this meta-analysis used data from some studies that were not either found in the literature or the mother studies itself did not have clear data on the patients using ACEIs/ARBs versus no-users of ACEIs/ARBs but adjusted with covariates, which makes come controversy regarding the outcomes of this meta-analysis.To date, only one meta-analysis has appeared in the literature but has several methodological limitations11. However, the severity of COVID-19 illness was not reported for the patients with or without ACEIs/ARBs users but was adjusted with other covariates in this analysis, which certainly renders lacking robust evidence regarding these conflicts. Most importantly, this analysis used the majority of the data from preprint articles which is not peer-reviewed making this analysis some concerns11.Another systematic review conducted by Mackey K et al. 2020 reported the association of ACEIs/ARBs users versus non-ACEIs/ARBs users with SARS-CoV-2 test positivity from limited perspectivesIt is evident from the perspectives of the current controversy that a robust study is needed showing the association of clinical manifestations of COVID-19 with or without ACEIs/ARBs users. Meanwhile, new studies may appear in the literature that need to be incorporated to update the literature. This study was therefore aimed to assess aggregated risk of mortality or severity of illness or SARS-CoV-2 test positivity in COVID-19 patients with or without ACEIs/ARBs users.20. It is pertinent to mention here that using combination of ACEIs and ARBs in COVID-19 patients not only lower blood pressure but also may maximize organ protection and was therefore used concurrently. Also, by stabilizing and internalization of ACE2–AT1R interaction, ACEIs/ARBs could prevent COVID-19 viral entry21. This may be another reason for using this combination in the management of COVID-19 patients.The selection process of included studies as undertaken following PRISMA guidelines for this meta-analysis is shown in Fig. 20 although only three studies18 reported outcomes with multiple comorbidities including hypertension and one study separately reported outcomes for hypertensive COVID-19 patients as well as patients with other comorbidities16. Out of 49,188 patients, 52.9% were male COVID-19 patients with an average age of 61.1 (IQR 50.8–70.1). Besides using ACEIs/ARBs, patients were taking other classes of drugs e.g. statins, antiplatelets, beta-blockers, antidiabetics, antidiuretics etc. Importantly, 35% COVID-19 patients had hypertension and other important comorbidities were diabetes, cardiovascular diseases, respiratory disease, kidney and liver disease, cancer etc. The important baseline characteristics of the included studies are summarized in Table Out of 10, eight studies provided data on mortality outcome, seven studies provided data on composite severe clinical manifestations outcome and only two studies provided data on SARS-CoV-2 test positivity as shown in Table P=0.48). This finding may be considered insightful since no study has measured the pooled effects of mortality for ACEIs/ARBs users against non-users of ACEIs/ARBs in COVID-19 patients using such a large sample size. Whilst controversial whether ACEIs/ARBs should or should not continue in these patients, this aggregated finding may be considered as the highest evidence and may reduce debating in this regard.The association of mortality was assessed in COVID-19 patients where 2,332 patients were taking ACEIs/ARBs and 6,743 patients were non-users of ACEIs/ARBs. As shown in Fig. P=0.28), as shown in Fig. The risk of composite severe clinical manifestations  was not significantly different between SARS-CoV-2 positive patients using ACEIs/ARBs versus non-users of ACEIs/ARBs , as shown in Fig. Many COVID-19 patients had multiple comorbidities and were using ACEIs/ARBs before SARS-CoV-2 testing, and many were started to use these medications after hospital admission. On the other hand, many patients were not using ACEIs/ARBs before or during hospitalization. It was of utmost importance to investigate whether prior or during hospitalization use of ACEIs/ARBs increased the magnitude of infections compared to those patients never using these medications. After pooled estimation, it was found that the magnitude of SARS-CoV-2 test positivity had no risk difference between the COVID-19 patients with or without prior or during hospitalization use of ACEIs/ARBs , figure not shown here. Although the number of studies included for assessing publication bias in this analysis was very small (n = 8), even after visual inspection of the funnel plot, there was no publication bias, as shown in Fig. In the sensitivity analysis, it was also found that there was no risk difference for severe clinical manifestations in COVID-19 hypertensive patients with or without ACEIs/ARBs users and  it was statistically insignificant  in ACEIs/ARBs users of SARS-CoV-2 positive patients compared to those not using these medications although it was statistically insignificant. These findings are consistent with other analyses carried out by independent researchers5. From the findings of the current study, it may be postulated that the organo-protective nature of upregulated ACE2 directly induced by the ACEIs/ARBs in COVID-19 patients was probably associated with the reduction of deaths compared to those not with ACEIs/ARBs. This is because, COVID-19 patients appeared to have elevated levels of plasma angiotensin II, which in turn caused lung injury22. It was also found that COVID-19 was particularly severe in patients with cardiovascular diseases, and many of these patients had active myocardial injury, myocardial stress and cardiomyopathy provoking the severity of illness and even death28. This may be due to the downregulation of ACE2 for SARS-CoV-2 infection and replication which in turn may be unable to exert protective effects in organs9. The ACEIs or ARBs may particularly use in COVID-19 hypertensive patients to manage hypertension and other cardiovascular diseases where it is assumed that using ACEIs/ARBs in COVID-19 patients may upregulate ACE2 enzyme which in turn may protect organ damage and may reduce associated death as well. Since no study has proven experimentally the association between ACEIs/ARBs use and reduction of mortality in COVID-19 patients found in this study, future experimental studies are warranted to firmly establish the exact molecular mechanism behind this scientifically novel outcome.Without elucidating the exact molecular mechanism, it would not be plausible to accurately explain governing the trends towards a slight reduction of mortality for using ACEIs/ARBs in patients with COVID-19 compared to those not with these medications. However, with accumulating existing evidence and data, it is reasonably believed that upregulation of ACE2 may act as a biological sword and is organ protective, e.g. protecting lung injury, myocardium and kidney, which is mainly executed by using ACEIs/ARBs in these patients29 conducted by Mancia et al. 2020 after multivariable adjustment indicated that neither ACEIs nor ARBs had a significant association with the risk of COVID-19, which is consistent with the findings of the present study.Although using ACEIs/ARBs in COVID-19 patients in general slightly increased the risk of composite severe clinical manifestations  but it failed to reach statistical significance. Importantly, when the association was assessed only for hypertensive COVID-19 patients, the risk drastically decreased, and there was almost no risk difference with or without ACEIs/ARBs users. This indicated that using ACEIs/ARBs in hypertensive COVID-19 patients actually had no significant risk differences compared to those not using these medications. A recent studyThe magnitude of infection positivity caused by SARS-CoV-2 was not found to be significantly different with or without ACEIs/ARBs users, which is scientifically exciting and may be considered new insights for COVID-19 patients. Again, future experimental studies are warranted to elucidate the molecular mechanism for these associations.17. Because the results of this meta-analysis pooled data from individual studies that were unadjusted for known confounders, e.g. age, sex, race, ethnicity and comorbidities such as diabetes, chronic kidney disease and heart failure, the findings of the current study cannot completely address the complexity of this question. However, a recent meta-analysis indicated that COVID-19 male patients and those with age over 50 years were associated with a significantly increased risk of mortality. Comorbidities were also associated with a significantly increased risk of mortality in patients with COVID-196. Although data about the impacts of ethnicity on the risk of COVID-19 in published literature remains limited, however, a systematic review conducted by Pan D et al. 2020 suggested that Black, Asian and Minority Ethnic (BAME) individuals were at an increased risk of SARS-CoV-2 infection and worse clinical outcomes from COVID-19 compared to White individuals30. Future clinical studies are warranted again to address these issues. However, with aggregating existing data in the literature to date, this study provides evidence that using ACEIs/ARBs in COVID-19 patients in general and in particular hypertensive patients were not associated with an increased risk for composite severe clinical manifestations or SARS-CoV-2 test positivity compared to those patients not taking these medications. Instead, using ACEIs/ARBs in these patients compared to non-using patients was associated with a statistically insignificant slight reduction in the risk of mortality. Although confounding factors e.g. age, sex, ethnicities, comorbidities were not adjusted for the current analysis, however, this evidence may be considered insightful findings in the current perspectives of controversy and lack of sufficient robust evidence in the literature.This is an important concern whether it should or should not continue ACEIs/ARBs in COVID-19 patients especially with hypertension because these medications are the most prevalent antihypertensive medications among all therapeutic classes used generallyAlthough this study has great findings in terms of providing evidence regarding whether ACEIs/ARBs should or should not continue with COVID-19 patients, there are some limitations of this analysis. First, it was not possible to extract data to examine the effects of age, sex, and comorbidities in ACEIs/ARBs users against non-users of ACEIs/ARBs, which may be accountable for the high level of heterogeneity. Second, due to the unavailability of data, it was not possible to estimate the dosage regimen effects of ACEIs/ARBs. Third, there was no information about the types of drugs used in users vs. non-users of ACEIs/ARBs in the majority of the included studies since there are many drugs falling into the classes of ACEIs/ARBs, which may increase the inconsistency of the outcomes. Fourth, since definition of ICU admission/mechanical ventilation/death collectively referred to as ‘composite severe clinical manifestations’ were varied slightly in the included studies, hence the risk of composite severe clinical manifestations may vary accordingly. It is therefore suggested to address these issues in future clinical studies.In conclusions, the findings of this analysis indicated that using ACEIs/ARBs in SARS-CoV-2 positive patients was not associated with an increased risk of mortality, rather, it had a trend towards a slight reduction in the risk of mortality, although results were not adjusted for other confounding factors, e.g. age, sex, comorbidities etc. Furthermore, it was also found that neither severe clinical manifestations nor magnitude of SARS-CoV-2 infectivity were significantly different with or without ACEIs/ARBs users. These findings may augment current professional society guidelines for not discontinuing ACEIs/ARBs in treating COVID-19 patients where it is clinically indicated.31. Keywords were searched as "2019 novel coronavirus with ACEIs/ARBs" or "2019-nCoV with ACEIs/ARBs" or "SARS-CoV-2 with ACEIs/ARBs" or "COVID-19 with ACEIs/ARBs" or "angiotensin-converting enzyme inhibitors (ACEIs) with COVID-19/SARS-CoV-2" or "angiotensin II receptor blockers (ARBs) with COVID-19/SARS-CoV-2" or "SARS-CoV-2 with ACEIs/ARBs" AND "clinical outcomes or death or severe illness or severe clinical outcomes or test positivity with SARS-CoV-2 or clinical features/characteristics". Additionally, different important journal websites e.g. New England Journal of Medicine, Lancet, JAMA and Nature were checked for the relevant articles.The Literature was searched in PubMed from inception to May 31, 2020 following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines as described elsewhereThe studies were included if they fulfilled the following criteria: (1) studies must have two arms of COVID-19 patients in which one arm used ACEIs/ARBs whereas the other arm did not use ACEIs/ARBs; (2) studies must have reported outcomes for at least one of the three clinical outcomes in both arms; (a) mortality (b) composite severe clinical manifestations which include a combination of patients admitted to the ICU/using mechanical ventilation/mortality (c) magnitude of SARS-CoV-2 test positivity; and (3) studies must be original peer-reviewed published research that had sufficient data to be included as described above.The studies were excluded based on the following criteria: (1) if the study had only one arm of treatment group using either ACEIs or ARBs but did not have another arm; (2) if the study did not clearly report outcome data for two selected arms of patients as described above; and (3) if the studies were reviews, systematic reviews, viewpoints, perspectives or correspondences.32 was used for primary selection of the studies after importing all the literature search histories in this software following inclusion and exclusion criteria. For final selection of the studies, full texts of all primary included studies were retrieved and checked one by one. When the articles were finally selected, the full text of the included studies was carefully checked for data synthesis and quality assessment purposes. All the selection process was carried out by two investigators independently, and at the end of selection, any disagreement was resolved by principal investigator. Data synthesis and entry were double checked and validated by a third independent reviewer. To determine the quality of the included studies, Newcastle Ottawa assessment scale guidelines were followed as described elsewhere33.Rayyan QCRI, a systematic review software tool34. Meanwhile, the I2 statistic was used to efficiently test for heterogeneity, where I2 < 25%, I2 = 25–50% and I2 > 50% indicate low, moderate and high degrees of heterogeneity, respectively35. A random effects model was used to estimate pooled effects if I2 > 50 and a fixed effects model was applied to calculate pooled effects when I2 < 50. Sensitivity analyses were carried out to measure any significant risk differences between the studies, especially if heterogeneity was found. This was accomplished by removing the included study one after another. In addition, publication bias was carried out by visual inspection of the funnel plot where symmetrical distribution of the plot indicated the absence of publication bias36. Funnel plot asymmetry was also assessed with Egger’s test37. Review-Manager software  was used for analyzing all data where the level of statistical significance was set as <0.05 (2-sided).Estimation of pooled effects was calculated as risk ratios (RRs) and 95% Confidence Intervals (CIs) using the Mantel–Haenssel (M-H) method following either a random or fixed effect model based on the levels of heterogeneity of the included studies. Heterogeneity in the forest plot was evaluated by using the Cochrane chi-square-based Q-test and regarded as significant if the p value was less than 0.1Supplementary Information


Background: SARS-CoV-2-infected subjects have been proven contagious in the symptomatic, pre-symptomatic and asymptomatic phase. The identification of these patients is crucial in order to prevent virus circulation. No reliable data on the sensitivity of nasopharyngeal swabs (NPS) are available because of the lack of a shared reference standard to identify SARS-CoV-2 infected patients. The aim of our study was to collect data on patients with a known diagnosis of COVID-19 who underwent serial testing to assess NPS sensitivity.Methods: The study was a multi-center, observational, retrospective clinical study with consecutive enrollment. We enrolled patients who met all of the following inclusion criteria: clinical recovery, documented SARS-CoV-2 infection (≥1 positive rRT-PCR result) and ≥1 positive NPS among the first two follow-up swabs. A positive NPS not preceded by a negative nasopharyngeal swab collected 24–48 h earlier was considered a true positive. A negative NPS followed by a positive NPS collected 24–48 h later was regarded as a false negative. The primary outcome was to define sensitivity of SARS-CoV-2 detection with NPS.Results: Three hundred and ninety three NPS were evaluated in 233 patients; the sensitivity was 77% . Sensitivity of the first follow-up NPS (n = 233) was 79%  with no significant variations over time. We found no statistically significant differences in the sensitivity of the first follow-up NPS according to time since symptom onset, age, sex, number of comorbidities, and onset symptoms.Conclusions: NPS utility in the diagnostic algorithm of COVID-19 should be reconsidered. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible of coronavirus disease 2019 (COVID-19) . The ideThe study was a multi-center, observational, retrospective clinical study with consecutive enrollment. Participating centers included IRCCS Fondazione Ca' Granda Ospedale Maggiore Policlinico and ASST Santi Paolo e Carlo, Università degli Studi di Milano, both based in Milan. All patients who sequentially referred to the two participating centers for follow-up outpatient testing with nasopharyngeal swabs between 05/03/2020 and 20/05/2020 were screened for enrollment. We enrolled patients who met all of the following inclusion criteria: clinical recovery , documented SARS-CoV-2 infection (≥1 positive rRT-PCR result) and ≥1 positive nasopharyngeal swab among the first two follow-up swabs. We excluded patients whose first two follow-up swabs delivered negative results .All patients, once clinically recovered, were to undergo the first follow-up swab after 14 days since hospital discharge. In case of a positive result, the test was to be repeated after 7 days; in case of a negative result, a second swab was performed 24–48 h later. If this was also negative, patient isolation was ended ; if posiA positive nasopharyngeal swab not preceded by a negative nasopharyngeal swab collected 24–48 h earlier was considered a true positive (TP). A negative nasopharyngeal swab followed by a positive nasopharyngeal swab collected 24–48 h later was regarded as a false negative (FN) .The primary outcome was to define sensitivity of SARS-CoV-2 detection with nasopharyngeal swabs. The secondary outcome was to evaluate nasopharyngeal swab sensitivity over time and the association between the sensitivity of the first nasopharyngeal follow-up swab and the patient's age, sex, comorbidities, and onset symptoms.We estimated that the enrollment of 230 patients with documented SARS-CoV-2 infection would enable to estimate a 63% sensitivity with an acceptable precision, as quantified by the 95% CI (56 to 69%). Patient data have been recorded on Microsoft Excel and analyzed with STATA 15 . Data were expressed as means ± standard deviation (SD) or medians with interquartile ranges (IQR) as appropriate and sensitivities were compared using the Chi-squared test.Seven hundred and six patients were screened for enrollment after referral to the two participating centers for serial nasopharyngeal swab outpatient testing between 05/03/2020 and 20/05/2020. Four hundred and seventy-three patients reached viral clearance after the first two follow-up nasopharyngeal swabs, and were therefore excluded. Two hundred and thirty-three patients met all inclusion criteria. All patients had ≥1 positive rRT-PCR test collected at the time of diagnosis of COVID-19. All patients received the first follow-up swab once clinically recovered, in most cases 14 days after hospital discharge. The patients' median age was 55.3 years , 39% of the patients were women, 74% were Caucasian, 15% were Hispanic, 7% were Maghrebian, Middle Eastern or Arab, and 3% were Asian. The ethnicity of 4 patients was unknown. Clinical data were available for 222 patients. Forty-nine patients (22%) had no comorbidities, while 169 (78%) had ≥1. One hundred and eighty-seven patients (84%) had pneumonia. The most frequent onset symptoms were fever (96%), cough (80%), dyspnea (47%), fatigue (45%), ageusia (41%), and anosmia (34%). All patients underwent ≥1 follow-up swab. The median number of TP and FN nasopharyngeal swabs per patient was 1 . Of the 233 patients included in our analysis, 182 (78%) reached viral clearance. At the end of the study period data collection was still ongoing for the remaining 51 (22%). The median time to viral clearance was 45.0 days  since symptom onset.n = 233) was 79%  with no significant variations over time . Sensitivity of the first follow-up nasopharyngeal swabs . Wang et al. evaluated SARS-CoV-2 detectability in different biological specimens in a similar cohort of COVID-19 patients and found a nasopharyngeal swab sensitivity of 63% . Two of Our sensitivity assessment might have been overestimated. Firstly, most of the patients included in our study had ≥1 positive nasopharyngeal swab at time of diagnosis. Secondly, positive follow-up nasopharyngeal swabs weren't followed by a second swab 24–48 h later, unlike negative swabs; this might have led to the underestimation of the number of FN nasopharyngeal swabs. On the other hand, we didn't perform further nasopharyngeal swabs once viral clearance was reached; this, too, might have contributed to the underestimation of total FN swabs.Our study has some limitations. The fact that nasopharyngeal swab sensitivity varies throughout disease course limits the external validity of our findings. Although we might have overestimated true nasopharyngeal swab sensitivity for the aforementioned reasons, sensitivity may be likely higher at the beginning of the disease, when viral shedding is greater. We did not investigate the association between the antiviral treatment received during hospitalization and sensitivity of the first follow-up swab. Lastly, as recommended by the WHO , we didnIn conclusion, in our large cohort of COVID-19 patients, sensitivity of SARS-CoV-2 detection with rRT-PCR on nasopharyngeal swabs was 77% . For the purposes of our study, we assumed specificity to be 100%. It is however safe to say that a positive nasopharyngeal swab indicates SARS-CoV-2 infection. Conversely, with consideration of the 77% sensitivity we found, a negative result alone cannot rule out infection when this is suspected on clinical or epidemiological grounds. This has led to serial retesting in clinical practice. We think that such a strategy should not be encouraged since it is time-consuming, requires complex organization and leads to facilities overcrowding and delay in treatment initiation. An alternative diagnostic strategy is urgently needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comitato Etico Milano Area. All subjects signed a written informed consent to personal data treatment, which allowed the anonymous use of clinical data for research purposes.AM, FB, DP, MO, GM, and VC provided clinical data and revised the manuscript. GMP conceived the study and wrote the manuscript. BC wrote and edited the manuscript. GCo conceived and designed the study and revised the manuscript. GCa performed the analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.