Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs. SARS-CoV-2 diagnosis was performed by a real-time PCR test targeting the nucleocapsid gene. PCR tests targeting SARS-CoV-2 nonstructural protein 10 (nsp10), nsp14, and envelope and three regions of the plasmid vector were performed to differentiate amplification of SARS-CoV-2 RNA from the plasmid vector’s DNA. Nasal swabs from four asymptomatic coworkers with positive real-time PCR results for the SARS-CoV-2 nucleocapsid targets were negative when tested for SARS-CoV-2 nsp10, nsp14, and envelope protein. However, nucleic acids extracted from these nasal swabs amplified DNA regions of the plasmid vector used by the coworkers, including the ampicillin and neomycin/kanamycin resistance genes, the promoter-nucleocapsid junction, and unique codon-optimized regions. Nasal swabs from these individuals tested positive repeatedly, including during isolation. Longitudinal detection of plasmid DNA with SARS-CoV-2 nucleocapsid in nasal swabs suggests persistence in nasal tissues or colonizing bacteria. Nonviral plasmid vectors, while regarded as safe laboratory reagents, can interfere with molecular diagnostic tests. These reagents should be handled using proper personal protective equipment to prevent contamination of samples or laboratory personnel.IMPORTANCE Asymptomatic laboratory workers who tested positive for SARS-CoV-2 for days to months were found to harbor a laboratory plasmid vector containing SARS-CoV-2 DNA, which they had worked with in the past, in their nasal secretions. While prior studies have documented contamination of research personnel with PCR amplicons, our observation is novel, as these individuals shed the laboratory plasmid over days to months, including during isolation in their homes. This suggests that the plasmid was in their nasal tissues or that bacteria containing the plasmid had colonized their noses. While plasmids are generally safe, our detection of plasmid DNA in the nasal secretions of laboratory workers for weeks after they had stopped working with the plasmid shows the potential for these reagents to interfere with clinical tests and emphasizes that occupational exposures in the preceding months should be considered when interpreting diagnostic clinical tests. Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be asymptomatic or cause mild, moderate, or severe disease. To determine the biomarkers predictive of these varied outcomes, we conducted a prospective study of incident SARS-CoV-2 infection. Biomedical researchers and/or household members were enrolled, and the study participants self-collected anterior nasal swabs and delivered them to the testing site weekly. Testing of the nasal swabs by PCR detected SARS-CoV-2 in 14 cases within the cohort, including four asymptomatic coworkers and one of their household members who had no known exposures to SARS-CoV-2-infected individuals. When this “outbreak” was noted, epidemiologic investigation revealed that the four coworkers within this research laboratory were using a plasmid vector to express the SARS-CoV-2 nucleocapsid in the month prior to enrollment. In this report, we describe the molecular studies performed to determine if these five case participants were infected with SARS-CoV-2 circulating in the community or with the laboratory plasmid.TC) values for N1 and N2 in the positive nasal swabs were 38.5  and 39.3 , respectively. A total of 14 specimens were positive for both N1 and N2, and 11 additional specimens had only N2 detected at TC values between 38.8 and 44.1  of all cases , nsp14, and envelope (E), were negative. However, multiple PCR assays conducted using nucleic acids without reverse transcription from the cases’ specimens amplified DNA sequences unique to the plasmid used in the coworkers’ laboratory. Specifically, the ampicillin and kanamycin/neomycin resistance genes were amplified from four of the five cases, and the cytomegalovirus (CMV) promoter-nucleocapsid junction, which included a codon-optimized synthetic sequence unique to this plasmid construct, was amplified from two of the five cases . The latll cases , indicatSARS-CoV-2 was amplified from longitudinally collected nasal specimens from five asymptomatic individuals, four coworkers and one of their household members, which suggested person-to-person transmission of pandemic coronavirus. However, after the study team learned that a plasmid vector had been used in the month prior to enrollment by all four case coworkers in the laboratory outbreak, additional studies were performed to differentiate SARS-CoV-2 RNA from virus circulating in the community from DNA from the laboratory plasmid. Our inability to amplify regions of SARS-CoV-2 RNA outside the nucleocapsid that serve as targets in other diagnostic assays  did not Escherichia coli containing the plasmid had colonized their noses.Unlike prior reports of false diagnostic test results due to contamination of specimens by PCR amplicons , 3, the The coworkers in our study used laboratory protocols to amplify and purify plasmids that included handling high-titer bacterial cultures and conducting procedures with the potential for generating aerosols , which may pose a risk of inhalation. Bacterial strains and reagents used for cloning are generally not considered to be infectious materials. Therefore, most of these procedures are performed on the “open bench” in biosafety level 2 (BSL2) laboratories, following standard laboratory practices and using proper personal protective equipment (PPE), including gloves, lab coats, goggles, and safety centrifuge cups. Additionally, solutions of the purified plasmid are highly concentrated and can contaminate gloves, equipment, and the research environment, similarly to surface contamination with PCR amplicons . SeveralThe nonviral SARS-CoV-2 DNA from the nasal specimens collected longitudinally from the coworker cases was amplified over weeks to months, and the household member who did not work in a laboratory had a single positive test , illustrating the potential for this type of reagent to be carried from the lab to the home. While the initial positive swabs were collected aseptically by study personnel, we cannot rule out whether these or subsequent self-collected nasal swabs were contaminated by plasmid-contaminated hands or surfaces. However, laboratory work with the plasmid encoding the SARS-CoV-2 nucleocapsid was performed between mid-April and 3 June 2020 and completed before the coworkers enrolled in the study. Additionally, the enrollment nasal swabs were collected by the clinical team in a dedicated health room away from the laboratories, and confirmatory samples were also collected by study staff at the participants’ homes while they were in isolation, which suggests that the plasmid persisted within the nasal cavity for days to weeks.Plasmid vectors, commonly used in research, can contaminate laboratory reagents  and biol2–The SARS-CoV-2 prospective cohort study (SARS2 Study) performed at the Seattle Children’s Research Institute enrolled participants between 3 April and 23 July 2020. Following informed consent, as approved by the Seattle Children’s Institutional Review Board, Seattle Children’s and University of Washington employees and their household members were screened for SARS-CoV-2 exposures and symptoms and risk factors associated with COVID-19 disease severity and had blood and nasal swabs collected aseptically by the study personnel. Subsequently, each participant completed a weekly survey reporting contacts with others and respiratory and other symptoms and provided a self-collected nasal swab sampling both anterior nasal cavities for SARS-CoV-2 testing over the course of 54 weeks. Participants testing positive for SARS-CoV-2 (cases) had home visits by the study team to collect nasal specimens to confirm infection and to document their vital signs and oximetry. After detection of SARS-CoV-2, these “case participants” collected nasal swabs twice weekly until testing negative on two sequential specimens, then resumed weekly collection.TC) of 40. Processing of the nasal swabs and diagnostic testing were performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory.SARS-CoV-2 diagnosis was performed using a laboratory-developed real-time PCR assay with Emergency Use Authorization from the Washington Department of Health. The assay amplified two regions of the SARS-CoV-2 nucleocapsid gene (N1 and N2) , followiTo determine if nasal swabs from the five cases indeed contained SARS-CoV-2 RNA and/or the plasmid, nucleic acids extracted from the nasal swabs were subjected to additional testing. These included reverse transcription and PCR to amplify the SARS-CoV-2 RNA encoding nonstructural protein 10 (nsp10), nsp14, and envelope (E) .


Background and Objectives: The revascularization strategy for percutaneous coronary intervention (PCI) in patients with multivessel (MV) acute coronary syndrome (ACS) remains controversial. Certain gaps in the evidence are related to the optimal timing of non-culprit lesion revascularization and the utility of instantaneous wave-free ratio (iFR) in the management of MV ACS intervention. The major benefits of iFR utilization in MV ACS patients in one-stage complete revascularization are: (1) the possibility to virtually plan the PCI, both the location and the extension of the necessary stenting to achieve the prespecified final hemodynamic result; (2) the opportunity to validate the final hemodynamic result of the PCI, both in culprit artery and all non-culprit arteries and (3) the value of obliviating the uncomfortable, costly, time consuming and sometimes deleterious effects from Adenosine, as there is no requirement for administration. Thus, iFR use fosters the achievement of physiologically appropriate complete revascularization in MV ACS patients during acute hospitalization. Materials and Methods: This pilot study was aimed to test the feasibility of a randomized trial research protocol as well as to assess patient safety signals of co-registration iFR-guided one-stage complete revascularization compared with that of standard staged angiography-guided PCI in de novo patients with MV ACS. This was a single-center, prospective, randomized, open-label clinical trial consecutively screening patients with ACS for MV disease. The intervention strategy of interest was iFR-guided physiologically complete one-stage revascularization, in which the virtual PCI planning of non-culprit lesions and the intervention itself were performed in one stage directly following treatment of the culprit lesion and other critical stenosis of more than ninety percent. Seventeen patients were recruited and completed the 3-month follow-up. Results: Index PCI duration was significantly longer while the volume of contrast media delivered in index PCI was significantly greater in the iFR-guided group than in the angiography-guided group . There were no significant differences in PCI-related major adverse cardiovascular events (MACE) between the groups during acute hospitalization and at 3-months follow-up. One-stage iFR-guided PCI requires fewer PCI attempts until complete revascularization than does angiography-guided staged PCI. Conclusions: Complete revascularization with the routine use of the virtual planning tool in one-stage iFR-guided PCI is a feasible practical strategy in an everyday Cath lab environment following the protocol designed for the study. No statistically significant safety signals were documented in the number of PCI related MACE during the 3-month follow-up. Approximately 40% of patients with ST-segment elevation myocardial infarction (STEMI) and up to 70% of patients with non-ST-segment elevation myocardial infarction (NSTEMI) have significant multivessel (MV) coronary artery disease (CAD), indicating the presence of significant lesions in addition to the culprit lesion ,2,3. ThiThis was a single-center, prospective, randomized, open-label clinical trial consecutively screening patients with ACS for MV disease. The study population included patients with sinus rhythm only, to precisely measure left chamber segmental myocardial deformation and peak mechanical dispersion. Simple randomization method has been used without cross-over between groups. Seventeen patients were initially enrolled in this study and completed the 3-month follow-up during the period of two years (2020–2021). Trial performed in Cardiology and internal diseases center of Daugavpils Regional Hospital, Latvia. First round patient selection was performed by intensive care unit and emergency department clinicians. Where NSTEMI patients received echocardiography prior to the acute coronary angiography. And STEMI patients received echocardiographic evaluation in the first 24 h after acute coronary angiography to re-check for exclusion criteria.The ‘all-comers’ study population comprised hemodynamically stable de novo patients with ACS and a stenosis of ≥50% by visual estimation in at least two separate coronary arteries, as well as a Thrombolysis in myocardial infarction flow ≥2 . All pat2), congenital heart defects, acute pulmonary artery embolism, isolated left main ostial stenosis, active oncologic treatment, toxic cardiomyopathy, and inability to perform revascularization by PCI due to comorbidities or patient refusal. The design of this trial required the mandatory use of drug-eluting stents in both treatment groups.Acute coronary syndrome diagnosis was confirmed using angina pectoris symptoms, electrocardiography changes, and/or hs-troponin I levels above the reference range. Exclusion criteria were inability to give consent, age <18 years, significant valve disease requiring surgical intervention, cardiogenic shock, reduced kidney function . In iFR technology, a wire-based pressure transducer measures the aortic and coronary pressures distal to the stenosis in an isolated period during diastole when the relationship between pressure and flow is linear and constant. iFR is subsequently calculated. After the distal point iFR is calculated based on five heartbeats, the pullback co-registration with the last angiogram is obtained. During pullback, a pressure wire is slowly withdrawn under fluoroscopic control to the tip of the guiding catheter, measuring the ratio of every millimeter. Following pullback, the software transposes the ratio results to the results of the last angiogram, allowing the operator to directly observe pressure drops in the coronary artery. This allows virtual PCI planning, which in turn allows the operator to see the final estimated results of distal iFR by placing a virtual stent in the SyncVision software. Patients with an iFR ≤0.89 were considered severely ischemic and were recommended to undergo PCI treatment. The mandatory PCI result evaluation by iFR was performed. In patients with a post-PCI iFR ≤0.89 further PCI optimization was recommended, if feasible. Further optimization feasibility was determined based on information from post-PCI pullback co-registration. The concept of iFR is like that of fractional flow reserve (FFR), but it is easier to perform and provokes less patient discomfort since hyperemic agents are not needed. Moreover, iFR allows PCI planning based on pullback co-registration results as multiple lesions do not interfere with result calculations. In this study, the patients treated using virtual PCI planning by iFR co-registration were assigned to the iFR-guided group.The primary safety endpoints of this study were PCI-related major adverse cardiovascular events (MACE), comprising cardiac death, non-fatal myocardial infarction (MI), unplanned revascularization, stent thrombosis, flow limiting coronary dissections, no-reflow phenomenon, coronary perforations, coronary distal embolization, vascular access site complications requiring blood transfusion or surgical intervention, and contrast-induced nephropathy requiring hospitalization. Non-fatal MI was defined by criteria for acute myocardial infarction by the Fourth universal definition of myocardial infarction. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and approved by the Ethics Committee of the Riga Stradins University. The ClinicalTrials.gov identifier is NCT05006183.® Statistics for Windows, version 22.0  was used for all statistical analyses. Patient characteristics were analyzed using descriptive statistical methods, with mean values as central tendency indicators and standard deviations as dispersion indicators. If the data were not normally distributed, median values and interquartile ranges were used. Data compliance with normal distribution was determined by the Shapiro-Wilk test. In case of normally distributed continuous data “t-test” was used to compare means of two groups. According to the number of patients in both groups, Fisher’s exact test was used to compare qualitative nominal variables, and the nonparametric Mann-Whitney U-test was used to compare quantitative linear variables in two samples. Statistical power is determined by the following variables: alpha cut-off of 5%, beta cut-off of 20%, power of 80%, iFR-guided group incidence of 1.8% and angiography-guided group incidence of 18.3% [IBM SPSSof 18.3% ,24. A sa2. There were no significant differences in baseline characteristics between the groups, except for age, as patients in the angiography-guided group were significantly older than were those in the iFR-guided group   were female, and 10 (58.8%) were male, with a median age of 64 years and median body mass index of 28.5 ± 4.6 kg/m= 0.046) .p = 0.004; and 360.0 ± 97.9 vs. 192.5 ± 52.8 mL, p = 0.003)  .p = 0.007)  there were no significant differences in: the amount and length of stents implanted; the total volume of contrast media delivered; total PCI duration; the length of overall hospital stay; and total PCI-related MACEs , unplanned revascularization, stent thrombosis, flow limiting coronary dissections, no-reflow phenomenon, coronary perforations, coronary distal embolization, vascular access site complications requiring blood transfusion or surgical intervention, and contrast-induced nephropathy requiring hospitalization) between groups. The complete simultaneous myocardial revascularization strategy executed in the iFR-guided group required a significantly smaller number of PCI attempts than did the staged approach of the angiography-guided group  . Four inIn this pilot study, the iFR-guided and virtual planning tool-optimized PCI strategy was tested for safety and feasibility as a revascularization strategy in patients with MV ACS. Despite its significantly longer procedure and higher volume of the contrast medium delivered at the index PCI, there were no significant differences in safety endpoints , unplanned revascularization, stent thrombosis, flow limiting coronary dissections, no-reflow phenomenon, coronary perforations, coronary distal embolization, vascular access site complications requiring blood transfusion or surgical intervention, and contrast-induced nephropathy requiring hospitalization) between the angiography and iFR-guided groups at the 3-month follow-up. Furthermore, except for two cases of distal embolization in the iFR group, and one case of flow-limiting coronary dissection in the angiography-guided group during the second revascularization stage, there were no cardiovascular deaths or non-fatal MIs, and no patients needed unplanned revascularization. Moreover, there were no cases of no-reflow phenomenon, coronary perforation, stent thrombosis, contrast-induced nephropathy requiring hospitalization, and vascular access site complications requiring blood transfusion or surgical intervention.Overall, using invasive physiological coronary blood flow indices to assess lesion functional significance improves clinical outcomes in patients with MV CAD  and in pBoth FFR and iFR methods showed comparable results and diagnostic accuracy for non-culprit lesion assessment in the WAVE study in patients with STEMI  and in tRegarding the treatment of non-culprit arteries, the currently available evidence indicates that complete revascularization PCI is better than culprit-only revascularization for MV CAD. The COMPLETE trial demonstrated that complete revascularization for STEMI significantly reduced coprimary outcomes, including cardiovascular death, MI, and ischemia-driven revascularization . These fIn this study angiography-guided staged PCI strategy had a tendency towards longer overall hospital stay needed for complete revascularization. Additionally, iFR-guided PCI enabled complete revascularization in a single stage, demonstrating a significantly reduced number of PCIs needed in achievement of complete revascularization. This indicates several potential benefits in terms of the overall PCI-related risk since each procedure is associated with potential risks of vessel dissection, perforation, and hemodynamic complications . Thus, tComplete revascularization with the routine use of the virtual planning tool in one-stage iFR-guided PCI is a feasible practical strategy in an everyday Cath lab environment following the protocol designed for the study. One-stage iFR-guided PCI requires fewer PCI attempts until complete revascularization than does angiography-guided staged PCI. No statistically significant safety signals were documented in the number of PCI related MACE both during acute hospitalization and at 3-months follow-up. Further studies will help assess the role of routine iFR co-registration guidance in one-stage complete revascularization PCI to treat patients with MV ACS. After the lack of safety signals in the pilot study, further trial progress commenced to gain statistical power.


In longitudinal designs, unadjusted retest effects can confound developmental change estimates. This study utilized a measurement burst design and three-level multilevel modeling to a) independently parameterize short-term retest and long-term developmental change and b) employ these estimates as predictors of cognitive status at long-term follow-ups. Using data from Project MIND , participants  were assessed across biweekly sessions nested within annual bursts . Cognitive impairment no dementia (CIND) status was classified at Years 4  and 8 (the study end date). Response time inconsistencies (RTI) were computed to index intraindividual variability across RT trials of a one-back response time (BRT) task. Three-level multilevel models simultaneously yet independently estimated BRT RTI change across weeks and years, indexing short-term retest and long-term developmental change, respectively. Individual slope estimates were extracted and utilized in multinomial logistic regression models contrasting short- vs. long-term RTI change as predictors of long-term cognitive status. Results from the three-level models indicated that retest and developmental slopes yielded non-redundant sources of variance, providing unique estimates of change that would otherwise be confounded. Further, short- and long-term RTI differentially predicted cognitive status at Years 4 and 8; failing to benefit from retest effects on the BRT task was associated with increased likelihood of cognitive impairment. This innovative approach to parameterizing retest effects can reduce systematic bias in estimates of long-term developmental change, as well as highlight the utility of retest effects as predictors of cognitive health.


Eye lens opacities and cataracts among physicians and healthcare workers occupationally exposed to radiation.A systematic review and meta-analysisAlhasan & Aalam evaluate the risk of developing eye lens opacities and cataracts among physicians and healthcare workers occupationally exposed to radiation. Healthcare workers exposed to radiation have a significantly greater risk of posterior subcapsular cataracts (PSCs), cataracts, and any lens opacities than those of the non-exposed participants. Subgroup analysis reveal that nurses have the highest risk for PSCs, followed by interventional cardiologists. They concluded that the risk of posterior subcapsular opacities and cataracts was significantly higher in healthcare workers with occupational radiation exposure than in non-exposed workers, highlighting the necessity to enhance and promote the wearing of protective measures with high safety levels.see page 665 Comparison of bone mineral density and Fracture Risk Assessment Tool in Saudi women with and without type 2 diabetes mellitus.A cross-sectional studyAlfadhli et al compare the bone mineral density and the fracture risks in Saudi women with and without type 2 diabetes mellitus (T2DM). The 10-year probabilities of major osteoporotic fracture (MOF) and hip fracture (HF) are calculated using the Abu Dhabi Fracture Risk Assessment Tool (FRAX) with and without adjustment for T2DM. There are no significant differences in mean age, menopausal status, height, weight, and body mass index between T2DM and non-diabetic women. Bone mineral density values and the unadjusted FRAX scores are comparable between the 2 groups. However, after adjusting FRAX for T2DM, the FRAX for MOF and HF becomes significantly higher in T2DM women. They concluded that in Saudi women with T2DM, unadjusted FRAX underestimated the risk of MOF and HF. Type 2 diabetes mellitus should be included as one of the clinical risk factors for fracture in future versions of the FRAX score.see page 700D-dimer as a predictive and prognostic marker among COVID-19 patientsElkhalifa examines D-dimer, coagulation profile, and platelet count among patients hospitalized with coronavirus disease-19 (COVID-19) and compare them to findings from non-COVID-19 subjects. The participants in this retrospective hospital-based observational study design included 112 confirmed diagnosed with COVID-19 and another 112 non-COVID-19 subjects as a comparative group. Pneumonia related to COVID-19 is classified as critical, severe, moderate, mild, and asymptomatic whereas thrombocytopenia is marked when the platelet count is <150.00×109/L. The D-dimer and activated partial thromboplastin clotting time mean values are significantly elevated. The international normalized ratio and platelet count mean values confirms a significant decrease. Thrombocytopenia is found 9 times in COVID-19 higher than in the non-COVID-19. D-dimer and prothrombin time mean values increase significantly among the COVID-19 patients with all patterns of symptoms on admission. He concluded that D-dimer mean values increased significantly in deceased COVID-19 and in hospitalized intensive care unit (ICU) wards patients, indicating a potential predictive and prognostic severity marker, particularly among COVID-19 patients in the ICU.see page 723 Hidradenitis supparativa complicated by penoscrotal lymphedema and renal amyloidosisBin Rubaian et al report a case of a 42-year-old Indian gentleman who has hidradenitis suppurativa (HS) for 9 years presenting with 2 rare complications: penoscrotal lymphedema and end-stage renal disease secondary to renal amyloidosis. The patient is treated with prednisolone and adalimumab to treat both his HS and renal amyloidosis, and is referred to general surgery to manage his genital lymphedema. They recommended following adult patients with moderate-to-severe HS and clinical duration of greater than 3 years and screening for amyloidosis before they reach end-stage organ disease, similar to what happened to their patient with end-stage renal disease.see page 723


Our case adds to the emerging evidence of SARS-CoV-2 as a potential etiological agent of AIN in children and also suggests that interstitial kidney injury may result from secondary inflammatory damage. Epidemiological history, serologic testing, and SARS-CoV-2 detection in biopsy should be considered in the work-up of children with AIN of unknown etiology.Acute interstitial nephritis (AIN) has been recently recognized as one of the infrequent kidney involvement phenotypes among adult patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although SARS-CoV-2 associated intrinsic kidney disease has been scarcely reported in children, only one case of AIN temporally associated with the infection has been described in the pediatric population so far. We presented a case of a 12-year old boy who presented with fatigue, anorexia, and polydipsia following an RT-PCR that confirmed SARS-CoV-2 infection seven weeks prior to admission. Initial workup revealed increased serum creatinine (235 μmol/L), glucosuria, low-molecular-weight proteinuria, mild leukocyturia, and microhematuria with hyaline and granular casts on microscopy. Antibodies against the SARS-CoV-2 S protein receptor-binding domain confirmed prior infection with high titers. Kidney biopsy showed diffuse active interstitial nephritis with negative immunofluorescence and positive immunohistochemistry for SARS-CoV-2 in the inflammatory cells within the interstitium. Electron microscopy revealed several SARS-CoV-2-like particles. Kidney function continued to deteriorate despite several days of supportive therapy only (peak serum creatinine 272 μmol/L); thus, treatment with methylprednisolone pulse-dose therapy was initiated and was followed by oral prednisolone with gradual tapering. Kidney function completely recovered after 3 weeks post-discharge and remained normal after 11 weeks of follow-up (last estimated glomerular filtration rate 106 ml/min/1.73 m Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been associated with a wide range of kidney involvement, encompassing tubular, vascular, glomerular, and interstitial injury . The mecAcute interstitial nephritis (AIN) has been recently recognized as a distinct phenotype of kidney injury in adults with COVID-19 but evidence about its underlying mechanisms and the clinical course remains limited . RecentlWe described a case of an adolescent boy who developed AIN following COVID-19, with histological evidence suggesting the presence of SARS-CoV-2 in the kidney tissue.The patient was referred to our tertiary care center due to general malaise, loss of appetite, increased thirst, and a significant increase in serum creatinine. The patient was an otherwise healthy 12-year-old boy who had a RT-PCR confirmed SARS-CoV-2 infection with mild respiratory symptoms 7 weeks before the admission. The aforementioned clinical symptoms started ~1 week after the infection. The patient had no history of non-steroid anti-inflammatory drugs or other medication use prior to the development of symptoms or admission to the hospital. On admission, the patient was afebrile, had normal blood pressure (BP) and clinical examination was otherwise unremarkable.His blood tests revealed severe impairment of kidney function (serum creatinine 235 μmol/ml), high erythrocyte-sedimentation rate, anemia, thrombocytosis, and increased C-reactive protein. Urine tests showed glucosuria, low-molecular-weight proteinuria, and microhematuria, mild leukocyturia with hyaline and granular casts on microscopy. Initial laboratory results are presented in The complement levels of the patient were normal and no evidence of recent Epstein-Barr (EBV) or cytomegalovirus (CMV) infections was found by serologic testing. Additional workup that included ophthalmological evaluation for uveitis, anti-nuclear antibodies, and total serum calcium levels were all normal .2) within 3 weeks after treatment initiation. At the last follow-up 11 weeks post-discharge from the hospital, the patient remains asymptomatic while finalizing steroid tapering with normal eGFR (106 ml/min/1.73 m2), isolated microhematuria, and normal BP. The time course of serum creatinine since the admission to the hospital is summarized in Kidney function continued to deteriorate despite several days of the watch and wait period with supportive therapy only (peak serum creatinine 272 μmol/L), and thus the boy was treated with methylprednisolone pulse-dose therapy . Laboratory testing immediately after treatment revealed significant improvement in kidney function (serum creatinine 124 μmol/L), increase in hemoglobin levels (11.3 g/dL) and decrease in proteinuria (1.5–0.25 g/L), but glucosuria and microhematuria remained. The patient was discharged with normal BP on daily prednisolone (60 mg/day for 4 weeks followed by dose tapering planned over 2 months) which resulted in complete recovery of kidney function (estimated glomerular filtration rate (eGFR) of 102 ml/min/1.73 mThis is the second case of SARS-CoV-2 associated AIN in the pediatric population and the first one reporting possible presence of SARS-CoV-2 in the kidney tissue by IHC and EM thus suggesting a possible direct involvement of the virus in the development of AIN.in situ hybridization, RT-PCR, IHC, and immunofluorescence, while viruria has been documented in some infected patients  and bacterial infections with direct and indirect (immune-mediated) effects of infectious agents implicated in its development . A recenPediatric patients typically exhibit a milder course of COVID-19 but are prone to develop unique delayed sequelae   and as sin situ hybridization to detect viral particles in lung tissue, while one suggested poor specificity of IHC with significant observer bias, the other reported similar diagnostic performance , while no positivity was detected in glomeruli or tubules. However, the detection of SARS-CoV-2 in kidney tissue has not been standardized to date. RT-PCR may be a relatively sensitive and specific tool to identify virus presence but precludes distinguishing the localization of virus , . Severalformance , 12. NotEven more, controversy exists for EM as a tool to detect SARS-CoV-2 particles in kidneys and other tissues. Several prior reports about viral particles in kidney biopsies received heavy criticism for falsely identifying normal cellular structures, such as clathrin-coated vesicles or multivesicular bodies, like SARS-CoV-2 , 13–15. Although the identification of the virus in the kidney tissue supports the evidence of SARS-CoV-2 role, it does not inform about the mechanisms of kidney injury. We observed strong IHC positivity in some cells in the interstitial inflammatory infiltrate. Inflammatory cells, such as monocytes-macrophages, have been suggested to play a key role in the development of pathological hyperinflammatory response among patients with COVID-19. SARS-CoV-2 nucleocapsid positive macrophages have also been observed in COVID-19 patients with acute kidney tubular damage. It is, however, unclear whether this positivity may reflect phagocytosis of infected cells or direct infection of macrophages as ACE2 expression has been observed in macrophages stimulated by inflammatory stimuli .Finally, it is important to highlight the time delay between the start of symptoms and kidney biopsy which was at least 6–7 weeks in our patient presenting with advanced kidney failure. Thus, it remains unknown whether viral infection could have been more extensive within kidney cells in the earlier period of the disease. The lack of IHC positivity in tubular cells, however, points toward secondary damage caused by an inflammatory response in the interstitium.In conclusion, our case report adds to the emerging evidence of SARS-CoV-2 as a potential etiological agent of AIN in children and as such highlights the spectrum of SARS-CoV-2 manifestations in children. The demonstration of potential viral presence in kidney interstitial cells by IHC against SARS-CoV-2 spike glycoprotein suggests secondary inflammatory damage with a good response to steroid therapy. In the light of evolving virus spread and potential asymptomatic course, epidemiological history, serologic testing, and SARS-CoV-2 detection in biopsy should be considered in the work-up of children with AIN of unknown etiology. Further research on the role and mechanisms of SARS-CoV-2 mediated kidney injury and the role of inflammatory cells is needed.The original contributions presented in the study are included in the article/Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.KA conceptualized the manuscript. JB and AL performed and interpreted histological investigations. KA and AJ collected and interpreted clinical data. All authors critically revised and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To maximize protection and prevent spreading to other patients, COVID-infected-individuals were isolated. This poster will describe treatment outcomes of COVID 19 in Psychiatric hospital Ugljan.The ongoing pandemic of coronavirus disease 2019 (COVID-19) has made a serious public threat worldwide. The first case in the Republic Croatia was reported on 25This rapid review summarizes outcomes of COVID-19 infected psychiatric patients with mild disease severity to provide synthesized evidence to support policy decision making.PubMed, Medline, PsychINFO were systematically searched from January 2021 for COVID-19, with studies describing epidemiology, treatment and outcomes in various long-term care facilities. Studies were excluded if they did not report clinical evidence.In the time of COVID-19 breakthrough in psychiatric hospital, in December 2020 328 patients were hospitalized of whom 44 tested positive for SARS-CoV-2 infection: of 307 hospitalized patients in January 2021 36 tested positive for SARS CoV-2. By that time, there were no treatment options available, so we focused on repurposing efficacy of the currently used drugs. Five patients needed admission to ICU, we reported one death from coronavirus disease 2019 in that period.The COViD-19 pandemic has highlighted extreme vulnerability of psychiatric patients who reside in long-term care psychiatric hospitals and there is an urgent need for evidence-based policy that can protect adequately psychiatric patients.No significant relationships.


The tumor PMN-MDSCs were more sensitive to ferroptosis inducers, and spontaneous death could be rescued by not only inhibitors of apoptosis, but also inhibitors of ferroptosis. Moreover, the levels of lipid-derived signals associated with ferroptosis and CD71, a well-known ferroptosis biomarker, were highly expressed in tumor PMN-MDSCs. These results indicate that tumor PMN-MDSCs are susceptible to ferroptosis and even undergo ferroptosis spontaneously.+ T cell proliferation, only ferroptosis inducers could achieve immunosuppressive activity, with this suppressive effect being mediated by soluble factors. Genetic inhibition of ferroptosis by Alox12/15 deletion or Acls4 deletion, or inhibitors of ferroptosis, erased the immunosuppressive effect of tumor PMN-MDSCs in different murine models and patient samples. Gene set enrichment analysis suggested that the blockade of ferroptosis by Alox12/15 deletion in tumor PMN-MDSCs caused the upregulation of genes associated with the classical activation of PMNs, including complement activation, neutrophil-mediated immunity, monocyte chemotaxis, and antigen processing and presentation. These results suggest that ferroptosis mediates the conversion of classical PMNs to PMN-MDSCs, but the mechanism requires further investigation.Second, to investigate the association between ferroptosis and immunosuppressive function in tumor PMN-MDSCs, the authors treated non-suppressive PMNs with ferroptosis and apoptosis inducers. As measured by the reduction in CD8+ T cell proliferation. These results suggest that the release of oxidized phospholipids can also contribute to the ferroptosis-mediated suppression of PMN-MDSCs, in addition to the secretion of PGE2. Unsurprisingly, other factors that may promote polyunsaturated fatty acid phospholipid (PUFA-PL) synthesis and peroxidation (such as FATP2 and MPO), or that may cause GPX4 inhibition (such as hypoxia in the tumor microenvironment), may also influence the inhibitory activity of tumor PMN-MDSCs. Arachidonic acid, in combination with T cell-derived IFNγ, has been reported to cause immunogenic tumor ferroptosis.3 It is unclear whether other secreted factors such as free fatty acids could promote tumor PMN-MDSC ferroptosis.Third, the authors identified two kinds of soluble factors that regulate the ferroptosis-mediated immune suppression of PMN-MDSCs, prostaglandin E2 (PGE2) and oxidized phospholipids. Consistent with the upregulation of genes involved in the biosynthesis of PGE2 after RSL3 (a ferroptosis-inducer) treatment, the production of PGE2 was substantially increased after ferroptosis induction in tumor PMN-MDSCs. However, neither blockers of PGE2 synthetic enzymes (cyclooxygenase-1 and cyclooxygenase-2), nor inhibitors of PGE2 receptors (EP2 and EP4) completely erased the suppressive effect of ferroptosis induction. Oxidized phospholipids, the major products of ferroptosis, can directly cause the inhibition of CD83, although suppressive activity of tumor PMN-MDSCs was substantially inhibited. However, tumor growth was markedly inhibited in immunocompetent (but not immunodeficient) mice when using liproxstatin-1 at an earlier time when tumors were palpable. Flow cytometric analyses showed that liproxstatin-1 treatment increased the proportion of resident memory, central memory, effector CD8+ T cells, and natural killers in tumor microenvironment. Moreover, T cell activation was observed by scRNA-seq, characterized by upregulation of IL-2 and TNFα signaling and inflammatory response, and downregulation of oxidative phosphorylation. These results indicate that T cell infiltrating and activation contribute to the tumor regression with liproxstatin-1 treatment. The authors also observed a synergism of liproxstatin-1 treatment with PD-1 blockade. Additionally, analysis of patient data from The Cancer Genome Atlas showed strong correlations between tumor PMN-MDSCs and ferroptosis signatures in multiple cancer types. High expression of the ferroptosis signature was associated with lower survival. The ferroptosis signature was positively associated with tumor immunogenicity, and ferroptosis inducers could inhibit tumor growth and sensitize the tumors to IFNγ and immune checkpoint blockade.4 Moreover, ferroptosis in tumor-infiltrating CD8+ T cells could impair anti-tumor immunity and promote tumor progression.5 Thus, ferroptosis induction for cancer therapy must consider the complex nature of ferroptosis in the tumor microenvironment.Finally, the authors investigated the effect of pharmacological inhibition of ferroptosis on xenograft mouse tumor models. Unexpectedly, liproxstatin-1 treatment did not reduce tumor growth when treatment was initiated when tumors were approximately 100 mm+ T cells and tumor-associated macrophages? What is the role of ferroptosis for other immune cells such as dendritic cells and Treg cells in the tumor microenvironment?In summary, the present findings not only reveal a previously unrecognized role of ferroptosis-mediated immune suppression in PMN-MDSCs, but also raise a paradox in the use of ferroptosis inducers in tumor therapy. However, the ferroptosis inducers or inhibitors used in the study were not cell-specific, which could affect the viability and activity of other cells in the tumor microenvironment and finally cause the bias and misinterpretation of results. Thus, cell-specific delivery of ferroptosis inducers or inhibitors presents a promising and contentious area for future research. Like any pioneering study, such an ingenious study also promotes additional, interesting questions for the future. Why does the immunosuppressive effect of ferroptosis in tumor PMN-MDSCs outweigh the tumor-killing effect of ferroptosis in tumor cells? How does ferroptosis in tumor PMN-MDSCs affect other immune cells in addition to tumor-infiltrating CD8


Background: In late September 2021, a cluster of patients with nosocomial COVID-19 was identified on a liver transplant unit at University of Nebraska Medical Center. Methods: The outbreak investigation included contact tracing via patient chart and employee health record reviews and serial prevalence testing for SARS-CoV-2 among potentially exposed patients and healthcare workers (HCWs). Routine admission and preprocedural screening for SARS-CoV-2 was performed, and involved patients had negative admission screening results with positive SARS-CoV-2 tests >5 days from admission. Mitigation strategies involved reinforcement of patient care and visitation procedures. Whole-genome sequencing of positive SARS-CoV-2 specimens was conducted. Results: The potential outbreak cluster included 6 patients in the same quadrant of the liver transplant unit, 1 visitor, and 11 healthcare workers  variant in a solid-organ transplant unit including patients, a visitor, and vaccinated healthcare workers with multiple introductions of the virus. Further transmission was not detected after enhanced infection control measures were introduced, including universal masking and eye protection, closing patient doors, and enforcement of visitor masking policy. We describe the difficulties tracing SARS-CoV-2 transmission in the hospital setting, even with advanced sequencing techniques. This outbreak highlights the importance of booster vaccination and strict infection control practices, especially in the setting of the δ (delta) variant.Funding: NoneDisclosures: None


The term non-neoplastic hypercortisolism (NNH) is used frequently nowadays, interchangeable with the pseudo-Cushing (pCS) state, in order to differentiate the functional non-neoplastic hypercortisolism that mirrors the neoplastic activation of the HPA axis, termed endogenous Cushing’s syndrome (CS). Diagnosis of neoplastic CS is often complex, especially when modest cortisol excess is present. Indeed, functional activation of the hypothalamic-pituitary-adrenal (HPA) axis can be caused by a variety of illnesses/conditions  . AlthougBefore ruling out NNH/pCS with second-line testing the possibility of a false-positive result from first-line testing for hypercortisolism should be considered. The low specificity of immunoassays may cause false-positive results from interfering circulating steroids (mainly cortisone) in late-night salivary cortisol and urinary-free cortisol. The use of estrogen-progesterone contraceptives or CYP3A4 inducing drugs may cause false-positive results at low-dose dexamethasone suppression test (LDDST). Special attention to the pre-analytical phase , use of mass spectrometry assays, and dexamethasone (dex) measurement may be useful tools to avoid false-positive results. In addition, physicians should also select the most appropriate test for the considered patient: avoid measuring circadian rhythm in shift workers, urinary cortisol in case of impaired renal function, and so on [Several second-line tests have been proposed to distinguish NNH/pCS from neoplastic CS Table , but theACTH-secreting adenomas may aberrantly express vasopressin receptor 2 (VR-2). As a result, the synthetic analog DDAVP could only elicit ACTH response in Cushing’s Disease (CD) patients, because the normal pituitary expresses VR-3, which has a low DDAVP affinity . A rise No single test among those proposed seems to guarantee a perfect distinction between CS and NNH/pCS patients. Furthermore, the wide variation in the population recruited, the NNH/pCS definition criteria, and the cut-offs used in different studies make it difficult to define a gold standard in this intricate differential diagnosis. To summarize, ruling out NNH/pCS-related disorders is required in cases of moderate hypercortisolism, equivocal clinical presentation, or suspected non-neoplastic hypercortisolism. According to the recent Pituitary Society consensus report, the patient’s clinical history and the duration of symptoms are of utmost importance in the differential diagnosis of NNH/pCS . The spe


All analyses were restricted to EDs that reported consistently more complete data throughout the study period ; 56% of EDs sharing data with NSSP met these criteria.NSSP collects deidentified electronic health record data from EDs and other health care settings. ED visit data are derived from a subset of approximately 71% of the nation’s nonfederal EDs . Diagnosis codes from the All ED visits, including seizure-related ED visits, decreased among all age groups and among both males and females during the pandemic period April 1–December 29, 2020, compared with the corresponding period in 2019 . The lar–In this study of trends in seizure-related ED visits during the COVID-19 pandemic, seizure-related ED visits during the initial COVID-19 waves declined among all age groups, especially among children aged 0–9 years. These findings are consistent with several other studies , but could not attribute the observed decrease in seizure-related ED visits to seizure type   or seizure sequelae , thereby reducing the need for ED care  visits. Data on seizure- or epilepsy-related ED visits during the COVID-19 pandemic are limited.Weekly seizure- or epilepsy-related ED visits decreased sharply during the early pandemic period among all age groups, especially children aged 0–9 years. The return to prepandemic baseline in this group was delayed until mid-2021, longer than other age groups.These findings reinforce the importance of understanding factors associated with ED avoidance among persons with epilepsy or seizure, the importance that all eligible persons be up to date with COVID-19 vaccination, and the need to encourage persons to seek appropriate care for seizure-related emergencies.


By integrating large-scale transcriptomic and genomic data, this study identified immune-related pathways with aging characteristics among 25 cancer types. We found that the perturbation of immune-related pathways showed a cancer-specific expression pattern. Moreover, the aging- and cancer-related dysregulated patterns of pathways were in the same direction in most cancer types. Furthermore, the study found that aging-related immune pathways with clinical relevance improved immunotherapy benefits in melanoma and non-small cell lung cancer patients. Using a network-based method, this study predicted potential drug targets for immunotherapy and treatment combinations. Collectively, this study may provide insight into cancer immunotherapy, thus improving treatment benefits.(1) Background: Perturbation of immune-related pathways can make substantial contributions to cancer. However, whether and how the aging process affects immune-related pathways during tumorigenesis remains largely unexplored. (2) Methods: Here, we comprehensively investigated the immune-related genes and pathways among 25 cancer types using genomic and transcriptomic data. (3) Results: We identified several pathways that showed aging-related characteristics in various cancers, further validated by conventional aging-related gene sets. Genomic analysis revealed high mutation burdens in cytokines and cytokines receptors pathways, which were strongly correlated with aging in diverse cancers. Moreover, immune-related pathways were found to be favorable prognostic factors in melanoma. Furthermore, the expression level of these pathways had close associations with patient response to immune checkpoint blockade therapy in melanoma and non-small cell lung cancer. Applying a net-work-based method, we predicted immune- and aging-related genes in pan-cancer and utilized these genes for potential immunotherapy drug discovery. Mapping drug target data to our top-ranked genes identified potential drug targets, FYN, JUN, and SRC. (4) Conclusions: Taken together, our systematic study helped interpret the associations among immune-related pathways, aging, and cancer and could serve as a resource for promoting clinical treatment. Evidence from accumulating studies has substantiated that aging is one of the most fundamental causes of a broad range of cancer ,2,3. TheImmunotherapy has proven a promising strategy for cancer treatment, for which impressive results have been achieved in various cancers . By inhiWe integrated genomic and transcriptomic data of 25 cancer types to refine these gaps further. In this study, we systematically analyzed the age- and cancer-associated differences in immune-related pathways in pan-cancer. We noticed that the perturbation of immune-related pathways during aging showed cancer-specific characteristics. Besides, the age-related alteration patterns of these pathways were investigated. We identified several immune-related pathways that could predict the patient response to immunotherapy in melanoma and non-small cell lung cancer. Potential drug targets of ICB therapies were predicted by a network-based method. Collectively, our results might help elucidate the age-associated roles of immune-related pathways in tumorigenesis and possibly inform immunotherapy options.http://xena.ucsc.edu/, accessed on 19 November 2020) (The transcripts per million (TPM) of RNA-seq data for 25 cancer types from the Cancer Genome Atlas data portal (TCGA) and non-cancerous tissues from Genotype-Tissue Expression (GTEx) consortium were downloaded from the UCSC Xena platform  (http://er 2020) . TCGA anCancer types included for further analysis were as follows: adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC) and uterine carcinosarcoma (UCS).https://www.cancerrxgene.org/, accessed on 22 May 2022). Gene expression profile of cancer cell lines was downloaded from Cancer Cell Line Encyclopedia (CCLE) database   value of drugs in tumor cell lines was obtained from Genomics of Drug Sensitivity in Cancer (GDSC) database  (https:/ay 2022) .http://www.immport.org, accessed on 11 March 2021). We applied single-sample gene-set enrichment analysis (ssGSEA) to calculate the immune-related pathways (IRP) scores across 25 cancer types based on ImmPort genesets  project  Identification of differentially expressed IRPs in tumor samplesp-value < 0.05 were considered as differentially expressed.R package ‘limma’ was applied to extract dysregulated IRPs in tumor samples . IRPs wi(2)Identification of IRPs differentially expressed with agep-value < 0.05 was determined as statistically significant. Moreover, we applied Analysis of Variance (ANOVA) analysis to compare the IRP scores differences across cancers.It must be mentioned that the actual age of samples (donors from whom the sample was collected) from GTEx was not provided; thus, we approximated the median for each age group to each sample, i.e., 25 for all samples from 20–29 . A lineahttps://www.gsea-msigdb.org/gsea/msigdb, accessed on 7 May 2022), Human Aging Genomic Resources [http://genomics.senescence.info/genes/, accessed on 7 May 2022) and a previously published study [Aging-related-genesets (ARG) were downloaded from Molecular Signatures Database   betweenThe somatic mutation data of the mutation annotation format (MAF) file was downloaded from the publicly available TCGA MC3 project . IRP altWe performed the Cox regression model and log-rank tests to identify IRPs related to overall survival in 25 cancer types. Besides, the multivariate-Cox regression model was performed in SKCM to identify IRPs that served as independent prognostic factors. KM survival curve grouped by the median of IRP scores were plotted by R package ‘survival’ and ‘survminer’. We constructed a nomogram by R package ‘rms’ to select independent prognostic factors and evaluated the predictive capability of nomograms by the calibration plot.p < 0.05 was considered significant). Moreover, we used the Gide cohort as the training set and applied the support vector machines (SVM) model to predict the response of patients from the TCGA SKCM cohort. We used the R package e1071 to train the SVM model.Five cohorts of SKCM patients received immunotherapy, including GSE91061, GSE96619, GSE35640, Gide dataset, and VanAllen dataset ,37,38, aFC1 is the fold change of IRG in tumor and FC2 is the fold change with age.A protein–protein interaction network, including 141,296 interactions among 13,460 genes, was compiled from a previous study . First, P0 is the initial probability vector in which “1” were assigned to the top 10% IRGs; γ is the restarting probability; W denotes the adjacency matrix of the network; tP is the final probability vector of other nodes after iteratively reaching stability. After obtaining a ranked list of IRGs, we projected the top 10% of genes to the PPI network in 25 cancers, separately. A random walk with a restart was employed to measure the association between all other genes in the network and the top 10% IRGs by the following equation ,43:(3)Pthttps://www.dgidb.org/, accessed on 12 June 2022). Furthermore, we used the drug sensitivity data from GDSC and CCLE to validate our drug prediction results.For each cancer type, genes that score in the top 1% after network propagation were extracted, then we integrated the top genes shared among 25 cancers and re-performed a random walk with a restart. Finally, we obtained a ranked gene list for drug discovery by the Drug Gene Interaction Database (DGIdb) database  .Functional enrichment analysis consists of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Reactome genesets and Wikipathways was carried out using Metascape platform  (https:/p < 0.05), suggesting cancer-type specific immune mechanisms  scores in 25 cancer types using the RNA-seq profile from the TCGA and GTEx database. Then we explored the differential expression of these IRPs between tumor and normal samples. We found that most IRPs were dysregulated in more than 19 cancer types A, exceptchanisms C. In conchanisms . These schanisms D. CancerAs we have identified differentially expressed IRPs in tumor samples among 25 cancer types, we then assessed the association between patients’ age and IRP score in pan-cancer. Using a linear regression model, we obtained a list of IRPs that showed significantly differential expression with age . The resp < 0.05) at all levels. IMM-AGE is a gene signature developed by multi-omic-based approaches to explore immune-aging dynamics at high resolution [Several studies have collected essential genes associated with age. Here, we obtained ten gene sets from different sources to evaluate the aging-related value of 17 IRPs ,29,30. FNumerous studies have proved the essential role of tumor mutation burden in aging. During the aging process, accumulated somatic mutations were associated with increased or decreased risks of tumorigenesis. Besides, ongoing studies have revealed the relevance of TMB and immune checkpoint blockade (ICB) therapy response . Herein,p-value < 0.05). 17 of 25 cancer types had at least one IRP associated with patient survival; among them, 15 out of 17 IRPs were favorable survival factors (HR < 1) in SKCM  and two cohorts of NSCLC patients who received PD-1 blockade immunotherapy. First, we computed the AUCs to evaluate the predictive ability of each IRP in discriminating between responder and non-responder in SKCM . As a reMoreover, we used the Gide cohort to predict the immunotherapy response of TCGA SKCM patients using the SVM model; a prolonged overall survival was found in predicted responders C. We alsNext, the same procedures were carried out in the two NSCLC cohorts. Antigen Processing and Presentation, Interleukins Receptor, Chemokine Receptor, Natural Killer Cell Cytotoxicity, TCR Signaling, and TGF-β family member receptors showed great predictive power (AUC > 0.7) by ROC curve analysis F. The scHerein, we applied a network-based guilt-by-association approach to calculate the proximity of each gene with dysregulated immune-related genes (IRGs) . After integrating cancer-specific results , we obtaAs a result, we obtained a list of potential compounds and their target genes shown in . AKT1 anFYN was found to regulate autoimmunity by binding to B-lymphocyte surface antigen CD19  and inteFunctional enrichment analysis of the 136 genes showed that top-ranked genes were enriched in the initial 17 IRPs  and other immune-related pathways; besides, several oncogene pathways were enriched, including Hippo, PI3K-AKT, WNT, etc. Moreover, aging- and longevity-related pathways, as well as drug-resist-related pathways, were also enriched in our prediction C.Accumulating evidence has linked immune-related pathways to cancer. However, what remains elusive is the effect of aging on immune-related pathways. Herein we comprehensively interrogated aging-related characteristics of IRPs and how these IRPs contribute to tumorigenesis. We found that tumors from related tissue origin showed a similar IRP expression pattern D. Most IThe prognostic value of immune-related genes  in cancers has been well-established ,70,71,72Unprecedented breakthroughs have been achieved in cancer treatment with the emergence of immune checkpoint blockade immunotherapy . DurableOngoing clinical tests have approved several drug combinations of checkpoint inhibitors, including ipilimumab ,84,85,86In this study, we used only 17 genesets from Immport, which curated immunologically relevant gene lists related to specific immune functions. In the future, applying more available datasets from other sources will further improve the research in related fields. Collectively, our systematic analysis of immune-related pathways in pan-cancer identified the aging-and tumor-related characteristics of these pathways and suggested that several pathways might help improve the response to cancer immunotherapy. Our results were instrumental in fully understanding the potential of immune-related pathways in cancer immunity and providing actionable strategies to broaden clinical tumor therapy.In conclusion, our work established a link between aging, immunity, and cancers by applying transcriptomic and genomic analysis to immune-related genes and pathways. This study found a perturbation of expression and mutation pattern of immune pathways with aging characteristics in human cancer. These pathways could serve as potential biomarkers for cancer immunotherapy. Our work highlighted the importance of immune-related genes and pathways with oncogenic roles in the aging process, which could further provide insights into immunotherapy for patients.


The myelodysplastic syndromes (MDS) represent a group of clonal disorders characterized by ineffective hematopoiesis, resulting in peripheral cytopenias and frequent transformation to acute myeloid leukemia (AML). We and others have demonstrated that MDS arises in, and is propagated by malignant stem cells (MDS-SCs), that arise due to the sequential acquisition of genetic and epigenetic alterations in normal hematopoietic stem cells (HSCs). This review focuses on recent advancements in the cellular and molecular characterization of MDS-SCs, as well as their role in mediating MDS clinical outcomes. In addition to discussing the cell surface proteins aberrantly upregulated on MDS-SCs that have allowed the identification and prospective isolation of MDS-SCs, we will discuss the recurrent cytogenetic abnormalities and genetic mutations present in MDS-SCs and their roles in initiating disease, including recent studies demonstrating patterns of clonal evolution and disease progression from pre-malignant HSCs to MDS-SCs. We also will discuss the pathways that have been described as drivers or promoters of disease, including hyperactivated innate immune signaling, and how the identification of these alterations in MDS-SC have led to investigations of novel therapeutic strategies to treat MDS. It is important to note that despite our increasing understanding of the pathogenesis of MDS, the molecular mechanisms that drive responses to therapy remain poorly understood, especially the mechanisms that underlie and distinguish hematologic improvement from reductions in blast burden. Ultimately, such distinctions will be required in order to determine the shared and/or unique molecular mechanisms that drive ineffective hematopoiesis, MDS-SC maintenance, and leukemic transformation. The myelodysplastic syndromes (MDS) are a group of clonal myeloid disorder characterized by ineffective hematopoiesis, disordered hematopoiesis evidenced by morphologic dysplasia, and frequent progression to acute myeloid leukemia (AML). Chemotherapy, stem cell transplantation, hypomethylating agents (HMAs) including decitabine (DAC) and azacytidine (AZA), and immunomodulatory drugs such as lenalidomide are used to treat MDS patients . AlthougMDS can be classified into different clinical risk categories based on hematopoietic features, age, and cytogenetic/genetic information at diagnosis. The International Prognostic Scoring System (IPSS) divides MDS into low-risk (Low and Intermediate-1 [Int-1]) and high-risk (Int-2 and High) disease, with high-risk MDS associated with higher blast counts, increased incidence of leukemic transformation, and poor clinical outcome . Studiesin vitro and maintain long-term engraftment in immunodeficient mice. Taken together, these studies demonstrate that MDS is a disease that is maintained and propagated by HSCs. In more recent studies exploring the role of the hematopoietic niche in MDS, studies have shown that HSPCs from low-risk MDS patients can reprogram healthy mesenchymal stromal cells (MSCs), and that the reprogrammed MSCs promote MDS HSC engraftment in xenografts  in AML immunophenotypically resemble committed progenitor cells such as lymphoid-primed multi-potent progenitors (LMPPs) (Lin-CD34+CD38-CD90-CD45RA+) or GMPs (CD34+CD38+CD123+/loCD110-CD45RA+) . MDS, innografts . In addinografts .in vivo , and transforming growth factor beta (TGF-b) pathways can promote the expansion of normal HSPC populations while increasing apoptosis in more mature populations . HSCs alin vivo . A recenin vivo .Several other innate immune signaling pathways have been implicated in MDS. The innate immune cytokine IL-8 and its receptor CXCR2 were found to be concomitantly overexpressed in high-risk MDS-SCs and AML LSCs when compared with healthy counterparts, suggesting that an IL-8/CXCR2 autocrine loop promotes MDS-SC and AML LSC function . CXCR2 iOne dysregulated non-inflammatory pathway thought to promote MDS HSCs self-renewal is the immunoglobulin-like and endothelial growth factor-like domains 1 (Tie2) pathway. Angiopoietin 1 (ANGPT1) promotes vascular development and angiogenesis by binding to the receptor tyrosine kinase TIE2, which supports normal HSC quiescence and self-renewal . Recent Defective ribosome biogenesis has been implicated in MDS pathogenesis, as loss of RPS14 expression contributes to del (5q) MDS . SimilarAlthough genetic loss of ribosomal proteins can directly contribute to decreased ribosome formation and MDS-like phenotypes, reduced ribosomal protein gene transcripts have been shown to be common in low-risk MDS. Reduced RPS14 transcripts were described in CD34+ HSPCs from MDS patients without del (5q), defining a subgroup of patients with prolonged survival . Other sStudies utilizing fluorescence in-situ hybridization (FISH) techniques demonstrated the presence of cytogenetic abnormalities in sorted HSPCs from MDS patients . StudiesA number of large studies have evaluated the presence of somatic mutations in MDS using whole genome- or targeted exome sequencing. Collectively, these studies have demonstrated that MDS shares many driver mutations with other myeloid neoplasms including AML. Genome-wide sequencing analysis of a large cohort of MDS patients showed a mean number of nonsynonymous mutations of approximately 11 per patient (or 0.34/Mb) for all MDS samples with a lower mutation rate in low-risk MDS (0.19/Mb) . Using tThese genetic studies revealed the prevalence and clinical significance of recurrent driver mutations in MDS. Multiple groups have shown that many of the hematologic features of MDS can be partial recapitulated in single-gene genetic mouse models, such as SRSF2, U2AF, SF3B1, and ASXL1 knock-in or knock-out mouse models . HoweverDelineating the hierarchical organization of clones in MDS HSCs is critical to understand mechanisms of disease progression and responses to drug therapy in MDS. Clonal structures and mutation hierarchy are inferred using MAF data, assuming mutations with the highest MAFs are clonal and likely occur early, while mutations with lower MAFs are subclonal and likely occur late in disease pathogenesis. Among different categories of oncogenic mutations, alterations in genes involving RNA splicing components including SF3B1, ZRSF2, SRSF2, and U2AF1 are predicted to represent earlier events that dictate disease evolution and are associated with distinct clinical phenotypes . AnotherNewer sequencing approaches now allow concomitant evaluation of genetic alterations and transcriptomes at the single cell level. Chen et al. performed deep targeted sequencing combined with single-cell sequencing on phenotypically defined malignant stem cells , premalignant stem cells , and blast populations in 7 patients with MDS who later progressed to sAML . They foSince MDS-SCs are critical for the initiation and propagation of MDS and are the presumed cell that undergoes additional genetic/epigenetic changes to mediate disease progression and relapse, they must be eradicated in order to cure the disease. Not surprisingly, much of the effort to identify therapeutic targets in MDS have focused on high-risk MDS/AML, with relatively few studies directly assessing effects on MDS HSCs in lower-risk disease. This distinction is important to keep in mind when interpreting studies and the potential utility of novel therapeutic strategies in lower-risk MDS. Ideally, therapies in MDS would allow targeting of malignant HSCs without affecting normal HSCs or hematopoiesis. A number of targets have been identified as potential therapeutic targets in MDS.Multiple studies demonstrated that current therapies including lenalidomide and HMAs treatment are insufficient to eradicate MDS-SCs. Lenalidomide can induce clinical and cytogenetic remissions in MDS patients with del (5q). However, all patients will eventually relapse. A study examined HSPCs from BM specimens obtained from seven MDS patients with del (5q) treated with lenalidomide showed persistence of HSC harboring del (5q), even in patients who achieved cytogenetic remissions and hematologic responses . AZA treRecently, the combination of AZA plus venetoclax, a BCL2 inhibitor, has been shown to be superior to AZA alone in several large clinical trials, inducing longer overall survival and higher incidence of remission in elderly AML and high-risk MDS patients . Analyziin vitro, and exhibit antileukemic activity in AML xenografts, without significantly depleting normal HSCs. CD99 may promote MDS-SC and AML LSC self-renewal ability by activating downstream pathways like SRC family kinases  is overexpressed on HSPCs in high-risk MDS, but not in low-risk MDS. IL1RAP expression is positively correlated with overall survival in MDS and AML , and antHigh levels of CD123  expression in immature HSPCs (CD34+/CD38−) has been reported in high-risk, but not lower-risk MDS . CD123 iCD47 is an antiphagocytic marker that is overexpressed in many human cancers, allowing them to evade immunosurveillance by providing a “don’t eat me” signal . CD47 isT-cell immunoglobulin mucin-3 (TIM3) is overexpressed on LSCs in AML compared to normal HSPCs. TIM-3 and its ligand, galectin-9 , form an autocrine loop critical for AML LSC self-renewal . In MDS,in vitro and leukemia burden in AML xenografts , an antagonistic IgG4 mAb targeting TLR2, induces differentiation of erythroid cultures . A phasenografts . IRAK4 inografts . Effortsnografts  , in order to better understand the molecular bases of these distinct disease processes.


This cohort study assesses whether influenza vaccination is associated with SARS-CoV-2 infection and related outcomes such as hospitalization or mortality in individuals aged 66 years or older. Is influenza vaccination associated with SARS-CoV-2 infection and related outcomes, such as hospitalization and mortality in individuals aged 66 years or older?In this cohort study of 2 279 805 patients, influenza vaccination was found to be associated with a 22% to 24% lower risk of SARS-CoV-2 infection; however, undertaking a periodic health examination (PHE) was also associated with a 15% lower risk of SARS-CoV-2 infection. The negative association between influenza vaccination and SARS-CoV-2–related outcomes differed considerably when the analysis was stratified by the history of PHE in the preceding year.The findings of this study suggest that a PHE may at least partially modify the association between influenza vaccination and SARS-CoV-2–associated outcomes in individuals aged 66 years or older owing to the healthy vaccinee bias. Vaccine effectiveness studies have rarely implemented strategies to reduce the healthy vaccinee bias arising from differences in health care–seeking behavior between vaccinated and unvaccinated individuals. Although previous observational studies suggest that influenza vaccination is associated with a reduced risk of SARS-CoV-2–associated outcomes, the healthy vaccinee bias may have led to overestimating the vaccination effect.To estimate the association between influenza vaccination and SARS-CoV-2–associated outcomes.This cohort study was conducted over 2 consecutive influenza vaccination campaigns (2019-2020 and 2020-2021), owing to the substantial COVID-19 burden and the greater validity of influenza vaccination data in the studied age group. The study population included community-dwelling adults aged 66 years or older in Ontario, Canada.Influenza vaccination for a given season.The outcomes of interest included SARS-CoV-2 infection, SARS-CoV-2–associated hospitalization, SARS-CoV-2–associated death, and a composite of SARS-CoV-2–associated hospitalization or death. Cox proportional hazards models were used to measure the association between influenza vaccination and SARS-CoV-2–associated outcomes, censoring individuals who moved into long-term care, received COVID-19 vaccines, or died before the observation period end date. Primary care periodic health examinations (PHEs) were explored as a negative tracer exposure  and as an effect modifier of the association between influenza vaccination and SARS-CoV-2 outcomes., 0.81; 95% CI, 0.76-0.87), and also SARS-CoV-2–associated hospitalization or death in vaccinated individuals who underwent PHE  and vaccinated individuals who did not undergo PHE .Of 2 922 449 individuals aged 66 years or older  living in Ontario, 2 279 805 were included in the study. Among these, 1 234 647 (54.2%) were female and 1 045 158 (45.8%) were male; their mean (SD) age was 75.08 (7.21) years. Those who had received influenza vaccination exhibited a lower incidence of SARS-CoV-2 infection than unvaccinated individuals for the 2019-2020 cohort  and the 2020-2021 cohort . This association was also observed for SARS-CoV-2–associated hospitalization or death . Similarly, undergoing a PHE was also associated with a lower incidence of SARS-CoV-2 infection  and SARS-CoV-2–associated hospitalization or death , and modified the association between influenza vaccination and SARS-CoV-2 infection for vaccinated individuals who underwent PHE  and for vaccinated individuals who did not undergo PHE (aHRThe findings of this cohort study suggest that undergoing a PHE may at least partially modify the association between influenza vaccination and SARS-CoV-2–associated outcomes in individuals aged 66 years or older, providing evidence of the healthy vaccinee bias that may affect vaccine effectiveness studies. A novel coronavirus now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered to have caused the COVID-19 pandemic. The pandemic response has placed a great emphasis on vaccination.1 observational studies suggest that influenza vaccines may confer some protection against SARS-CoV-2–related outcomes.5 A meta-analysis of 16 observational studies involving 290 327 participants showed that the receipt of influenza vaccine was associated with a lower risk of SARS-CoV-2 infection .2 Data from Brazil on more than 50 000 patients hospitalized because of COVID-19 demonstrated that influenza vaccination was associated with a 7% lower odds of admission for intensive care treatment, 17% lower odds of requirement of mechanical ventilation, and 16% lower odds of death.3 Ecological analyses of large-scale data from the US and Italy have observed an inverse association between influenza vaccine coverage and COVID-19 mortality rates.5 Some potential mechanisms to support the biological plausibility for the inverse association observed between influenza vaccination and SARS-CoV-2–related outcomes have been offered, including induction of innate immunity,6 trained immunity,7 vaccine-associated viral interference,2 “original antigenic sin,”8 heterologous effects,10 and common pathogenesis features.11Although clinical trials have demonstrated the efficacy of novel vaccines specifically targeting the SARS-CoV-2 spike protein,13 vaccine effectiveness studies are susceptible to the “healthy vaccinee bias,” which may exaggerate the apparent protective effects of vaccines.14Because receipt of vaccines is generally voluntary and is largely sought by motivated, health-conscious individuals who are more likely to observe health-promoting behaviors ,15This study assessed the associations between influenza vaccination and SARS-CoV-2 infection and related outcomes, such as hospitalization and mortality. We examined primary care periodic health examinations (PHEs) as a negative tracer exposure  and as an effect modifier.STROBE) reporting guideline  of all adults aged 66 years or older. The start date of the follow-up for the 2019-2020 cohort was October 1, 2019, and included influenza vaccinations received until March 31, 2020, while the follow-up for the 2020-2021 cohort started on October 1, 2020, and included influenza vaccinations received until March 31, 2021. The maximum follow-up dates for the 2 cohorts were September 30, 2020, and April 30, 2021, respectively. All individuals had free and universal access to influenza vaccines, hospital and physician care, and SARS-CoV-2 testing using real-time reverse transcription–polymerase chain reaction (RT-PCR). We excluded non-Ontario residents, individuals ineligible for the Ontario Health Insurance Plan (OHIP) and those with no recorded sex or date of birth, persons with no contact with the health care system in the 3 years prior to follow-up, and people living in long-term care , public health unit (PHU) region, rural vs urban residence, and census dissemination area–level measures of household income, household density, apartment building density, recent immigration, self-identification as visible minority, uncoupled adults, educational attainment, and essential work status. Comorbidities included asthma, chronic obstructive pulmonary disease, diabetes, active cancer, chronic kidney disease, advanced liver disease/cirrhosis, autoimmune disorder, immunocompromised status , rheumatoid arthritis, inflammatory bowel disease, frailty, dementia, hypertension, ischemic heart disease, congestive heart failure, and transient ischemic attack/stroke. Because we hypothesized that health care use may likely be associated with increased access to testing and may also be a marker for comorbidities, we measured health care use by the number of acute-care hospital admissions in the past 3 years and physician office visits in the preceding year.We compared the baseline characteristics between vaccinated and unvaccinated individuals in each cohort. We used multivariable Cox proportional hazards models to estimate cause-specific hazard ratios (HRs) (reported with 95% CIs) for the association between influenza vaccination and SARS-CoV-2–related outcomes. Influenza vaccination was modeled as a binary time-varying covariate. Individuals were censored if they died, moved into long-term care, or received a COVID-19 vaccine.In all analyses, we calculated adjusted HRs (aHRs) after adjusting for age and sex, and subsequently fully adjusted HRs accounting for age, sex, comorbidities, past health care use, receipt of the previous-year influenza vaccination, neighborhood income, rurality, PHU region, and census variables. To examine the association between influenza vaccination and having an RT-PCR test result for SARS-CoV-2, we used logistic regression to calculate adjusted odds ratios (AORs), accounting for the same factors.To examine the presence of the healthy vaccinee effect, we evaluated the association between having a PHE in 2018-2019 and SARS-CoV-2–related outcomes in 2019-2020, essentially treating PHE as a negative tracer exposure because there is no biological plausibility to expect that having a PHE should reduce the risk of SARS-CoV-2 infection and related outcomes. We subsequently examined the association between influenza vaccination and SARS-CoV-2–related outcomes stratified by PHE, thereby treating it as a potential effect modifier.For the 2019-2020 cohort, we evaluated the influence of repeated influenza vaccination by comparing individuals who did not receive influenza vaccination in either 2018-2019 or 2019-2020  vs those who received influenza vaccination in both 2018-2019 and 2019-2020 (YY), those who received influenza vaccination in 2018-2019 but not in 2019-2020 (YN), and those who did not receive the vaccination in 2018-2019 but received it in 2019-2020 (NY). We did not include the 2020-2021 cohort in this analysis because experiencing a SARS-CoV-2 outcome during 2020-2021 prior to influenza vaccination may alter the estimates for the YN group .P < .05 defined as the level of statistical significance. We performed our analyses using SAS, version 9.4 software (SAS Institute Inc).All statistical tests were 2-tailed, with We identified 2 922 449 individuals aged 66 years or older  living in Ontario at the start of the study period . After the exclusions  . We obseFor the 2019-2020 cohort, the frequency of SARS-CoV-2 testing was significantly higher in influenza-vaccinated individuals . For the 2020-2021 cohort, we observed a lower frequency of SARS-CoV-2 testing in influenza-vaccinated individuals  .We examined the association between PHE in 2018-2019 and SARS-CoV-2 infection in the 2019-2020 cohort. A total of 342 800 individuals (15.0%) in this annual cohort underwent a PHE, and undergoing a PHE was associated with a lower risk of SARS-CoV-2 infection in the next year  . SimilarIndividuals who underwent a PHE in 2018-2019 were more likely to have received an influenza vaccine in the 2019-2020 cohort (209 563 of 342 800 [61.1%]) compared with those who did not . Owing to the significant association between PHE and influenza vaccination, we stratified our analysis of influenza vaccines and the risk of study outcomes by PHE. We observed that the aHR for the association between influenza vaccination and SARS-CoV-2 infection  was considerably decreased for vaccinated individuals who underwent a PHE  than for those who did not undergo PHE  . This diThe YY group had a significantly lower incidence of SARS-CoV-2 infection than that of the NN group  . The NY Similarly, the YY group had a significantly lower incidence of SARS-CoV-2 hospitalization/death compared with the NN group . The association between influenza vaccination and SARS-CoV-2 hospitalization/death was not statistically significant for the NY and YN groups , respectively.We demonstrated that influenza vaccination during the 2019-2020 and 2020-2021 vaccination campaigns was associated with a 22% to 24% lower risk of SARS-CoV-2 infection, a 17% to 32% lower risk of SARS-CoV-2–associated hospitalization, and a 27% to 42% lower risk of SARS-CoV-2–associated mortality during the first 2 years of the COVID-19 pandemic. However, we also found that undergoing a PHE during 2018-2019 was associated with a 15% lower risk of SARS-CoV-2 infection, a 21% lower risk of SARS-CoV-2–associated hospitalization, and an 18% lower risk of SARS-CoV-2–associated mortality during 2019-2020. We subsequently showed that the negative association between influenza vaccination and SARS-CoV-2–related outcomes differed considerably when the analysis was stratified by a history of PHE in the preceding year. This finding suggests that the negative association between influenza vaccination and SARS-CoV-2–related outcomes was at least partially overestimated because of the healthy vaccinee bias. In addition, we showed that aHRs for the associations were decreased for individuals who had received the influenza vaccine during 2 consecutive seasons than for those who had received it during the current season only, which further suggests the presence of healthy vaccinee bias.Observational studies are susceptible to potential sources of bias associated with selection of the study population, misclassification, and confounded measurements. In this large-scale, population-based study, we showed the importance of considering the simultaneous effects of variables, which may alter the estimation of vaccine effectiveness. The frequency of SARS-CoV-2 testing was greater in influenza-vaccinated vs unvaccinated individuals for the 2019-2020 cohort, while this association was negative for the 2020-2021 cohort.17 This negative association was also demonstrated by estimating the odds of testing positive for SARS-CoV-2 in patients who received the influenza vaccine compared with those who did not.18 Although these observational studies showed that individuals who received influenza vaccines were at reduced risk of experiencing SARS-CoV-2–related outcomes, potential sources of healthy vaccinee bias have not been investigated.19Other observational studies have reported a negative association between influenza vaccination and SARS-CoV-2–related outcomes.21 Experimental studies have suggested that influenza vaccines may provide a temporary preventive effect against noninfluenza respiratory infections by inducing innate immunity through various mechanisms.23 The expression of interferon genes was found to be induced by influenza vaccines immediately after vaccination.24 In addition, a study has hypothesized that exposure to the influenza virus confers short-term immunity against other respiratory viruses, at least for a few weeks, because of the activation of a nonspecific immune response mediated by the release of interferons and other cytokines.10 Although this phenomenon was suggested to provide a protective effect against several respiratory viruses,25 this protection should only be transient.22 Thus, the protective effect of influenza vaccination against infection with noninfluenza respiratory viruses does not appear biologically plausible. Moreover, this “heterologous immunity” against other viruses may cause harm  rather than benefits.26The protective effect of influenza vaccination against SARS-CoV-2 infection and severe outcomes would appear implausible. Some studies hypothesized that “trained immunity” following influenza vaccination may alter the SARS-CoV-2 infection risk.14 explained the likelihood of influenza vaccine effectiveness overestimation during influenza seasons secondary to healthy vaccinee bias. According to this concept, individuals with health-promoting behaviors are more likely to adhere to the annually recommended vaccination. Subsequently, Remschmidt et al12 showed that the healthy vaccinee bias could have influenced multiple influenza vaccine effectiveness studies. To explore this bias, we initially calculated the HRs of SARS-CoV-2–related outcomes adjusted for comorbidities and other potential confounders. The estimated aHRs showed that this strategy might not overcome the risk of healthy vaccinee bias, as the negative association between influenza vaccination and SARS-CoV-2–related outcomes remained statistically significant, with fully adjusted HRs. In some epidemiologic studies that assessed the effectiveness of influenza vaccines in older adults, the individuals who were classified as “healthy” because they lacked diagnostic codes for comorbidities  were more likely to be cognitively or functionally impaired based on hospital medical record reviews.27 Hence, although healthy adherers may truly have a lower rate of comorbidities, hazard estimates adjusted for comorbidities recorded in health administrative databases may not successfully eliminate the risk of healthy vaccinee bias.Nelson et alhealthy in this bias refers to health consciousness and not necessarily a lack of comorbidities. The healthy vaccinee bias may be more conspicuous for influenza vaccination compared with other immunizations.14 Although individuals with health-promoting behavior are more likely to receive the influenza vaccine, reduced adherence to vaccination by the individuals with deteriorating health status may lead to overestimating mortality risk reduction.28Of note, the term Receiving the influenza vaccine is voluntary, and individuals who are more cautious about their health are more likely to receive vaccines. Regardless of their comorbidities, these individuals are likely more motivated and inclined to engage in health-promoting and disease-preventing behaviors. Therefore, to overcome the healthy vaccinee bias, vaccine effectiveness studies should consider some strategies to identify these individuals.29 Thus, population-based studies are needed to estimate SARS-CoV-2 vaccine effectiveness in real-world conditions.30 The exact influence of the vaccines cannot be estimated unless potential sources of bias, such as the healthy vaccinee effect, are corrected. Most vaccine effectiveness studies did not implement specific strategies to reduce the bias arising from differences in health care–seeking behavior between vaccinated and unvaccinated individuals.36 Some studies measured the weight of an observation by the inverse of the propensity to be vaccinated.31 However, none of these strategies specifically targeted the healthy vaccinee effect to reduce the risk of bias. Although multivariable adjustment for demographic variables and comorbidities likely decreases the risk of confounding bias, the risk of healthy vaccinee bias may not be eliminated.38 Studies can implement different strategies to detect potential proxies of the healthy vaccinee bias, such as frequent health care system contact to monitor health status, insurance condition, functional limitations, living alone, awareness of vaccine recommendations by health authorities, and mobilization status.41The study population in SARS-CoV-2 vaccine trials is typically restricted to healthy volunteers, and their well-defined end points are usually measured under ideal settings.42 Fourth, as with all observational studies, unmeasured confounding may persist.This study has limitations. First, we were not able to identify undiagnosed patients who were not tested for SARS-CoV-2. Second, 14-day hospitalization and 30-day mortality after a positive SARS-CoV-2 test result are all-cause outcomes, and individuals may have been admitted to hospitals or died for reasons unrelated to SARS-CoV-2 infection. Third, measurement of influenza vaccination using physician and pharmacist billing claims has imperfect sensitivity and specificity; thus, some misclassification of the exposure variable may have occurred.In this cohort study, we observed a lower risk of SARS-CoV-2 infection and associated hospitalization and mortality in older adults who received influenza vaccination in 2 provincial, annual cohorts. We found a similar negative association between undertaking a PHE in the preceding year and occurrence of SARS-CoV-2–related outcomes. The association between influenza vaccine and SARS-CoV-2 outcomes was at least partially modified by whether an individual had undergone a PHE, suggesting that PHE may be a proxy for the presence of healthy vaccinee bias. Our findings may have implications for other influenza vaccine effectiveness studies in adults aged 66 years or older.