Are new-generation tyrosine kinase inhibitors associated with a higher risk of hepatotoxicity than imatinib in patients with chronic myeloid leukemia?In this meta-analysis of 9 studies including 3475 patients, bosutinib, nilotinib, and ponatinib were associated with an increased risk of hepatotoxicity compared with imatinib. No significant difference was found with dasatinib.These findings suggest that hepatic function should be monitored frequently in patients treated with bosutinib, nilotinib, and ponatinib. This systematic review and meta-analysis examines the relative risk of hepatotoxicity of new-generation BCR-ABL fusion oncoprotein tyrosine kinase inhibitors (TKIs) vs imatinib among patients with chronic myeloid leukemia. Although BCR-ABL fusion oncoprotein tyrosine kinase inhibitors (BCR-ABL TKIs) can substantially improve the survival rate of chronic myeloid leukemia (CML), they are clinically accompanied by severe hepatotoxicity.To compare the relative risk (RR) of hepatotoxicity of new-generation BCR-ABL TKIs with that of imatinib, and to provide an overall assessment of the clinical benefit.PubMed, Embase, Cochrane library databases, and ClinicalTrials.gov were searched for clinical trials published between January 2000 and April 2020.Study selection was conducted independently by 2 investigators according to the inclusion and exclusion criteria published previously in the protocol: only randomized phase 2 or phase 3 clinical trials that compared bosutinib, dasatinib, nilotinib, or ponatinib with imatinib were included. Among the 2666 records identified, 9 studies finally fulfilled the established criteria.Two investigators extracted study characteristics and data independently using a standardized data extraction form. Data were extracted according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines. When substantial heterogeneity was observed, pooled estimates were calculated based on the random-effect model; otherwise, the fixed-effect model was used.Data extracted included study characteristics, baseline patient information, interventions and data on all-grade and high-grade (grades 3 and 4) elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, overall survival, and major molecular response (MMR). The RRs and 95% CIs were calculated using the inverse variance method.P < .001) and grades 3 and 4 ALT elevation  compared with those receiving imatinib. Patients receiving new-generation TKIs were also more likely to experience all grades of AST elevation  and grades 3 and 4 AST elevation  compared with those receiving imatinib. New-generation TKIs were associated with a significantly higher rate of MMR at 1 year compared with imatinib . No statistical difference in overall survival at 1 year was found between new-generation TKIs and imatinib .Nine trials involving 3475 patients were analyzed; the median (range) age was 49 (18-91) years; 2059 (59.2%) were male patients. Increased risks were observed for each new-generation TKI except for dasatinib. Patients receiving new-generation TKIs were more likely to experience all grades of ALT elevation (pooled RR, 2.89; 95% CI, 1.78-4.69; When compared to imatinib, bosutinib, nilotinib, and ponatinib had higher relative risks of hepatotoxicity. Treatment with new-generation TKIs was associated with a higher MMR rate at 1 year but not with 1-year overall survival. Severe liver injury was also reported in patients with CML treated with nilotinib and dasatinib.11 However, among several studies comparing imatinib with the other BCR-ABL TKIs,19 only one mentions hepatic effects without stratification study.19 Hence, it is necessary to perform a meta-analysis focusing specifically on the relative risk of hepatotoxicity associated with each BCR-ABL TKI.Drug-induced hepatotoxicity is a major safety concern in clinical therapy, because nearly 50% of oral drugs on the market are associated with hepatotoxicity.The aim of this study was to compare the risk of all grades and high grades (grades 3 and 4) hepatotoxicity associated with 4 new-generation BCR-ABL TKIs  vs the first generation BCR-ABL TKI imatinib in patients with CML. Stratification by BCR-ABL TKI treatment was performed to provide drug-specific risk assessments. Overall survival (OS) and major molecular response (MMR) were also analyzed to provide global risk-benefit evaluations.The primary outcome of interest was hepatotoxicity, including all grades and grades 3 and 4 of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. Each variable of all grades and grades 3 and 4 was analyzed to compare the hepatotoxicity of a new generation of BCR-ABL TKI with that of imatinib. Adverse events were defined as the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0 or 4.0. Data on the OS and the MMR reported at 12 months of TKI treatment were also extracted.PRISMA) reporting guideline.20 We searched the following databases using keywords and medical subject headings from January 2000 to April 2020: PubMed, Embase, and Cochrane library databases  and any disagreements were resolved by consensus. Data extracted for each study were: first author’s name, year of publication, trial phase, number of enrolled participants, drugs used in the experimental and standard arm, dosage, frequency, type of cancer, stage of disease, median age, sex, and data on ALT levels, AST levels, OS, and MMR. Some of these data were found at ClinicalTrials.gov to ensure data integrity. Race/ethnicity data were not collected because too many countries and regions were involved.Q statistic and 2I statistic were applied to evaluate the heterogeneity among the various trials. When substantial heterogeneity was observed (P < .10 for Q-test or 2I > 50%), the pooled estimate was calculated based on the random-effect model, otherwise, the fixed-effect model was used. The relative risk (RR) and 95% CI were calculated using the inverse variance method. Additionally, funnel plots and the Egger test were used to assess the publication bias of the enrolled studies. A 2-sided statistical test with P < .05 was considered significant. Statistical analysis was performed from April 2020 to March 2021.All calculations were performed using RevMan software version 5.3 (Cochrane Library). The 20 After the first screening (reading titles and abstracts), 72 articles and abstracts fulfilled the previously established criteria and were evaluated in their entirety. Of these, 30 studies were identified as duplicate trials; 12 studies were conference abstracts and posters; another 12 were excluded because of the absence of hepatic toxicity data. Of remaining articles, 4 were single-group trials, and 2 were non-imatinib controlled trials. At the end of the selection procedure 3 studies were excluded because of the limited sample sizes. Finally, a total of 9 studies with 3475 patients were included for statistical analysis , and 1743 remained after deduplication.The studies included in the meta-analysis were published between 2010 and 2018. TKIs included in the analysis were bosutinib (n = 2), dasatinib (n = 3), nilotinib (n = 3), and ponatinib (n = 1). As per inclusion criteria, all included studies were randomized, imatinib-controlled trials. Only one study was conducted in a single country (China), and 6 of 9 studies (67%) were conducted in the US or Canada in partnership with other countries. All 3475 patients included in this meta-analysis were diagnosed with the chronic phase of CML (CP CML); 2059 (59.2%) were male patients; and the median (range) patient age was 49 (18 to 91) years. Other characteristics and Jadad score of the included studies are shown in eTable 2 in the The results of the fixed- or random-effects model RR for the elevation of ALT and AST levels, OS, and MMR were summarized in P < .001) (P < .001), nilotinib , and ponatinib  were associated with an increased risk of all grades of ALT elevation, but dasatinib was not associated with an increased risk of all grades of ALT elevation .All grades of ALT elevation occurred in 654 of 1840 patients (35.5%) treated with new-generation TKIs vs 151 of 1564 patients (9.7%) treated with imatinib. The use of new-generation TKIs was associated with a statistically significant overall increase in the risk of developing all grades of ALT elevation compared with imatinib  A. When sP < .001) compared with controls (P < .001) and nilotinib  were associated with an increased risk of grades 3 and 4 ALT elevation, whereas dasatinib  and ponatinib  were not.Grades 3 and 4 ALT elevation was observed in 150 of 1718 patients (8.73%) treated with new-generation TKIs compared with 24 of 1440 patients (1.67%) treated with imatinib. Patients who received new-generation TKIs were more likely to develop grades 3 and 4 elevation of ALT levels  (P < .001), nilotinib , and ponatinib , whereas no difference was found for dasatinib .All grades of AST elevation occurred in 440 of 1739 patients (25.3%) treated with new-generation TKIs vs 144 of 1461 patients (9.9%) treated with imatinib. New-generation TKIs were also associated with a significantly increased risk of all grades of AST elevation compared with imatinib  A. SubgroP < .001) (P < .001) but not the other TKIs .Grades 3 and 4 AST elevation was observed in 64 of 1617 patients (3.9%) treated with new-generation TKIs vs 20 of 1338 patients (1.5%) treated with imatinib. Compared with imatinib, new-generation TKIs were associated with a significantly increased risk of high-grade AST elevation  B. SignifP = .33). Stratification by treatment did not change the results .As shown in P < .001). Similar results were observed for each TKI, although the increase of MMR rate for ponatinib did not achieve statistical significance .As shown in 32 Previously published meta-analyses also found that the use of vascular endothelial growth factor receptor (VEGFR) TKIs, epidermal growth factor receptor (EGFR) TKIs, and anaplastic lymphoma kinase (ALK) TKIs significantly increased the risk of developing liver toxicities.36 A recent meta-analysis indicated that patients treated with new-generation BCR-ABL TKIs were associated with more frequent hepatic events than imatinib,19 but it did not make stratified meta-analyses of these drugs.Hepatotoxicity is one of the serious safety issues signaled in preapproval clinical trials with TKIs. The FDA has approved more than 60 TKIs for human use as of June 2020, with 5 having black box warnings for hepatotoxicity in product labeling . Clinical trials indicated a low-grade elevation in serum liver enzyme (ALT and/or AST) in 25% to 30% and a high-grade elevation in approximately 2% of patients treated with TKIs.To our knowledge, this is the first meta-analysis focusing specifically on hepatotoxicity associated with new-generation TKIs vs the first generation TKI imatinib in patients with CML. Overall survival and efficacy were also analyzed to provide global risk-benefit evaluation. We were able to demonstrate that the treatment with bosutinib, nilotinib, or ponatinib was associated with a higher risk of hepatotoxicity than that of imatinib, whereas no significant increased risks were found for dasatinib. New-generation TKIs were associated with improvement in the rate of MMR but not for 1-year overall survival.37 It has been demonstrated previously that cytochrome P450-mediated bioactivation of gefitinib, erlotinib, lapatinib, pazopanib, and sunitinib can generate reactive quinone-imine and epoxide compounds.42 These reactive metabolites can covalently bind to nucleophilic macromolecules in target cells such as endogenous protein, lipids, or DNA, causing irreversible cellular damage and even cell death. Immune mediated response and drug metabolizing enzyme-based drug-drug interactions have also been proposed to be a mechanism of toxicity.44 Although little is known about the underlying mechanisms of BCR-ABL TKIs–induced liver injury, it has been reported that the direct and indirect mitochondrial toxicity may represent a toxicological mechanism of dasatinib and ponatinib.46 Furthermore, UGT1A1 is the sole physiologically relevant enzyme participating in the metabolic elimination of bilirubin, an endogenous toxic metabolite. The inhibition of UGT1A1 may lead to accumulation of bilirubin to toxic levels, which may be the mechanism of atazanavir-, indinavir-, erlotinib-, and nilotinib-related hepatic toxicities .49 Further studies will be needed to investigate the mechanism of BCR-ABL TKIs–associated hepatotoxicity.The precise mechanism of TKI-related hepatotoxicity is still not well clarified. It has been hypothesized that the formation of reactive metabolites during metabolism plays a key role in TKI-induced hepatotoxicity.50 For older patients with the goal of the treatment of improving survival, imatinib is the first choice. If intolerance or resistance occurs, it is recommended to switch to dasatinib. For patients with newly diagnosed CP CML without baseline hepatic impairment comorbidities and for whom achieving a deep molecular response is the predominant objective, dasatinib remains to be an excellent choice. In cases of treatment failure, bosutinib and nilotinib are preferred. Particularly, the use of bosutinib and nilotinib as first-line treatments requires a screening for potential risk factors . Ponatinib should be reserved for patients with advanced disease, with the T315I variant, or for whom other treatments cannot be used.To provide a personalized medication strategy, the physician should consider the patient’s characteristics and the efficacy and safety profiles of the different TKIs. Imatinib, nilotinib, and dasatinib are currently registered for the first-line treatment of CP CML.A limitation in this analysis concerns the lack of access to individual data that might have provided important additional information, including the number of deaths due to liver failure and the baseline hepatic risk factors in each study group. Reporting of hepatic adverse events was also lacking in many studies, and the exclusion of these studies might have introduced bias into the analysis. Moreover, apart from elevation of ALT and AST, other biochemical markers of liver injury include bilirubin, alkaline phosphatase, albumin concentration, and the prothrombin time should be further analyzed to improve sensitivity or specificity of the evaluation of liver injury. Furthermore, the exclusive reliance on English-language studies may not represent all the evidence.29 further studies are needed to clearly define the dose regimen of each BCR-ABL TKI, which will provide the best clinically relevant benefit-risk profile.This meta-analysis found a significant increase in the risk of hepatotoxicity associated with the use of bosutinib, nilotinib, and ponatinib compared with imatinib. Treatment with these TKIs should be considered with frequent hepatic function monitoring. As the risk of hepatotoxicity of nilotinib seems to be associated with the dose administered,


Mortality and morbidity associated with COVID-19 continue to be significantly high worldwide, owing to the absence of effective treatment strategies. The emergence of different variants of SARS-CoV-2 is also a considerable source of concern and has led to challenges in the development of better prevention and treatment strategies, including vaccines. Immune dysregulation due to pro-inflammatory mediators has worsened the situation in COVID-19 patients. Inflammasomes play a critical role in modulating pro-inflammatory cytokines in the pathogenesis of COVID-19 and their activation is associated with poor clinical outcomes. Numerous preclinical and clinical trials for COVID-19 treatment using different approaches are currently underway. Targeting different inflammasomes to reduce the cytokine storm, and its associated complications, in COVID-19 patients is a new area of research. Non-coding RNAs, targeting inflammasome activation, may serve as an effective treatment strategy. However, the efficacy of these therapeutic agents is highly dependent on the delivery system. MicroRNAs and long non-coding RNAs, in conjunction with an efficient delivery vehicle, present a potential strategy for regulating NLRP3 activity through various RNA interference (RNAi) mechanisms. In this regard, the use of nanomaterials and other vehicle types for the delivery of RNAi-based therapeutic molecules for COVID-19 may serve as a novel approach for enhancing drug efficacy. The present review briefly summarizes immune dysregulation and its consequences, the roles of different non-coding RNAs in regulating the NLRP3 inflammasome, distinct types of vectors for their delivery, and potential therapeutic targets of microRNA for treatment of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease, 2019 (COVID-19), which first originated in the Chinese city of Wuhan and has since claimed the lives of over 5.1 million individuals worldwide . The inf2+) from the Golgi apparatus, consequently activating the inflammasome. Additionally, non-structural accessory proteins of SARS-CoV-2, including open reading frame 3a (orf3a) and orf8b proteins, trigger inflammasome activation and pyroptosis by potentially causing ionic imbalance along the cell membrane, mitochondrial dysfunction producing reactive oxygen species (ROS), and activating intracellular stress pathways . M. M147]. Inflammasome activation is important for host defense against viral infections, because these molecular sensors can trigger an innate immune response against invading pathogens. Emerging evidence has shown how different viruses regulate inflammasome activity via miRNAs. One such example is Epstein–Barr virus (EBV) miR-BART15 targeting the miR-223 binding site in NLRP3 3’ UTR site. Secretion of this miRNA from EBV-infected B cells was observed using exosomes as vehicles for delivery into non-infected cells . NLRP3 cLike miRNAs, lncRNA can maintain the homeostasis of the immune system by preventing dysregulation of the NLRP3 inflammasome. These transcripts regulate NLRP3 in both positive and negative manners under various cellular conditions  Figure . The lncThe lncRNA KCNQ1OT11 targets and binds miR-214, and hence prevents the binding of miR-214 to caspase 1, while miR-214 is reported to confer an inhibitory effect on caspase 1 activity . In brieA number of lncRNAs also modulate the immune response by negatively regulating the NLRP3 inflammasome or its components . For exaRecent clinical and computational studies on COVID-19 patients reported the presence of miRNAs and predicted their role in the disease pathophysiology. The prediction showed that both virus and host miRNAs might play essential roles in the pathogenesis of COVID-19. Upregulation of miRNAs, such as miRNA-16-2-3p, miRNA-6501-5p, and miRNA-618, and downregulation of miRNA-627-5p in peripheral blood, were reported in COVID-19 patients . SARS-CoN gene along with 5′ end of the viral genome (5′UTR), orf1ab, orf2 , orf5 , orf7a, and orf10 of SARS-CoV-2, which mainly include the coding regions for structural and accessory proteins [In addition to host miRNAs, studies also showed the presence of SARS-CoV-2-derived miRNAs. Recently our research group have demonstrated how miRNAs derived from the SARS-CoV-2 genome regulate host metabolic pathways . In thisproteins . We haveproteins .CASP 1 and IL-1β [These microRNAs and lncRNAs could be used to target specific SARS-CoV-2 motifs. According to a study, the SARS-CoV-2 genome contains motif sequences in the 5′ UTR leader sequence that can be selectively recognized by specific human miRNAs and lncRNAs, such as H19, Hotair, Fendrr, and LINC05 . Our stund IL-1β . AdditioNcRNAs present a novel therapeutic target for respiratory diseases and viral infections. However, several challenges need to be addressed for the effective utilization of ncRNAs as a potent therapeutic tool . These tNew concepts in human therapeutics are emerging as a result of advances in our understanding of RNA biological processing and regulation with functional implications in the therapies for respiratory viral infections . VariousThe application of RNA viruses to transiently deliver genetic information for cell manipulation in basic research and clinical therapy is an increasingly common practice. Multiple viral vectors have been employed for gene transfer into a particular target site. Viral vectors are viruses that have been modified for the purpose of gene delivery, including adenoviruses, adeno-associated viruses (AAVs), and retroviruses. These viruses lack replication properties but can ensure targeted delivery and constant expression of their cargo ,190.Adenoviral vectors (ADV) are the most commonly used viral vectors for clinical trials of gene therapeutic agents. ADVs have an icosahedral protein capsid and a 36-kb linear duplex DNA genome that can carry around 8–30 kb foreign cargo with the ability to transfect both dividing and non-dividing cells with high expression levels ,192. ADVAAVs can also deliver small RNA payloads, owing to their small ssDNA genome containing only two genes that can be replaced with foreign genetic material, and require adenoviruses for replication and vector production. However, engineered AAVs can perform these functions on their own ,194. AAVRetroviruses represent another class of vectors that perfectly integrate their genomes into the host, ensuring the long-term expression of the delivered payload. The viral genome comprises a minimum of three genes and can be replaced with the gene of interest because viral entry into the host is independent of viral proteins . To dateBiodegradable polymers are more widely used as a delivery system for miRNAs than viral vectors, owing to their low toxicity and versatile chemical nature . PolyethOn the other hand, the RNA payload conjugated with PEI only was separated in the lungs and rapidly excreted . Polyes. This stChitosan is another natural polymer with diverse biological properties and can effectively deliver ncRNAs or their mimics into host cells . ChitosaNanomaterials are at the forefront of the fast-paced growth of nanotechnology. These materials are superior and indispensable in several human activities due to their unique size-dependent properties. An NP, also known as an ultrafine particle, is a small particle with a diameter of 1 to 100 nm. NPs also provide a valuable option for the controlled delivery of ncRNAs for therapeutic purposes. These particles facilitate targeted and protected delivery of their payloads, thus showing enhanced therapeutic potential . ANP-basLNPs are also commonly used delivery systems, since lipid is the main component of the cell membrane. Interactions of lipid with the cell membrane facilitate uptake of the RNA payload by target cells . The strExosome-based carriers have emerged as a new class of vehicles that exhibit high safety and bioavailability along with low toxicity and immunogenicity. Exosomes represent a class of endogenous NPs secreted by many different cell types. These molecules are characterized by size, chemical composition, and the ability to carry various cargo types (such as RNA and proteins) and are involved in regulating intracellular communications. Alterations in exosome functions are associated with several disorders and are of significant value as diagnostic and therapeutic tools . ExosomeBryniarski and colleagues successfully demonstrated T-cell tolerance in allergic cutaneous contact hypersensitivity mice through miR-150 transfer by exosome-like nanovesicles regulating T-cells . In anotThese studies clearly support the potential of exosomes as a reliable vehicle for the delivery of RNA-based therapeutics. However, their use is limited by the lack of an efficient method to load RNA cargo into the exosome . SpecifiGiven mounting evidence that the most harmful SARS-CoV-2 reactions occur predominantly in the respiratory system, locally targeted therapy delivery via proper delivery vehicles is critical. Experiments are currently underway to test various delivery vehicles for vaccine development and other treatments. Viral vectors or virus-like particles are being used in a number of ongoing COVID-19 vaccine candidate trials . AdenoviThe COVID-19 pandemic has emerged as the worst health crisis the modern world has ever witnessed, causing substantial human and economic losses. The disease’s severity ranges from mild pneumonia cases to life-threatening conditions such as ARDS. SARS-CoV-2, along with its different variants, can activate the NLRP3 inflammasome either via altering the expression of ACE2 protein or the production of ROS and ionic imbalance in the cell membrane. NLRP3 inflammasome activation could trigger a cytokine storm in the lungs as well as other organs, ultimately leading to organ failure and death. Regulation of inflammasome is one avenue that could significantly reduce COVID-19-inflicted damage. NcRNAs and their synthetic counterparts represent one of the most efficient tools to regulate inflammasomes at different levels. These transcripts act via different mechanisms and could relieve severe symptoms, with potential to save human lives if used as therapeutic agents. However, the utility of ncRNAs is limited, owing to various challenges, such as short half-life, low stability in fluids, immune reaction, and unintended toxicity. The lack of an efficient delivery system also limits their use as a potent and safe therapeutic option. Recently, exosome-derived carriers have been investigated as a new type of vector for delivering biological cargo into target sites. These carriers are less toxic because they are secreted by different types of cells in the body, but their utility as a biological vector is yet to be established. Further research is warranted to fine-tune these vectors for optimizing their therapeutic effects. In conclusion, considering ncRNAs, coupled with efficient and safe delivery systems, should provide an effective targeted therapeutic option for managing COVID-19-associated immune dysregulation and future global health challenges.


The systemic autoinflammatory diseases are rare conditions; to date, data on coronavirus disease 2019 (COVID-19) infection and vaccination safety are scarce. Agents targeting innate immune pathways have transformed the management of affected patients, and their outcomes are of wider interest given the role of inflammation in both viral clearance and severe COVID-19 disease. We surveyed patients with systemic autoinflammatory disease on biologic therapy to determine the prevalence and outcomes of COVID-19 infection and to gather early safety data on vaccination. Electronic medical records of 248 patients with systemic autoinflammatory disease on biologic therapy at a national centre were reviewed. Patients were then surveyed in clinic or using a Web-based survey.In the cohort of 248 patients, no deaths were recorded. One hundred and seventy-five survey responses were received. Among the respondents, 27 reported suspected COVID-19 infection, of which 14 were confirmed by testing (8.0%). Two patients required hospital admission owing to dehydration. No patient required respiratory support or intensive care. One hundred and thirty-eight doses of COVID-19 vaccine had been administered to 130 patients. Side effects were reported after 71 of 138 (51.4%) administrations and were consistent with a flare of the underlying disease in 26 of 138 (18.8%) instances. No serious adverse events or hospital admissions were reported after vaccination.These data, including the largest published series of patients on anti-IL-1/6 biologics to receive any adenoviral vector or messenger RNA vaccine, show no serious early concerns regarding vaccination and will provide an urgently needed resource to inform decision-making of these patients and their clinicians.  Coronavirus disease 2019 (COVID-19) infection occurred in patients with systemic autoinflammatory diseases at population expected rates and was generally uncomplicated.COVID-19 vaccination was generally well tolerated in this series, with no serious adverse events reported.This includes the largest report of patients on anti-IL-1/6 biologics receiving any adenoviral vector or mRNA vaccine.A recurring issue raised by the coronavirus disease 2019 (COVID-19) pandemic is the role of inflammatory cytokines in the balance between viral clearance and hyper-inflammation mediating severe disease. Cytokine-modulating agents have been assessed in clinical trials in COVID-19 infection, and IL-6 (but not IL-1) blockade is now part of the standard care of patients requiring respiratory support [For patients with these conditions and their clinicians, there is currently an almost complete absence of data to guide decision-making on COVID-19 vaccination and on continuation of biologic therapy where risk of COVID-19 infection is high or proven. Problematically, pre-pandemic consensus guidelines and biologic drug product information do not contain guidance pertaining to the classes of vaccine, mRNA and non-replicating adenoviral vector, currently approved for use for COVID-19 , 4. Morehttps://www.R-project.org). Informed written consent was provided by all patients, and the study was conducted in accordance with ethical approval from the Royal Free Hospital and University College Medical School Research Ethics Committee (REC 06/Q0501/42).Electronic medical records of 248 patients with SAID on biologic therapy and under active follow-up at a national centre for management of autoinflammatory diseases were reviewed for diagnosis, treatment, hospital admission data and mortality data. These patients were then surveyed on experiences of COVID-19 infection and vaccination using a Web-based survey that was completed by the patient or their carer, or by their clinician during a clinic appointment, between 4 March 2021 and 26 March 2021 inclusive. Data analysis and visualization were performed in R statistics  patients reported suspected COVID-19 infection, of which 14 cases were confirmed by laboratory testing (8.0%), including one asymptomatic infection detected by surveillance screening. No patients required supplemental oxygen, ventilatory support or intensive care unit admission. Two patients on anakinra required hospital admission because of vomiting and dehydration. Anakinra was discontinued temporarily in the two patients admitted to hospital by the admitting teams. All patients with confirmed COVID-19 infection who did not require hospital admission continued their biologic therapy without interruption. Two patients reported persistent, otherwise unexplained symptoms >12 weeks after COVID-19 infection, consistent with a diagnosis of post-COVID syndrome as currently defined [In the cohort of 248 patients, no deaths were recorded between 29 January 2020 and 26 March 2021. Survey responses were received from 175 of 248 patients (70.6%). Diagnoses and biologic therapy of respondents are summarized in One hundred and thirty of 175 (74.3%) patients had received a first vaccine, and eight had received the second dose in line with national guidance on dose intervals. The characteristics of vaccinated patients and reported vaccine reactions are summarized in The 8.0% prevalence of confirmed COVID-19 infection in our series of patients with SAID on biologic therapy is broadly in line with that reported for the general UK adult population. Compared with the reported Parisian experience of familial Mediterranean fever, our patients have had relatively mild disease, with no deaths or requirement for respiratory support . Taken tAs the vaccination programme proceeds, there is an urgent need for real-world clinical data to facilitate informed decision-making on the part of clinicians and patients with SAID. Recent EULAR guidance recommend the use of any approved COVID-19 vaccine in these patients, although safety and efficacy data pertaining specifically to patients with SAID or on biologics are yet to be published . In our The study has a number of limitations. This is a real-world observational study with a short follow-up time after vaccination and, given that SAIDs are rare, the number of COVID-19 infections reported is small. Additionally, although our survey response rate was high (70.6%), we cannot exclude a non-response bias, which would be expected to cause over-estimation of rates of COVID-19 infection and vaccine reactions. Finally, a key question remains regarding vaccine the efficacy of COVID-19 vaccination and, as such, there is a need for further studies investigating the degree of humoral and cellular response to vaccination in this patient group.Nonetheless, there is a pressing need for timely sharing of data sets like this in a global pandemic, particularly in a rare patient population where there are legitimate concerns owing to historical reports of vaccine reactions and inconsistent guidance . Moreoverkab043_Supplementary_DataClick here for additional data file.


Vaccine reluctance among healthcare workers (HCW) can have widespread negative ramifications, including modeling behavior for the general population and challenges with maintaining a healthy workforce so we can respond to a resurgence of the pandemic. We previously reported that only one-third of HCW were willing to take the vaccine as soon as it became available prior to its Emergency Use Authorization (EUA). Here, we re-examine the attitude toward COVID-19 vaccines among HCW several months after the vaccines have been made widely available. In this study, only 7.9% (n = 107) of respondents were hesitant to take the first or second dose of the vaccine. Younger age (18–40 years) and lower level of education attainment (GED or less) were associated with higher vaccine hesitancy, whereas self-identified Asian racial identity was associated with greater acceptance of COVID-19 vaccination. Among the vaccine-hesitant group, more respondents noted mistrust of regulatory authorities (45.3%), government (48.6%), and pharmaceutical companies (50%) than mistrust of doctors (25.4%). Nearly two-thirds of respondents were concerned that vaccination may be ineffective against new strains and booster doses may be required; however, vaccine-hesitant respondents’ acceptance of a hypothetical booster dose was only 14.3%. Overall, vaccine hesitancy was observed to have demographic predictors similar to those previously reported; the hesitancy of some US HCW to receive booster doses may reflect a general hesitancy to receive other forms of vaccination.  The World Health Organization (WHO) declared COVID-19, the disease caused by SARS-CoV-2, a pandemic on 11 March 2020 . Since tHealthcare workers (HCW) are a priority population for vaccination against COVID-19. As a high-risk population, HCW were prioritized as one of the first groups to receive the vaccine in keeping with recommendations by the Advisory Council on Immunization Practices . HCW areDesign: We conducted a cross-sectional survey to assess vaccine hesitancy among US HCW toward COVID-19 vaccination. An online English language survey tool was created using the REDCap electronic data capture tool hosted at the University of New Mexico. This survey was modified from our previously published study to gather more pertinent information regarding vaccine uptake among HCW. All responses were anonymized and no personally identifying information was collected. The study was conducted according to the guidelines of the Declaration of Helsinki and approved on 19 September 2020 by the Institutional Review Board of the University of New Mexico Health Sciences Center (study ID 20-515).Sampling: The snowball sampling method was utilized. In this method of sampling, researchers initially reach out to their networks of contacts to recruit participants; agreeable participants may then recommend other potential participants from among their respective networks. This method of sampling was chosen for ease of recruitment with limited resources, as our populations of interest of HCW in the US were likely to be linked through professional contacts.The survey tool was distributed via links emailed to employees of a large academic medical center in the southwestern US and posted to HCW groups on social media. Data were collected between 1 February 2021 and 31 March 2021.Population: All adults (>18 years of age) working in a healthcare setting in the US, regardless of role or patient contact, were eligible to participate in the study. Informed consent was obtained prior to enrollment in the study. The main outcome of the study was identification of vaccine hesitancy among HCW with access to vaccination. Vaccine hesitancy was measured with a set of two questions. First, we asked respondents whether they had been offered any of the COVID-19 vaccines currently available in the US. Those who answered in the affirmative were then asked whether they received the vaccine(s). Their answers were captured as responses to one of the following questions: “Yes, I already took the first dose and plan to take the second dose/already taken the second dose”; “Yes I took the first dose but do not plan to take the second dose”; “No, I will wait to review real-world safety data”; “No I do not plan to take the vaccine”; and “Not sure”. Those who responded that they received both doses or were planning to receive both doses of the vaccine were identified as the vaccine non-hesitant group. Participants who did not accept either dose or responded that they were waiting or unsure were identified as the vaccine-hesitant group.The primary analysis was performed by comparing these two groups (vaccine-hesitant and non-hesitant) to identify factors associated with vaccine hesitancy. The comparison was based on major demographic predictors: age, gender, race, ethnicity, education, income, occupation, primary area of work in healthcare , type of healthcare facility , political identity, co-morbidity, self-perceived risk of contracting COVID-19, and experience of caring for patients with COVID-19. Based on occupation, the respondents were grouped as either direct medical providers (DMP) or direct patient-care providers (DPCP). DMP included physicians, resident physicians, medical students, and advanced-practice providers such as nurse practitioners (NPs) and physician assistants (PAs). DPCP included RNs, rehabilitation therapists, radiology technicians, nursing aides, social workers, care coordinators, administrative staff, and others without direct patient contact. Respondents’ attitudes toward COVID-19 vaccination in these groups, in terms of vaccine safety, efficacy, and trust, were assessed via their agreement to statements on a Likert scale. A descriptive analysis was performed by frequency distribution. A secondary outcome of the study was an assessment of the attitudes of HCW toward potential additional booster doses of COVID-19 vaccines. These attitudes were assessed with a set of two questions. First, we assessed the perception of need for booster doses by asking, “Are you worried that the current vaccine might not be effective against new strains, and you might require a booster dose or repeat vaccination?” We then assessed acceptance of booster doses with the question, “Would you be willing to take the vaccine/booster dose every year if required for sustained immunity?”.Descriptive analyses were conducted on all study variables. Multiple logistic regression models were applied to test for plausible associations of vaccine hesitancy with age, gender, self-identified racial identity, ethnicity, highest level of educational attainment, income, occupation, primary area of work in healthcare , type of healthcare facility , history of taking influenza vaccine, co-morbidity, self-perceived risk of COVID-19 infection, and experience in taking care of patients with COVID-19. Each statistical test was conducted at α = 0.05, unless otherwise indicated. Data analysis was conducted using statistical software R (version 4.0.4).The survey was completed by 1374 respondents. Twelve of the respondents had not yet been offered the vaccine, and data on vaccine acceptance were unavailable for four respondents. Therefore, these respondents were excluded from the analysis.Most of the respondents were between 30 and 60 years of age; female; white; held a bachelor’s degree or higher level of education; and identified as Democrat .GED: general educational development, indicating high school level of study; DMP: direct medical providers, including physicians, residents, fellows, medical students, NPs, PAs, and others; DPCP: direct patient care providers, including nurses, nursing aides, rehabilitation therapists, and others. A larger proportion of the respondents reported working in medical specialties as DMP, and in urban settings. Most participants reported no co-morbid conditions and perceived themselves to be at risk of slight-to-severe COVID-19 disease. A majority had directly taken care of COVID-19 patients. Of the 1358 participants who were offered the vaccine and whose full data were available, 107 (7.9%) were hesitant to receive either the first or second dose. Of those who were hesitant, 60 (56.1%) were waiting to review more data, 13 (12.1%) were not sure about their intention to vaccinate, and 31 (29.0%) did not plan to take the vaccine. Only three respondents (2.8%) were hesitant to take the second dose after receiving the first dose. All respondents who had received the first dose and were hesitant to take the second dose identified side effects with the first dose as the reason. DMP: direct medical providers, including physicians, residents, medical students, NPs, PAs, and others. DPCP: direct patient care providers, including nurses, nursing aides, rehabilitation therapists, and others.Younger age  and lower level of education attainment  were associated with higher vaccine hesitancy. Self-identified Asian racial identity  was associated with lower levels of vaccine hesitancy.Lack of trust was more common in the vaccine-hesitant group across all demographic domains .Among the vaccine-hesitant group, lack of trust was reported in pharmaceutical companies (50%), government (48.6%), and regulatory authorities such as CDC/FDA (45.3%), with a much lower percentage reporting mistrust of doctors (25.4%). Those who were hesitant to receive COVID-19 vaccination noted concerns about vaccine safety (33.7%) and efficacy (19.8%) at higher rates than those in the non-hesitant group .On the need for additional booster doses, most respondents (63.6%) were worried that current vaccination may not be effective against new strains and that additional booster doses or new vaccines may be needed for protection against emerging variants of the virus. These concerns were similar for the vaccine-hesitant (68.8%) and non-hesitant (63.1%) groups. When asked about acceptance of a hypothetical yearly booster vaccine to maintain immunity, overall acceptance was 83.6%. However, this acceptance was widely divergent among the two groups, with acceptance of a hypothetical annual booster dose at 13.8% among the vaccine-hesitant group and 89.9% among the non-hesitant group. A,B.Demand for COVID-19 vaccine has slowed in recent months ,7,8,9,10In the past year, hesitancy to receive vaccination against COVID-19 has been documented in the US and globally ,12,13,14Few demographic factors seem to be associated with vaccine hesitancy, although vaccine hesitancy is lower among higher-education-level and older-age HCW. These factors have been consistently noted in multiple studies of the general US population ,14, as wGiven the rise in cases of COVID-19 among young adults  and repoHCW who are hesitant to be vaccinated also state concerns about vaccine safety at much higher rates compared to non-hesitant HCW. While misinformation about adverse effects after COVID-19 vaccination has gained traction in segments of social and mass media, the robust monitoring systems under VAERS (vaccine adverse events reporting system) have shown very few serious adverse events causally linked to vaccination. Rates of serious adverse events such as anaphylaxis (2 to 5 people per million vaccinated), thrombosis with thrombocytopenia syndrome (46 reports after more than 14.5 million doses of Johnson & Johnson/Janssen vaccine and 2 after more than 362 million doses of mRNA COVID-19 vaccine), myocarditis, or pericarditis (854 confirmed reports after mRNA vaccine) are low. Death has been reported in 0.002% of people after COVID-19 vaccination, although a causal link with COVID-19 vaccine has not been established after review . The datHCW who are hesitant to be vaccinated have much higher levels of distrust in information provided by pharmaceutical companies, government, or regulatory authorities such as the CDC/FDA. Respondents who were hesitant to accept vaccination were more trusting of vaccination information provided by doctors and healthcare professionals than by these other sources of information; however, they were less trusting of doctors overall than were the respondents who were not hesitant to receive the COVID-19 vaccine. A perception of the beneficial role of healthcare providers has previously been suggested as a reason for vaccine uptake among children . Thus, iMounting evidence suggests a role for booster doses of COVID-19 vaccine in select populations. The FDA has issued EUA for the third dose of mRNA vaccines among solid organ transplant recipients and others who are similarly immunocompromised. Anticipating that, in the future, the scope for booster doses may broaden, we asked our respondents about their attitudes toward booster doses of COVID-19 vaccines. Their responses produced interesting findings. Nearly two-thirds of the respondents in each group (vaccine-hesitant and non-hesitant) were concerned that current vaccines may have decreased efficacy against new variants and booster doses may be required. However, when asked whether they would accept a yearly vaccine against COVID-19 to maintain immunity, acceptance was widely different between those who had taken the current vaccine and those who were hesitant to complete current vaccination. We hypothesize that hesitancy about initial COVID-19 vaccination may be a strong predictor for hesitancy about booster doses of the vaccine. This is unsurprising, as concerns over vaccine safety and mistrust of authorities, which were prevalent among hesitant HCW, are likely to extend to booster doses of the vaccine. Prior refusal of vaccination for other illnesses such as influenza has also been noted as a predictor of COVID-19 vaccine hesitancy ,14. It iThis study has limitations. As we utilized snowball sampling, our study population may not be representative of all US HCW, which limits the generalizability of our findings. The survey questionnaire was available only in an English online format; therefore, HCW with limited English proficiency and/or with limited internet access may be under-represented in our study. Social desirability bias may also affect the interpretation of our study, although the responses were anonymized to minimize this factor. The reluctance of some US HCW to receive COVID-19 vaccination is ongoing and is more likely to be prevalent among those of younger ages and lower levels of educational attainment. It is less likely among those with self-identified Asian racial identity. Concerns about vaccine safety, vaccine efficacy, and lack of trust were possible underlying causes of vaccine hesitancy. Physicians are more likely than pharmaceutical companies, regulatory authorities, or government to be trusted sources of information among HCW with COVID-19 vaccine hesitancy. Acceptance of COVID-19 vaccine booster doses may mirror a general hesitancy of some US HCW to receive other types of vaccination.


Several comorbidities have been shown to be associated with coronavirus disease 2019 (COVID-19) related severity and mortality. However, considerable variation in the prevalence estimates of comorbidities and their effects on COVID-19 morbidity and mortality have been observed in prior studies. This systematic review and meta-analysis aimed to determine geographical, age, and gender related differences in the prevalence of comorbidities and associated severity and mortality rates among COVID-19 patients. We conducted a search using PubMed, Scopus, and EMBASE to include all COVID-19 studies published between January 1st, 2020 to July 24th, 2020 reporting comorbidities with severity or mortality. We included studies reporting the confirmed diagnosis of COVID-19 on human patients that also provided information on comorbidities or disease outcomes. We used DerSimonian and Laird random effects method for calculating estimates. Of 120 studies with 125,446 patients, the most prevalent comorbidity was hypertension (32%), obesity (25%), diabetes (18%), and cardiovascular disease (16%) while chronic kidney or other renal diseases , cerebrovascular accident , and cardiovascular disease  patients had more COVID-19 severity and mortality respectively. Considerable variation in the prevalence of comorbidities and associated disease severity and mortality in different geographic regions was observed. The highest mortality was observed in studies with Latin American and European patients with any medical condition, mostly older adults (≥ 65 years), and predominantly male patients. Although the US studies observed the highest prevalence of comorbidities in COVID-19 patients, the severity of COVID-19 among each comorbid condition was highest in Asian studies whereas the mortality was highest in the European and Latin American countries. Risk stratification and effective control strategies for the COVID-19 should be done according to comorbidities, age, and gender differences specific to geographical location. The current strategies for managing COVID-19 are mainly with treatments that showed promising results in observational or preliminary clinical trial studies or recommended for an earlier outbreak of SARS-CoV-1. Given the nature of transmission of SARS-CoV-2 and the lack of any available antiviral therapy or vaccine, the optimal approach to treatment remains unclear. Rapidly evolving data provides guidance on therapeutic approach, including risk stratification utilizing comorbidities and laboratory features associated with severe COVID-19, prevention of venous thromboembolism, empiric treatment for bacterial pneumonia, addressing hypoxia and use of immunomodulatory agents on a case-by-case basis5.The world health organization (WHO) officially declared the novel coronavirus (COVID-19) infected by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as a global pandemic on March 11th 2020 because of the alarming level of transmission, the evolving disparities among cases, and rising mortality rates10. Prior systematic reviews and meta-analyses confirmed that diabetes mellitus (DM) and hypertension (HTN) are strongly associated with mortality in COVID-19 patients11 and most frequently observed conditions in COVID-1912. However, malignancy and DM were found to be associated with disease severity in prior meta-analyses14. Other meta-analyses15 showed that chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), cardiovascular disease (CVD), DM, and HTN are the major risk factors predicting severity in COVID-19 patients. In contrast, a large study identified CKD as the major risk factor for mortality while COPD as the risk factor for the severity of COVID-199. Another meta-analysis16 showed that cerebrovascular disease (CVA) is strongly associated with the severity and mortality in COVID-19. Most of these reviews were based on the analysis of studies where the majority of participants were from China. Recently, several observational studies from the US, Europe, and Latin America also demonstrated some coexisting diseases associated with the severity and mortality among COVID-19 patients19. Some studies22 reported COPD, CKD, malignancy, HTN, and DM as the prominent risk factors for disease severity or mortality in COVID-19 patients while other studies29 showed no association between these comorbidities and the disease outcomes. These studies at times reported rather inconsistent and conflicting findings. Rapid accumulation of data on comorbidities associated with poor prognosis of COVID-19 warrant updating the evidence.Numerous studies have identified comorbidities associated with the adverse prognosis of COVID-19. Age, gender, and at least a few comorbidities have been shown to be the strongest predictors of prognosis of COVID-19 patients31. A proper understanding of coexisting diseases associated with COVID-19 severity and mortality is paramount to effectively allocate healthcare resources, endorse appropriate preventive and containment measures, and guide emerging treatment protocols. We sought to perform a comprehensive systematic review and meta-analysis to determine the differences in the prevalence of major comorbidities associated with COVID-19 and the severity and mortality of COVID-19 associated with each of the comorbidities according to the geographical regions. In addition, we also aimed to determine the impact of age and gender on the distribution of different comorbidities, severity, and mortality among COVID-19 patients.The continental differences in the distribution of COVID-19 comorbidities, disease severity and mortality are more likely due to the variation in the timing of COVID-19 spread, population size, and prevalence of comorbidities among COVID-19 patients, age and gender distribution, implementation of mitigation strategies, diagnostic testing, capacity, COVID-19 management strategies, and reporting accuracy32 guidelines and SAMBR33 checklists for conducting and reporting this study. We conducted a systematic literature search using multiple electronic databases including PubMed, Scopus, and EMBASE between Jan 1st, 2020 to July 24th, 2020. The search strategy was designed to retrieve all published articles on COVID-19 reporting comorbidities and outcomes in the reported comorbidities. We applied various combinations of Boolean operators by using the following keywords for our search: [(SARS-Cov-2 OR 2019-nCOv OR COVID-19 OR coronavirus) AND (comorbidities OR comorbid OR comorbidity) AND ]. In addition, the references from review or other systematic review studies were cross-checked to retrieve any additional articles that were missed at the initial search. Three persons  reviewed all articles to eliminate any duplicated study. Studies with similar authors, the duration of data collection, and the location of the study were strictly matched to further identify any duplicated study. All the duplicates were omitted from the analyses.We followed the preferred reporting in systematic reviews and meta-analyses (PRISMA)Studies were eligible for inclusion in our systematic review if they met the following criteria: (1) originally published in the English language; (2) included confirmed diagnosis of COVID-19 through laboratory examination; (3) conducted on human patients; (4) provided information about comorbidities; (5) contained information on the disease outcomes: severity or mortality within comorbidity; and (6) published as an original investigation. Studies without diagnostic information, involved the pediatric population, and performed surgical procedures were excluded from the analysis. In addition, any studies published as case reports and case series with smaller numbers (≤ 10) were also excluded from the analyses.All relevant information from the eligible studies were extracted and recorded in an excel datasheet. The following information was extracted from each study: study characteristics involved the first author of the study; geographical region ; sample size ; the average age in years with standard deviation or interquartile range; gender ; status of the comorbidities , the type of comorbidity that included HTN, DM, CVD, obesity, CVA, lung disease, cancer/malignancy, either chronic or acute kidney or other renal diseases (CKD) and liver disease, and the outcomes of COVID-19  within each comorbid condition. The detail information on the inclusion of comorbidities, outcomes including the criteria for COVID-19 severity assessment, and comparing variables is provided in an additional .34. Three authors  independently graded the quality of the included studies using Sundemo et al. 2019 methods35. Consensus discussions were carried out among the three authors to resolve any disagreements. Additional information on quality assessment and publication bias is included in the additional file  scale was used for critical appraisal of study qualitynal file .36. A priori random effects model was applied due to considerable heterogeneity across the studies. We applied the Freeman-Tukey double arcsine method for computing a 95% confidence interval (CI) for each proportion to obtain a reasonable range. The pooled proportion of each comorbidity, severity, and mortality was estimated according to average age groups  and percentage of the female population  using random effects meta-analysis of proportions with the D&L method. Meta-regression analyses with Knapp-Hartung modification were also performed to determine associations of demographic data with disease outcomes. All the estimated proportions were summarized with their 95%CI and I2 statistic37. An I2 greater than 50% was considered as an indication of presence of heterogeneity. All statistical analyses were conducted using Stata 15.1.We estimated the pooled proportion of underlying medical conditions, severity, and mortality among each comorbidity separately by geographic region  using a random effects meta-analysis of proportions with DerSimonian and Laird (D&L) methodFigure Of the total, 72 studies reported from Asia, 26 from Europe, 16 from the USA, five from Latin America, and one from South Africa. These studies yielded a total sample size of 125,546 patients . The female proportion was estimated as 45%  with the highest in South Africa followed by Asia, USA, and Latin America and the lowest in Europe. The mean age of the patients was 56.2 years (95% CI: 52.6 – 59.8 years) with the highest in Europe (64.8 years), followed by the USA (60.4 years), Asia (54.4 years), and Latin America (50.2 years) . The history of liver disease and CVA were frequently reported in Asian studies while rarely reported in other country studies. In contrast, obesity was rarely reported from Asian studies. The most prevalent comorbidity was HTN  followed by obesity , DM , CVD , lung disease , and CKD  while other underlying medical conditions were estimated to be 4–5%  whereas a similar level of severity of COVID-19 cases was observed in CVD (44%), CVA (43%), DM 42%), and lung disease (42%) conditions followed by HTN (39%) and malignancy (37%) conditions. The least proportion of disease severity was estimated in liver disease (23%) and obese (33%) patients. COVID-19 severity remained similar in patients with at least one comorbidity (41%) or multiple comorbidities (44%) but substantially higher than COVID-19 cases without any comorbidity (19%) , CKD , and CVD . The other underlying conditions were associated with similar levels of mortality (range: 30–33%) except for obesity 27%)  receptor by interaction with viral spike protein which allows the virus to enter into host cells41. Furthermore, several medications for the treatment of COVID-19 may yield cardiovascular adverse events, and COVID-19 may also directly cause CVD or aggravate underlying CVD43. Interaction of SARS-CoV-2 with the renin–angiotensin–aldosterone system (RAAS) may likely to observe in HTN and DM patients reflecting as the most prevalent conditions in COVID-19 patients9. Furthermore, immunocompromised patients are at high risk for developing any infectious diseases and immune dysfunction has been linked with HTN, DM, obesity, CVD, lung disease, and CKD indicating these medical conditions as more common in COVID-19 patients as well44. Patients with CVD, CKD, and a history of CVA have been observed at high risk for mortality in COVID-19 in our study. These conditions share common risk factors including increased allostatic load, and COVID-19 may either induce or aggravate these conditions by impacting ACE2 receptor, RAAS function, immune, hemostatic and nervous system damage, and major physiological systems45. Our study suggests that COVID-19 patients with chronic diseases regardless of geographic location should be given proper health care attention and prioritized for vaccination.In our meta-analysis, HTN, obesity, DM, and CVD were more frequently observed in COVID-19 patients whereas mortality was more frequently observed in COVID-19 patients with CVD, CVA, and CKD. The presence of CVD and their most prominent risk factors such as HTN and DM have been shown to be associated with COVID-19 disease9. In another meta-analysis10, the mortality rate in COVID-19 has also been reported to be highest in the European region compared to the US or other regions. Our meta-analysis also estimated the highest rate of mortality in Latin American studies despite having younger patients than studies from other regions. Although the specific reason for this finding is yet not clear, a limited number of studies from Latin American regions might have amplified the proportion estimates of mortality among different comorbid conditions. Other potential reasons for having high mortality in Latin American countries could be the differential approaches to COVID-19 testing and timing and implementation of mitigation strategies compared to other geographic regions along with resource constrained health systems, allocation strategies of resources, inequality and poverty, and internal political dynamics46. Similar to another meta-analysis10, our study also confirmed the lower rate of mortality in the Asian region and a South African study than the European and American regions. It appears that the varying immune profile of individuals from different racial groups can modulate their risk of disease infection and autoimmunity and may respond differently to novel infections47. Preliminary evidence showed a trend towards the slower spread of COVID-19 in countries where the national Bacillus Calmette–Guérin vaccine policy is current for all48. Based on distributions of age and gender with differential prevalence of comorbid conditions associated with disease outcomes specific to geographic regions, we suggest the joint evaluation of country-specific demographic and comorbid factors for stratifying risk for COVID-19 disease severity and mortality.The highest mortality among all comorbidities was observed in European studies compared to the US and Asian studies despite lower prevalence of comorbidities compared to the US studies and lower severity prevalence in each comorbidity compared to Asian studies. This mostly occurred due to more elderly and male patients compounded with the second highest proportions of underlying medical conditions and disease severity in some comorbidities in European studies. In addition, an early hit of the pandemic in European regions especially in the elderly population might have also created a surge in these populations. In contrast, a single study from South Africa yielded the lowest mortality rate even with high proportions of DM (40.4%) and HTN (42%) patients. This could be due to the inclusion of predominantly females in the study compared to other region studies. Older age, male gender, and presence of at least one comorbidity have been shown to be independently associated with mortality in COVID-19 patients51. COVID-19 severity in the US remained lower than Asian and European studies even with high proportions of HTN and multiple comorbidities. This empirical observation indirectly suggests that RAAS inhibitors may not adversely impact COVID-19 patients with underlying medical conditions, although this needs further validation52. The low rate of mortality within each comorbidity in the US studies even with the high prevalence of underlying disease conditions is likely due to optimal management of COVID-19 cases with underlying disorders, timely and strategic implementation of mitigation strategies, avoiding unnecessary medication use, and proper reporting of public health practices30. Our stratified analyses clearly showed that higher COVID-19 severity was more likely to occur in all underlying medical conditions in the Asian region compared to other regions. Although a direct comparison of severity among each of the comorbid conditions is not available in COVID-19 across geographic locations, socio-demographic, cultural, behavioral, and biological factors might explain the differential prevalence of the disease severity in Asian studies53. Our study suggests developing COVID-19 management policies specific to geographic regions. For example, some regions need proper implementation of mitigation strategies and vaccination policy to minimize COVID-19 spread among at-risk individuals while some regions require adequate resources with proper guidelines, and management strategies for minimizing disease severity.Our study estimated the highest proportion of all comorbidities in US studies compared to studies from other countries. The prevalence of comorbid conditions have been reported to be higher in the overall US population which may be reflecting the higher prevalence of each comorbid condition in COVID-19 patients as well55. Similar to our study, a study also confirmed that older adults with underlying diseases including DM, HTN, CVD, liver diseases, and malignancy have been more likely to develop a critical illness56. Our meta-analysis also confirmed that there was no substantial difference in the burden of comorbidities between male or female-dominant studies. In consistent with a single-center retrospective study29 and another meta-analysis57, we also observed higher rates of disease severity with most of the underlying medical conditions in female-dominant studies while higher mortality rates were observed in male-dominant studies. Gender differences in mortality rates in all underlying medical conditions could be due to the sex hormones and sex chromosome genes associated with different immune responses59. Like we observed geographical differences in COVID-19 severity and mortality, the geographical differences in COVID-19 therapeutic practices have also been observed60. COVID-19 prevention and treatment strategies should be effectively implemented to elderly population and individuals with underlying disease conditions.This study has confirmed higher mortality in older adults (> 65 years) with all the underlying medical diseases except for CKD patients. This could be due to the inclusion of Latin American studies involving the youngest age patients and demonstrating a higher rate of mortality in CKD and obese patients. Mortality risk in older adults with comorbidities was also confirmed in other studiesWe have some limitations in our study. Although we have used random effects models to estimate proportions, high heterogeneity across the studies and publication biases in few subgroup analyses might produce biased estimates. Given the variations in study populations, we estimated prevalence estimates within each comorbidity stratified by geographic regions. However, the variation in racial/ethnic and socioeconomic populations might further yield heterogeneity in the proportion estimates. Limited studies from Africa and Latin America may produce higher estimates in these subpopulations. Furthermore, we could not explore the regional differences in disease outcomes in Asian countries due to limited studies from South and West Asia. Since the majority of the studies included in our study were retrospective and conducted in different countries, missing, misclassification, and variation in reporting of medical conditions may be present. While our study is the most update-to-date and comprehensive, our restriction to studies with only English language and upcoming evidence as COVID-19 is evolving might impact the findings of our study. Despite these limitations, to the best of our knowledge, our study is the first comprehensive systematic review and meta-analysis that produced estimates of major underlying medical conditions with COVID-19, and COVID-19 severity and mortality within each comorbidity by including a large number of high-quality studies from the US, Europe, Asia, and other countries with large sample sizes. Our methodological rigor and stratified analysis of the study population by geographic regions yielded reliable and regional specific findings to develop an effective control policy for this COVID-19 pandemic.In conclusion, we observed high prevalence estimates of major comorbid conditions in US studies. Among patients with any comorbidities, the highest disease severity was estimated in Asian region while the highest mortality in European and Latin American regions. The mortality among those with underlying medical diseases was estimated to be high in mostly elderly and predominantly male patients with considerable mortality in older patients without any comorbidities. COVID-19 severity and mortality were highly variable based on medical conditions specific to geographic regions. These findings clearly suggest that country-specific comorbidities should ideally be used to evaluate and stratify risk for the COVID-19 disease severity and mortality. We suggest developing policies across the globe with particular attention to specific geographic/ethnic populations such as avoiding COVID-19 spread to individuals with underlying medical conditions especially in the US population, protecting elderly male and sick individuals from SARS-CoV-2 infection mainly from European regions, and managing life-threatening COVID-19 complications particularly in Asian patients. In addition, at-risk individuals according to geographic regions should be given a priority for vaccination. Our study prioritizes comorbid conditions associated with the disease severity and mortality and provides insights for targeted risk stratification and effective control management of this pandemic.Supplementary information


In ovarian cancer, often diagnosed at an advanced stage and associated with poor overall survival, with just a third of women surviving five years, the controversial prognostic value of circulating tumor cells (CTCs) is still discussed. Currently CTC diagnostics use either molecular approaches or immunofluorescent staining. Our study shows that, given the heterogeneity and extreme scarcity of CTCs, a multifactorial analysis of CTCs is key. Combining both approaches, qPCR and IF, increased sensitivity and may better capture a treatment-related shift in the molecular phenotypes of CTCs. In addition to a long progression-free interval, the absence of CTCs after treatment was an independent predictor of an excellent outcome in patients who had already survived for five years. Thus, a multifactorial CTC approach can identify patients who have elevated risk of recurrence and death and who may require risk-adapted treatment strategies.combo), in particular for the patients who had survived more than five years. Material and Methods: Blood samples taken at baseline and six months after adjuvant treatment (follow-up) were assessed by quantitative PCR (qPCR) measuring PPIC transcripts and immunofluorescent staining (IF). A positive result with either IF or qPCR was classified as CTCcombo-positive. Further, PPIC was assessed in the primary tumor tissue. Results: The concordance of IF and qPCR was 65% at baseline and 83% after treatment. Results showed that 50.5% of the baseline and 29.5% of the follow-up samples were CTCcombo-positive. CTCscombo after treatment were associated with increased mortality after adjusting for FIGO stage , a higher risk of recurrence after adjusting for peritoneal carcinosis , and increased mortality after five survived years. Discussion: The two-sided analytical approach revealed CTC subpopulations associated with ovarian cancer progression and may illuminate a potential treatment-related shift in molecular phenotypes. That approach can identify patients who have elevated risk of recurrence and death due to ovarian cancer and who may require risk-adapted treatment strategies.Introduction: We previously reported the prognostic impact of circulating tumor cells (CTCs) in a multicenter study on minimal residual disease in primary ovarian cancer. With additional follow-up data, we evaluated the combined CTC approach (CTCs Ovarian cancer is the second most frequent and the deadliest malignancy of the female genital tract. The poor prognosis is largely attributed to late diagnosis of the disease when it has already spread beyond the pelvis and is difficult to treat. In Europe, only every third woman diagnosed with ovarian cancer survives five years . Yet proThe majority of ovarian-cancer-related deaths are attributed to intra-abdominal metastases, which may have formed through the direct shedding of malignant cells from the primary site into the peritoneal cavity. Thus, it has long been assumed that hematogenous spread plays a minimal role in ovarian cancer metastasis; nonetheless, the presence and prognostic relevance of circulating tumor cells (CTCs) in the blood was shown by our group amongst others (reviewed by ), implyiIn an earlier report on the results of a multicenter translational research study investigating predictive markers for the early detection of minimal residual disease in ovarian cancer , we demoSince these previous reports, the observation period of our patient cohort has been prolonged reaching a median follow-up time of more than ten years. Thus, in the present study, we (1) re-evaluated the prognostic significance of CTCs as assessed by qPCR and (2) asked whether a comprehensive CTC approach combining both techniques (IF and qPCR) could generate additional evidence for prognosis estimates in comparison to the single methods. Furthermore, we (3) added data on PPIC protein expression in primary tumor tissue and in CTC samples. Finally, due to the long follow-up time, (4) we evaluated prognostic factors for the likely survival of patients having already survived for five years.2 test p = 0.072).In the original analysis showing the impact of CTC-related gene markers on the outcome of patients, the median follow-up for still-living patients was 4.3 years (range of 1 to 69 months). At the time of this updated analyses, a further 56 patients have succumbed to their disease. The death rate of the OVCAD study population was 71.2%, with a median follow-up time of 11.1 years (range 4 months to 13.4 years), with 62 patients still alive. Disease recurrence was observed in 172 (80.0%) patients, while 43 patients (20.0%) did not experience recurrence within a median follow-up period of 9.9 (IQR 5.7–12.0) years. Recurrence was not significantly associated with the histological type/grade of the disease (chip = 0.289) or PFS . The clinicopathological characteristics of the patients with baseline blood samples available are shown in [Since our initial report, the number of deceased patients almost doubled from 61 (38%) to 115 (72%) among the 165 PPIC-negative patients and from 15 (47%) to 24 (71%) among the 34 PPIC-positive patients. In contrast, recurrences increased to a smaller extent, from 112 (68%) to just 128 (78%), among the PPIC-negative patients and from 24 (71%) to 29 (85%) among the PPIC-positive patients. In line with our initial report (9), PPIC-positivity at baseline still did not have an impact on OS .Among the 79 PPIC-negative patients by PCR, the number of deceased patients increased from 31 (39%) to 52 (66%) since the initial analysis. All 13 PPIC-positive patients have succumbed to their disease, and the median OS of PPIC-negative patients further increased by more than half a year from 64 months in the previous analysis to 71 months at present (log-rank p < 0.001). The clinicopathological characteristics of the patients with follow-up blood samples available are shown in [Likewise, since the initial analysis, a further 15 of 79 PPIC-negative patients relapsed, resulting in a total number of 65 patients with recurrent disease. The median PFS of PPIC-positive and PPIC-negative patients still differed significantly .The concordance rate of qPCR and IF at baseline was 65% (Cohen’s κ = 0.009). Thereby, 5 and 52 out of the 89 baseline blood samples were positive or negative by both methods, respectively. Additionally, 13 samples (15%) were assigned as CTC-positive by qPCR only, and 19 (21%) as IF-positive only a. The CTp = 0.345), none of the samples was scored as CTC-positive by both methods , the overall detection sensitivity increased from 20.2% (18/89 qPCR-positive cases) and 27.0% (24/89 IF-positive cases) to 50.5% (53/105) in the baseline samples. Similarly, in follow-up samples, the detection sensitivity increased from 15.8% (9/57 positive samples by PCR) and 8.8% (5/57 positive samples by IF) to 29.5% (18/61). CTCcombo-positivity at baseline was associated with the presence of ascites (chi2-test p = 0.007), while at follow-up, it was more likely in patients with advanced stage at diagnosis , the presence of peritoneal carcinosis , and of PPIC-positive CTCs at follow-up .Low PPIC protein expression was found in 19 (11.9%) of the cases, medium levels in 63 (39.4%), high levels in 45 (28.1%), and very high levels in 33 (20.6%) of the tumor tissues. However, PPIC gene expression levels were not statistically different between these four groups see . Neverthcombo occur significantly more often in patients with a short progression-free interval of less than 18 months (chi2-test p = 0.008) and those who would succumb to their disease within five years after the primary diagnosis  or six months thereafter (chi2-test p = 0.001), progression-free interval less than 18 months (chi2-test p < 0.001), and death within five years after diagnosis . LTS was significantly associated with lower age, less advanced disease, response to adjuvant treatment, and to the absence of CTCcombo at any time (Seventy-six (35.3%) patients survived for more than five years (LTS), with 27 (12.6%) of them being still alive ten years after the primary diagnosis. The median PFS was significantly longer in the LTS group as compared to the 139 non-LTS patients  with nonproportional hazards affecting OS. Less advanced disease at diagnosis, the absence of peritoneal carcinosis, a progression-free interval of at least 18 months, and the absence of CTCs six months after completion of the adjuvant treatment were significantly associated with an excellent prognosis in these patients . Even insurvival .combo-positivity six months after completion of the adjuvant treatment remained independently associated with worse prognosis after adjusting for the histological type and grade, both strongly related with outcome. The absence of CTCcombo at the previous follow-up examination six months after completion of chemotherapy was an independent predictor of long-term survival in ovarian cancer patients who had already survived five years after the initial diagnosis.CTCs are typically detected either by their specific protein expression or by their nucleic acid content. Here we demonstrate for the first time the complementary information provided by different methodological approaches, such as IF staining of the target cells and mRNA gene expression analysis by qPCR. By pooling positive findings from both techniques, the overall number of positive samples more than doubled compared to each single approach. CTCGiven their heterogeneity and scarcity , our stuThe discrepancy between qPCR and IF findings in our study is consistent with observations by others who have compared these technologies for the detection of CTCs in blood or bone marrow. High concordance rates along with low κ values are also reported by others ,15, mainWe already reported the considerable variability of CK and EpCAM expression in an individual CTC sample characterized by the large number of 187 CTCs per ml of blood ,19. The combo approach in in the present study.In recent years, a plethora of further label-free technologies have been developed, such as size-based filtration, dielelectrophoretic field-flow fractionation, microvortices, and devices based on deterministic lateral displacement, inertial focusing, or acoustic wave separation (reviewed by ). In conMolecular CTC profiling by PCR has unveiled myriad biomarkers of potential diagnostic relevance (among them the PPIC gene). Being aware of the advantages of label-free technologies in this regard, our subsequent studies suggested that the microfluidic enrichment of CTCs could be more appropriate for CTC gene expression analyses than density gradient centrifugation ,22. The chaperone activities. Among them, cyclophilin A is overexpressed in various cancer types and can protect cancer cells against cellular stress induced by cisplatinum [So far, only sparse information exists on PPIC in the context of cancer. PPIC belongs to the cyclophilins, a protein family with peptidyl-prolyl cis–trans isomerase and molecular platinum . The proplatinum  and in oplatinum . In our platinum . Being aplatinum . Of noteplatinum . In our combo-negative six months after the completion of the adjuvant treatment. Moreover, the absence of CTCcombo after treatment was an independent predictor of an excellent outcome in patients who had already survived for five years. Our study is characterized by an exceptionally long median observation period, more than 10 years. The percentage of patients surviving more than five years since their primary diagnosis in our study is broadly in line with the 5-year survival rates reported for European countries ,29. Givecombo approach was not applicable to all samples, because—as already mentioned above—some samples had been analyzed using a modified protocol with low sensitivity [Unfortunately, the small sample size gradually lowers the significance of our study. The reasons are that paired baseline and follow-up samples were not taken from all patients and that that the CTCsitivity . Furthersitivity .From January 2006 to December 2008, 276 patients with confirmed primary epithelial ovarian cancer were prospectively enrolled within the multicenter OVCAD study, a 6th Framework Program Project (LSHC-CT-2005-018698) of the European Union. The overall goal of the study was to investigate new predictors for the early detection of minimal residual disease. The study protocol was approved by the local ethics committees of the participating OVCAD partners . Detailed inclusion and exclusion criteria, together with clinical data have already been presented elsewhere [Blood samples were taken in six 9 mL-Vacuette EDTA coated blood collection tubes (Greiner Bio-One) after having obtained written informed consent before primary surgery or neoadjuvant chemotherapy , and six months ± 21 days after completion of the adjuvant chemotherapy . All samples had been processed on the same day, using a two-layer density gradient centrifugation as described previously  to obtaicombo-positive group, whereas patients who were CTC-negative by both methods were assigned to the CTCcombo-negative group. Blood samples, which were CTC-negative by just one method but had not been processed using the other one due to technical reasons, were omitted, because the negative result had not been confirmed by both approaches.To investigate whether a combination of both approaches—qPCR  and IF —would inPPIC protein expression was assessed on tissue microarrays (TMA) created from formalin-fixed paraffin-embedded primary tumor tissue samples as described . ImmunohPPIC expression levels were scored semiquantitatively based on staining intensity and the percentage of positive tumor cells and combined with the immunoreactive score (IRS) as described ,37: IRS 2O were used. The reaction mixture was preincubated at 50 °C for two minutes and at 95 °C for ten minutes, followed by 40 cycles of two-step incubation at 95 °C for 15 s and at 60 °C for one minute. Only samples with GAPDH Ct-values ≤ 26 indicating sufficient RNA quantity and quality were included. Replicate Ct-values showing a standard deviation of >0.5 indicating poor quality of the quantitative value were excluded. The ddCt algorithm was used to assess the PPIC gene expression relative to the reference gene and a cell line calibrator sample [Total RNA was extracted from fresh frozen tumor tissue and further transcribed into cDNA as described . Brieflyr sample .combo, respectively, and the clinicopathological characteristics of the patients. CTC counts were compared using the independent samples’ Mann–Whitney Tests. The linear-to-linear association was used to evaluate the correlation of the PPIC protein immunoreactive score (IRS) in the tumor tissue with the clinicopathological characteristics of the patients and PPIC-positive CTCs, respectively. Nonparametric tests  were used to assess the association of PPIC gene expression levels with clinicopathological characteristics, PPIC-positive CTCs, and PPIC IRS. The concordance of positive findings by qPCR and IF was calculated by dividing the number of concordant samples with the total number of analyzed samples and by computing Cohen’s κ values [Pearson’s chi-square and Fisher’s exact test were used to assess the relationship between the presence of PPIC-positive CTCs and CTCsκ values .combo . A landmark analysis at five years was performed to identify prognostic factors for the survival of patient having already survived for five years. Statistical analysis was performed by SPSS version 19.0 . The level of significance was set at p < 0.05.Clinical endpoints were calculated as follows: progression-free survival (PFS) between the time of blood collection (at primary diagnosis and six months after completion of the adjuvant treatment) and first recurrence, or overall survival (OS) between the time of blood collection (as above) and death due to any cause. Patients without a documented date of recurrence were excluded. Patients surviving for at least five years after the primary diagnosis were regarded as long-term survivors (LTS) according to the definition of Hoppenot et al. . Kaplan–In conclusion, our results indicate that a multiple approach is needed to detect rare CTCs, which express heterogeneous phenotypes, per se, and additionally, may undergo a treatment-related shift in molecular phenotypes. Such an approach can identify patients who have an elevated risk of recurrence and death and who may require risk-adapted treatment strategies. Our data set the stage for future studies of the longitudinal analysis of CTCs in serial blood samples and the evaluation of the effect of novel treatment strategies on patient outcome.E.O. and R.Z. filed a patent application for using PPIC as a novel tumor marker in ovarian cancer.