The role of three-dimensional (3D) TOF-MRA in patients with cognitive impairment is not well established. We evaluated the diagnostic yield of 3D TOF-MRA for detecting incidental extra- or intracranial artery stenosis and intracranial aneurysm in this patient group.This retrospective study included patients with cognitive impairment undergoing our brain MRI protocol from January 2013 to February 2020. The diagnostic yield of TOF-MRA for detecting incidental vascular lesions was calculated. Patients with positive TOF-MRA results were reviewed to find whether additional treatment was performed. Logistic regression analysis was conducted to identify the clinical risk factors for positive TOF-MRA findings.In total, 1,753 patients  were included; 199 intracranial aneurysms were detected among 162 patients . A 3D TOF-MRA revealed significant artery stenoses (>50% stenosis) in 162 patients . The overall diagnostic yield of TOF-MRA was 16.8% . Among them, 92 patients  underwent either medical therapy, endovascular intervention, or surgery. In total, eighty-one patients with stenosis were prescribed with either antiplatelet medications or lipid-lowering agent. In total, fifteen patients  were further treated with endovascular intervention or surgery. Thus, the “number needed to scan” was 19 for identifying one patient requiring treatment. Multivariate logistic regression analysis showed that being female (odds ratio [OR] 2.05) and old age (OR 1.04) were the independent risk factors for intracranial aneurysm; being male (OR 1.52), old age (OR 1.06), hypertension (OR 1.78), and ischemic heart disease history (OR 2.65) were the independent risk factors for significant artery stenosis.Our study demonstrated the potential benefit of 3D TOF-MRA, given that it showed high diagnostic yield for detecting vascular lesions in patients with cognitive impairment and the considerable number of these lesions required further treatment. A 3D TOF-MRA may be included in the routine MR protocol for the work-up of this patient population, especially in older patients and patients with vascular risk factors. Brain magnetic resonance imaging (MRI) plays a crucial role in the diagnostic work-up and follow-up of patients with cognitive impairment , 2. WithTOF (time of flight)-MRA is the most commonly used MRA technique . One of Risk factors for intracranial aneurysm and vascular stenosis in general population are well established in the previous studies. Female sex, hypertension, and smoking are the well-known risk factors for intracranial aneurysm –16. In aTherefore, we aimed to explore the clinical utility of TOF-MRA in patients with cognitive impairment by evaluating its diagnostic yield for detecting incidental extra- or intracranial artery stenosis and intracranial aneurysm. Furthermore, we aimed to identify the clinical risk factors for positive TOF-MRA findings in this patient population.This retrospective, observational, single-institution study was approved by the institutional review board of Asan Medical Center, and the need for informed consent was waived. We reported our results according to the guidelines of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) .From January 2013 to February 2020, patients complaining cognitive impairment who underwent a dedicated brain MRI protocol for dementia at our institution were consecutively enrolled through a retrospective review of our electronic database. Whether to perform TOF-MRA or not was determined according to the referring physicians' preference considering patients' medical history such as hypertension, diabetes mellitus, or coronary artery disease. Patients were excluded if (1) they did not undergo TOF-MRA; (2) their images were of poor quality; and (3) they already knew the existence of aneurysms or stenoses.Magnetic resonance imaging was performed using a 3.0-T system  with an eight-channel head coil. The dementia protocol at our institution included the following sequences: a three-dimensional (3D) T1-weighted image, two-dimensional T2-weighted image, two-dimensional fluid-attenuated inversion recovery image, susceptibility-weighted image, diffusion tensor image, and separate 3D-TOF-MRA image for carotid and intracranial vessel. The scan time for TOF-MRA for the head and the carotids was approximately 4 min and 30 s in total. Gadolinium-enhanced T1-weighted imaging was performed if necessary. The detailed parameters are presented in the Brain MRI with TOF-MRA was reviewed by two authors . Only saccular aneurysms were counted and their number, size, and location were documented. Aneurysms were categorized based on the size into the groups of <3, 3–5, 5–7, 7–10, and >10 mm according to the previous studies based on the risk stratification for spontaneous rupture , 22. DegThe primary outcome was the diagnostic yield of TOF-MRA for detecting incidental vascular lesions in patients with cognitive impairment. Diagnostic yield was defined as the proportion of patients with positive findings of incidentally detected intracranial aneurysm or extra- and intracranial artery stenoses among those who underwent brain MRI with TOF-MRA for cognitive impairment work-up. Patients with positive TOF-MRA findings were reviewed to identify whether additional treatment was performed and the “number needed to scan (NNS)” was calculated. The number needed to scan is a similar concept to “number needed to treat,” and it indicates the number of TOF-MRA examinations needed to be performed to identify one patient who requires subsequent vascular intervention. The secondary outcomes were the clinical risk factor identification and risk stratification for positive TOF-MRA findings in patients with cognitive impairment.p < 0.1 in the univariate analysis were then entered into multivariate analysis using backward elimination method based on the maximum partial likelihood estimation. At each step, variables with p > 0.1 were removed. Vascular risk factors were chosen based on the previous large cohort studies  and R Statistical Software version 4.0.5.The diagnostic yield of TOF-MRA was calculated as the number of patients with positive MRA findings divided by the total number of enrolled patients. In addition, the diagnostic yield of TOF-MRA was calculated in a subset of the patients with Clinical Dementia Rating (CDR) ≥ 0.5. The cognitive scales [Mini-Mental State Examination (MMSE) and Global Deterioration Scale (GDS)] were compared between the patients with and without vascular lesions on TOF-MRA. Logistic regression analysis was performed to identify the risk factors for positive MRA findings. Sex, age, and vascular risk factors including hypertension, dyslipidemia, diabetes mellitus, smoking, alcohol, obesity, and stroke or ischemic heart disease history were used as variables in the univariate analysis. Variables with  studies , 18. Hypobtained . The adjA total of 7,749 patients underwent our dedicated MRI protocol for dementia between January 2013 and February 2020. Among them, 5,897 patients were excluded because they did not undergo TOF-MRA. Furthermore, 73 patients with known vascular lesions before MRI and 26 patients of duplicate data were excluded from the analysis. Finally, 1,753 consecutive patients  were included . The demThe TOF-MRA features of 199 aneurysms from 162 patients are summarized in The TOF-MRA features of significant extra- and intracranial artery stenoses in 162 patients are presented in p < 0.001). In addition, the proportion of moderate dementia (GDS ≥ 4) was significantly higher in the patients with vascular lesions .Among the 1,753 patients with cognitive impairment, TOF-MRA revealed incidental intracranial aneurysms in 162 patients  and significant extra- or intracranial artery stenoses in 162 patients . A total of thirty patients had both aneurysms and significant stenoses. The overall diagnostic yield of TOF-MRA for detecting incidental vascular lesions in patients with cognitive impairment was 16.8% ; furthermore, the diagnostic yields for detecting incidental intracranial aneurysms and extra- and intracranial artery stenoses were both 9.2% . In patients with CDR ≥ 0.5, similar results were obtained with the overall diagnostic yield of 18.3% ; the diagnostic yields for intracranial aneurysms and stenoses are 9.0%  and 11.6% , respectively. Patients with vascular lesions showed worse cognitive function than patients without vascular lesions  or lipid lowering agent  once significant stenoses were detected on TOF-MRA. Of note, 26 patients underwent maximal medical therapy for atherosclerosis (aspirin + clopidogrel + statin). In total, fifteen patients  were further treated with endovascular intervention (coiling) or surgery   received either medical, interventional, or surgical treatment after TOF-MRA. A total of eighty-one patients were prescribed with either antiplatelet medications (rectomy) , 3. The p = 0.001]. Conversely, being male was an independent risk factor for significant extra- or intracranial artery stenosis . Old age was associated with both aneurysm  and significant stenosis . Among the vascular risk factors, hypertension  and ischemic heart disease history  were associated with significant artery stenosis. Hypertension showed association with intracranial aneurysm and diabetes with significant stenosis in in univariate analysis but not in multivariate analysis after adjusting for other variables .p < 0.001)] (p < 0.001].When the age cutoff was set at 70 (mean age of the study population), the diagnostic yield of TOF-MRA for incidental vascular lesions was significantly higher in older patients (age ≥ 70)  . Likewisp < 0.001). Nearly a third of the patients (92/294) required an additional treatment due to the positive findings on TOF-MRA.Time-of-flight magnetic resonance angiography is currently an optional sequence for the work-up of patients with cognitive impairment , 7. Our The number of patients visiting dementia clinics is increasing due to increased life expectancy . HoweverEvidence regarding the association between cognitive impairment and intracranial aneurysm is lacking. Few studies focusing on unruptured intracranial aneurysms reported that a non-negligible proportion of patients had cognitive impairment even before undergoing treatment for aneurysm , 13. AssCompared to healthy subjects, patients with cognitive impairment are more likely to exhibit significant extra- or intracranial artery stenosis –10. It ip < 0.001). This might have caused bias in our study results. However, no significant difference was observed between the two groups regarding sex, intracranial aneurysm, and significant stenosis proportion. Third, there may be a potential risk of selection bias in our study because whether to perform TOF-MRA or not was determined by the physicians' preference considering the patients' vascular risk factors such as hypertension, diabetes mellitus, or coronary artery disease. Indeed, 82.3%  of the included patients had at least one vascular risk factors. This may have resulted in overestimation of the diagnostic yield of TOF-MRA for detection of significant arterial stenosis. Additionally, high proportion of the included patients had hypertension (58.7%) and history of smoking (27.1%), which may have resulted in high incidence of intracranial aneurysm as well. However, reviewing the patients with cognitive impairment who did not undergo TOF-MRA, we found the similar proportion of the patients with at least one vascular risk factors . Although the proportion of the patients with hypertension, diabetes, dyslipidemia, or obesity was significantly higher in the group that underwent TOF-MRA, the absolute difference was not that large to fully explain the high diagnostic yield of TOF-MRA in this group  when there was any ambiguity in distinguishing between the two entities on projection images. Finally, other less common vascular lesions were not included in the study. On retrospectively reviewing the radiologic reports, we found three patients with suspected dural arteriovenous fistula and one patient with a small arteriovenous malformation. However, none of the patients underwent transfemoral cerebral angiography for confirmatory diagnosis.In conclusion, our study demonstrated the potential benefit of 3D TOF-MRA, given that it showed high diagnostic yield for detecting vascular lesions in patients with cognitive impairment and the considerable number of these lesions required further treatment. A 3D TOF-MRA may be included in the routine MR protocol for the work-up of this patient population, especially in older patients and patients with vascular risk factors.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board of Asan Medical Center. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.HP: curated and analyzed data and drafted manuscript. CS: design and conceptualized study and revised manuscript. WS, HH, and WK: collected and constructed database. J-SL, J-HL, HK, and SK: provided conceptual feedback and revised manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Research Foundation of Korea and Korea Health Industry Development Institute (NRF-2021R1C1C1014413 and HI18C2383).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.


Lung cancer remains one of the most common and difficult cancers to treat. However, excellent progress in identifying the genetic causes of lung cancers has revealed many of the mutant proteins driving progression of this cancer. This in turn has led to development of new precision medicines that target these proteins providing significant improvement to patient outcomes. Unfortunately, this benefit tends to be relatively short-lived as the cancers develop resistance to these targeted agents. In this review, we look specifically at cancers driven by the EML4-ALK protein that is present in around 5% of lung cancer patients, occurring more frequently in young non-smokers. We consider the recent evidence for how resistance develops to current clinically approved targeted ALK inhibitors in these patients. Furthermore, we explore how additional research is revealing exciting alternative treatment options that may lead to a more sustained response and increased long-term survival for these patients.EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants.EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the  Lung cancer is the leading type of cancer in males and among the top three for females around the globe. Regardless of gender, it is also the leading cause of cancer-related mortality worldwide . Lung caEGFR and KRAS mutations being ranked as the most common followed by ALK rearrangements [EGFR mutation or ALK rearrangement are more likely to be non- or light-smokers, suggesting that the proportion of these will increase as smoking-related changes reduce [Several genetic changes have been identified as the main drivers of NSCLC, with ngements . The disngements . Interess reduce . A recens reduce .anaplastic lymphoma kinase (ALK) gene was initially discovered in 1994 in non-Hodgkin’s lymphoma, as a result of a chromosomal translocation involving the nucleophosmin (NPM) gene that led to the formation of the NPM-ALK oncogenic fusion protein [ALK is situated on the short arm of chromosome 2 and encodes a transmembrane receptor tyrosine kinase (RTK). It is highly expressed in neuronal cells during the early stages of cellular development, but expression is almost completely absent in mature cells [ALK rearrangements involving a variety of genes have been reported in several types of malignancies, including colorectal, breast and NSCLC [ALK with the EML4 gene, which encodes the echinoderm microtubule (MT)-associated protein (EMAP)-like 4 (EML) protein to create the EML4-ALK fusion protein, is found in approximately 5% of NSCLC cases [The  protein . ALK is re cells . Since 1nd NSCLC ,15. MostLC cases .EML4 gene is the most common fusion partner of ALK and EML4-ALK was first identified in 2007 in ~7% of Japanese NSCLC patients [The patients ,17. The patients ,19. A nupatients ,23,24,25EML4 to that encoding the C-terminal kinase domain of ALK [EML4 gene give rise to distinct EML4-ALK variants , which was isolated from unfertilised sea urchin eggs in 1993 . EML1 toEML4 gene. For example, V1, V2 and V4 possess the EML4 NTD and part of the TAPE domain and are known as the long variants, while V3 and V5 have some or all of the NTD, but none of the TAPE domain and are referred to as the short variants [The ALK protein comprises an extracellular domain required for ligand binding, a transmembrane sequence, and an intracellular domain, which contains the tyrosine kinase . All EMLvariants   NSCLC patients ,41. A grTo date, several different ALK mutations have been identified that promote resistance to ALK inhibitors, with the majority sitting within the ATP-binding pocket of the ALK catalytic domain. Interestingly, these can confer differential sensitivity to distinct ALK TKIs . For insALK catalytic site mutations, additional resistance mechanisms have been described. For example, ALK amplification is also able to promote crizotinib resistance [ALK-independent growth through activation of “bypass” signalling pathways for growth and survival. Some of the most common bypass pathways involve EGFR, MAPK or IGF-IR [MYC. Indeed, overexpression of MYC, a transcriptional regulator of multiple cancer-related processes, has been linked to resistance to crizotinib and alectinib [Other than sistance ,57. Furtr IGF-IR ,59. Finalectinib ,61.There have been many excellent review articles that summarise the oncogenic signalling pathways activated in NSCLC by the EML4-ALK fusion proteins as well as the mechanisms of action of ALK inhibitors and acquired resistance to these inhibitors e.g., ,30,62,63Where it is found that resistance has arisen through mutations in the ALK catalytic domain, the most obvious alternative approach to ALK TKI monotherapy would be to target the ALK-dependent signalling pathways that occur downstream of ALK activation. The current evidence suggests that ALK signalling operates through three well-characterised pathways: the JAK/STAT, RAS/MAPK, and PI3K/AKT pathways . The JanSignalling through the RAS/MAPK pathway is also activated in ALK+ NSCLC cells. Treatment of the V3 patient-derived cell line, H2228, with a combination of an experimental ALK TKI (TAE684) and AZD6244, a MEK inhibitor, blocked both the STAT3 and MAPK pathways. This led to a simultaneous increase in pro-apoptotic BIM and decrease in anti-apoptotic survivin expression . SimilarThe PI3K/AKT pathway is another important ALK-dependent signalling pathway . In factAn alternative approach to using inhibitors of the three signalling pathways mentioned above would be to target other less well studied mediators of ALK signalling. An et al. (2016) reported that the Crk-like (CRKL) adaptor protein is a mediator of ALK signalling in EML4-ALK NSCLC cells. They demonstrated that CRKL knock-down led to attenuated ALK signalling and reduced cell viability and migration . Given tOf course, these signalling pathways can also be turned on by other receptors to bypass the requirement for ALK. For example, Tanizaki et al. (2012) reported not only an increase in the activation of three additional RTKs , but also an increase in secretion of their ligand, epidermal growth factor (EGF), in EML4-ALK V1 patient-derived cells following administration of the ALK TKI, TAE684. Furthermore, they demonstrated that this led to activation of both the MAPK and JAK/STAT pathways, suggesting that the switch to EGF/EGFR/HER-dependent signalling results in TAE684 resistance. Indeed, treatment with an EGFR inhibitor induced apoptosis in these TAE684-resistant EML4-ALK V1 cells . In addiInterestingly, in one case, EML4-ALK expression served as a resistance mechanism to treatment of EGFR-mutant NSCLC with the EGFR inhibitor, osimertinib. Here, EGFR-dependent signalling switched to ALK-dependent activation, leading to acquired resistance to EGFR inhibition. This resistance was reversed when a combination of osimertinib and the ALK inhibitor, alectinib, was administered . With thIt is clear that cells expressing distinct variants of the EML4-ALK fusion oncogene have different sensitivities to ALK inhibitors ,79,80,81As indicated earlier, it is assumed that TD-induced ALK oligomerization underlies the constitutive activation of the EML4-ALK fusion by enabling trans-autophosphorylation. Hirai et al. (2020) used competitive peptides to test the hypothesis that disrupting ALK oligomerization would result in reduced ALK activation. For this, they introduced synthetic peptides analogous to the EML4 TD coiled-coil sequence into cells to prevent trimerization of EML4-ALK . The pepThe “short” EML4-ALK variants that lack any part of the TAPE domain are more stable than the “long” variants that contain a truncated portion of the TAPE domain . The preLong non-coding RNAs (lncRNAs) regulate many pathological and physiological processes, including lung cancer ,96. DiffLoss of cell cycle or transcriptional control is also likely to contribute to acquired resistance to ALK TKIs. For example, upregulation of a variety of cyclin-dependent kinases  occurs upon treatment with crizotinib or alectinib. Selective inhibition of CDK9 with alvocidib or dinaciclib, and CDK7 and CDK12 with THZ1, led to induction of apoptosis in patient-derived cells expressing V1 or V3 through downregulation of anti-apoptotic genes, such as survivin or MYC . CDK7 anEML4 not only binds to microtubules via its NTD but also promotes microtubule stabilization ,101. As It was further demonstrated that cells expressing EML4-ALK V3 recruit the NEK9 and NEK7 serine/threonine kinases, which are normally activated in mitosis, to interphase microtubules through binding to the NTD of EML4-ALK V3 . IntriguMoreover, as EML4-ALK V3 recruits NEK7 and NEK9 to microtubules and promotes microtubule stabilization, it is plausible that microtubule association of the short EML4-ALK variants is required for activation of this pathway. If so, targeted agents that block microtubule association of these variants could also provide additional alternatives to ALK TKI monotherapy, although the rationale for this remains to be experimentally determined.The large majority of cancer patients receive chemotherapy at some point during their treatment. The chemotherapeutic combination of platinum and pemetrexed was approved as first-line therapy for ALK+ NSCLC patients prior to the introduction of targeted ALK inhibitors but did not benefit all patients. However, the progression-free survival of ALK+ NSCLC patients treated with crizotinib was significantly higher (~8 months) than those treated with pemetrexed chemotherapy (~3 months) and a greater proportion of patients responded to crizotinib (~65%) than to chemotherapy (~20%) ,103,104.In a novel experimental approach, Li et al. (2018) used gold nanoparticles to deliver doxorubicin as well as reagents to simultaneously knock-down ALK to EML4-ALK V3 patient-derived cells. This system enables enhanced drug-loading capacity as well as tumour-specific targeting and led to a synergistic response both in vitro and in vivo .EML4-ALK V3 not only associates with the microtubule network but also promotes microtubule stabilization, whereas V1 is found in the cytoplasm and does not obviously affect microtubule dynamics . MicrotuIn a complementary study, Lucken et al. (2022) reported that paclitaxel, a microtubule stabilizing agent, gave a synergistic response in terms of loss of cell viability when combined with the ALK TKIs, alectinib and ceritinib, in cells expressing both V1 and V3. However, the response was greater in cells expressing V3, consistent with the increased microtubule stability that this protein causes. Furthermore, they found that the EML4-ALK V3 protein localised to the mitotic spindle microtubules with cells exhibiting stabilized K-fibres and increased mitotic defects including multipolar spindles, lagging chromosomes in metaphase and misaligned chromosomes in anaphase compared to cells expressing V1. Finally, the activity of the spindle assembly checkpoint was found to be reduced in cells expressing V3 compared to V1 . Taken tFor patients with significant comorbidities, contraindications, or those who decline surgery, radiotherapy (RT) is a viable alternative, particularly with the introduction of stereotactic ablative radiotherapy (SABR) . A combiA case study also reported a higher PFS in a patient diagnosed with EML4-ALK V3-driven NSCLC following treatment with stereotactic body radiotherapy (SBRT) plus alectinib compared to lorlatinib alone . This inApart from determining suitable combination treatments, the order in which different therapies are administered must be considered. Antoni et al. (2020) used two different TKIs, crizotinib and PF-06463922 (a ROS inhibitor), together with RT to determine which therapy should be given first to allow maximal efficacy of the combination. They demonstrated that giving the TKIs after irradiating the tumour can increase the DNA damage in both EML4-ALK V1 and V3 patient-derived cells . In factThe advent of immunotherapy has allowed for novel treatment avenues to be explored that encourage the immune system of a patient to promote tumour eradication . Immune However, more research is needed to better understand when such combinations might be of use. For example, Pyo et al. (2020) demonstrated that anti-PD-1 therapy had no effect on murine models of ceritinib-resistant EML4-ALK, and that combination of anti-PD-1 monoclonal antibodies plus ceritinib conferred similar results to ceritinib monotherapy and was associated with severe side effects. It was observed that the level of intratumoral regulatory T cells was increased in ceritinib-resistant mice . RegulatMultiple studies have shown that the number of tumour-infiltrating cytotoxic T cells is reduced and their function impaired following PD-1/PD-L1 inhibition therapy ,130. HumFinally, cancer vaccination is an increasingly attractive immunological approach for prevention in those with a family history of cancer, or as an additional form of treatment through generation of anti-tumorigenic antibodies . Codony-ALK inhibitor monotherapy is the standard of care treatment for patients with EML4-ALK-driven NSCLC. However, even with the generation of more efficacious ALK inhibitors, most patients inevitably relapse, highlighting the need for novel and alternative treatments. An incomplete understanding of the mechanisms underpinning EML4-ALK-driven cancer progression and how resistance develops presents a challenge to the search for alternative treatments and emphasizes the need for further research. In this review, we have summarized efforts to identify alternative treatment options to ALK TKI monotherapy that overcome intrinsic or acquired resistance in ALK+ NSCLC patients. This knowledge may be beneficial not only for patients with EML4-ALK fusions but also those with less common fusions of ALK to other partner proteins.The use of targeted agents against pathways that are either dependent or independent of ALK activation, in combination with ALK TKIs, could prevent the emergence of resistance through the activation of bypass pathways or downstream effector molecules. Given that some EML4-ALK variants can bind and alter microtubule dynamics, it might be possible to use microtubule poison chemotherapies in combination with ALK TKIs to interfere with mitotic progression and promote cancer regression through the activation of apoptotic pathways, whilst inhibiting proliferative and survival pathways. There is a lot of debate about whether the combination of ALK inhibition and RT can be effective in preventing resistance. Nonetheless, most in vitro studies agree that RT can, on some level, enhance the anti-tumorigenic properties of ALK inhibitors; thus, their combination with RT should be further explored. Finally, immunotherapy represents an important avenue to pursue, either alone or in combination with ALK TKIs. Although some types of immunotherapy have proven to be ineffective so far , more research is required into other approaches, such as immunization.This review highlights the significant amount of pre-clinical data that suggests that identification of the specific EML4-ALK variant each patient possesses would be helpful in predicting response to ALK inhibitor therapy, as distinct variants give dramatically different responses to both ALK TKI monotherapy and combination treatments. Hence, introduction of variant identification into routine clinical practice will enable more informed decisions to be made on treatment selection. However, despite our growing understanding of the resistance mechanisms to ALK inhibitors in cultured cells, there remains insufficient data regarding their contribution to resistance in patients. Therefore, we propose that future research should focus on elucidating the importance of these and other resistance mechanisms in the clinic, further guiding selection of the most effective medicines for these patients.


The pandemic had a significant impact on those with underlying chronic health conditions being at risk of developing a more severe disease with rapid progression, significant complications, and with increased risk of mortality.This was also expected in the pulmonary vascular community owing to the vulnerable nature of this population, who are characterized by an increase in the pulmonary vascular resistance leading to right heart failure.This study is aiming to identify the incidence of COVID-19 infection among pulmonary hypertension patients receiving specific therapy as well as the predictors of the COVID-19 disease severity and outcome in those patients.n = 21). Seven patients (33.3%) required hospitalization. Mortality rate is 14.3% (3/21).Data analysis of 197 PAH and CTEPH patients, showed that the incidence of SARS-CoV-2 infection is 10.66% , ferritin , CRP , acute renal failure , and hypoxemia .Severity of COVID19 disease in those patients has statistically significant moderate to strong correlation with higher values of d-dimer , hypoxemia , higher values of ferritin  and d-dimer .Mortality from COVID-19 show moderate to strong statistically significant correlations with acute renal failure (COVID-19 in PAH and CTEPH patients is challenging, higher COVID-19 infection rate is present in those patients and is associated with increased disease severity and higher mortality. From the time of emergence of COVID-19 in December 2019, [The pandemic had a significant impact on those with underlying chronic health conditions being at risk of developing a more severe disease with rapid progression, significant complications, and with increased risk of mortality .This was also expected in the pulmonary vascular community owing to the vulnerable nature of this population, who are characterized by an increase in the pulmonary vascular resistance leading to right heart failure .Putting into consideration the lung injury inflicted via the COVID-19 disease  highlighHowever, data collected early in the pandemic from pulmonary arterial hypertension (PAH) centers worldwide observed paucity in reported PAH patients infected with COVID-19, and a perceived low risk for severe COVID-19 in PAH patients with an unexpectedly favorable clinical outcome. Multiple clarifications were proposed, some elucidated that the disease itself might be protective against COVID-19, this is suggested by autopsy findings of SARS-CoV-2 infecting endothelial cells with associated vascular injury, thrombosis, and the reduced angiotensin converting enzyme 2 (ACE2) expression that plays a role in reducing the cytokine storm and decreasing the viral entry in PAH patients, others attributed it to PAH-specific medications that showed beneficial effects on COVID-19 pneumonia along with the anticoagulation used in chronic thromboembolic pulmonary hypertension (CTEPH) that reduced the prothrombotic mishaps of the virus –9.Up to date, the available data regarding COVID-19 in pulmonary hypertension is limited, this lack of formal data available regarding COVID-19 infection in pulmonary hypertension (PH) patients, lead to limited scientific-based evidence to guide pulmonary hypertension providers to implement an efficient management approach, as well as to predict the disease course, hence improving survival .This study is aiming to detect the incidence of COVID-19 infection among PH patients receiving specific therapy as well as the prognosticators of the COVID-19 disease severity and outcome in those patients.A retrospective cohort study of 21 PAH and CTEPH patients infected with COVID-19 seeking medical advice at the Pulmonary Hypertension Centre, Pulmonology Department, Kasr Al-Ainy Hospital from March 2020 to October 2021.Patients infected with SARS-COV 2 proved positive via real-time polymerase chain reaction (RT-PCR) with or without radiological evidence of COVID-19 pneumonia were included in the study.Demographic data, presenting symptoms, comorbidities, oxygen saturation, laboratory data, imaging results, medical treatments, hospitalization requirement, disease outcome (death or cure), and risk assessment of pulmonary hypertension during the last scheduled visit are collected from the medical records.The research ethics committee, Cairo University, Egypt (N-3-2022) has reviewed and approved this study.The World Health Organization (WHO) classifies COVID-19 severity into mild COVID-19-infected patients showing no clinical signs of pneumonia or hypoxemia, moderate COVID-19 patients showing clinical signs of pneumonia but no hypoxemia, severe COVID-19-infected patients showing clinical signs of severe pneumonia with respiratory rate more than 30 breaths/min, or severe respiratory distress; or hypoxemia with oxygen saturation less than 90% on room air and Critical COVID-19: patient with acute respiratory distress syndrome (ARDS) criteria, septic shock, or requires life-sustaining therapies as mechanical ventilation (invasive or non-invasive) or vasopressor therapy .Pulmonary hypertension patients show an increase in the mean pulmonary artery pressure equal or more than 25 mmHg at rest when measured by the right heart catheter .Assessing the risk in PH patients to estimate 1-year mortality using the prognostic determinants showing that patients with low risk have less than 5% 1-year mortality whereas patients with intermediate risk have from 5 to 10% 1-year mortality and patients with high risk have more than 10% 1-year mortality .Data is collected in Excel sheet, tabulated, and statistically analyzed via SPSS version 21, qualitative data is presented by number and percentage, quantitative data is presented by mean and standard deviation.Odds ratio is calculated, spearman correlation analysis is done, and chi-square test of significance is done for qualitative data.p equal to or below 0.05.Level of significance is set at A total of 197 PAH and CTEPH patients registered in the records of Pulmonary Hypertension Centre, Pulmonology Department, Kasr Al-Ainy Hospital, Cairo University, during the period of March 2020 to October 2021, that yielded 21 confirmed cases of COVID-19, with a percentage of 10.66%. Their mean age was 43.5 ± 11.07 (23-62). Other demographic data, characteristics, clinical presentations and lab findings of the studied patients are summarized in Tables There is no statistically significant relation between risk assessment in the last scheduled visit and the severity and outcome of COVID-19 as shown in Table The presence of fever, high serum ferritin, and d dimer levels had moderate positive significant correlation with the risk assessment of PH in the last scheduled visit as shown in Table The severity of COVID-19 disease in PH cases has statistically significant moderate to strong correlation with higher values of d-dimer, ferritin, and CRP, as well as hypoxemia and acute renal failure as shown in Table The mortality of COVID-19 disease in our study population is 14.3%.Mortality in COVID-19 PH cases has moderate to strong statistically significant correlations with hypoxemia, acute renal failure, higher values of ferritin, and d-dimer as shown in Table Mortality among COVID-19 PH cases has statistically significant odds ratio with fever, absence of fatigability, hypoxemia, and hospitalization as well as patients who did not receive prednisolone as shown in Table The incidence of COVID-19 infection among our study population is 10.66%. (106.6 case per 1000 patients) which when compared to the incidence of COVID-19 infection in the general Egyptian population during the same period (3.3 per 1000) confirms a higher rate of COVID-19 infection among PAH and CTEPH patients .This might be partially explained by realizing that PAH and CTEPH patients are more in contact with the health care system and are medically educated enough to seek help upon recognizing any new symptom, so the probability of them being tested at a higher rate as compared to the general population is present.Our finding refuted prior studies that reported a relatively limited or similar incidence of COVID-19 infection in comparison to the general population , 14.These studies were conducted in the early phases of the pandemic when both medical knowledge and widespread COVID-19 testing were lacking as well as home isolation and the disrupted follow-up to the patients, all contributing to the underestimation of cases .The authors also noted that morbidity and mortality are discreetly worse in our study population with case fatality rate of 14.3% and hospitalization rate of 33.3% denoting unfavorable disease outcome.These findings echo the latest reports published via Belge et al. and Lee et al., both reported high COVID-related mortality of 19% and 12% respectively in PAH and CTEPH patients along with higher rates of hospitalization of 70% and 30% respectively , 14.Also, a retrospective study on PAH patients suffered from COVID-19 reported an unusually high mortality rate of 36.36% and hospitalization rate of 81.81% .Comparing the mortality of COVID-19 disease in our study population (14.3%) from March 2020 to October 2021 to those reported for the Egyptian population at large (5.5%) during the same period, reinforces the allegation that PH is a risk factor of mortality in COVID-19 patients with a relative risk of 2.6 .Our results contradict the earlier reports that suggested an attenuated disease course and favorable outcomes, assuming that specific pulmonary arterial hypertension therapy, low ACE2 levels, and anticoagulants provide protection from the pathological changes occurring from the disease –9.These data are based on early information when the COVID-19 was still evolving, so thorough analysis to avoid misleading the pulmonary hypertension community should be done to prevent putting those patients in a greater risk.Mortality in PH patients with COVID-19 infection is attributed to the observed pathophysiological impacts of the virus on different body organs.Acute hypoxemic respiratory failure as a sequel to COVID-19 pneumonia in PH patients is a well-recognized outcome that in our experience so far, is associated with a more severe disease and increased mortality.These findings echo the latest reports that management of COVID-19-infected PAH patients associated with hypoxemia is difficult  and thatPetrilli et al. stated that impaired oxygen status upon hospital admission despite supplemental oxygen is present among the critically ill patients and is coupled with increased mortality .Moreover, hypoxemia might overload the already compromised right ventricle in a patient with decreased cardiorespiratory reserve predisposing to right heart failure .Bearing in mind the risk of developing ARDS in the setting of pulmonary vascular disease as suggested via prior studies , 22, posEven though the pathogenesis of COVID-19-induced acute kidney injury is still not precisely clear, yet several underlying mechanisms including direct viral injury, ischemia-reperfusion insults, unwarranted cytokine release, thrombotic events, and drug-induced renal injuries have been recognized .Several prior studies have already identified renal failure as a risk factor associated with increased mortality in patients with sepsis and septic shock irrespective of the etiological cause , 25.Our finding that acute renal failure escalates disease severity and its existence among critically ill COVID-19 patients is associated with increased mortality is consistent with other report .Prior studies established that advanced age , 27 and In this study, only 19% of our patients presented with critical illness, this percentage is higher when compared to the multicenter retrospective study, performed on 2724 COVID-19 patients, of whom 423 (15.52%) were critically ill, denoting that PH is a risk factor contributing to COVID-19 disease severity .Higher level of D-dimer is a pivotal predictor of disease severity and mortality indicating that the severe inflammation triggered in the critically ill COVID-19 patients  results Our analysis demonstrates a significant increase in the D-dimer levels in the severe and critically ill COVID-19 patients and that disease mortality is significantly associated with higher D-dimer levels.These findings are in line with Tian et al and Zhou et al. found, both implied that elevated D-dimer level is associated with higher COVID-19 mortality , 34.Also, Zhang et al. concluded that a fourfold increase in D-dimer level on hospital admission is a predictor of disease mortality in COVID-19 patients .Recognizing the biomarkers that mirrors the level of inflammation, ferritin was first in line.Our data suggests that higher ferritin levels are encountered in the severe and critically ill patients with higher mortality risk.These results come in line with the meta-analysis conducted by Cheng et al. that revealed that elevated ferritin level is coupled with unfavorable outcome .Similarly, Para et al. states that higher ferritin level is associated with the COVID-19 disease progression , advocatLikewise, it is observed in this study that the CRP level directly correlates with disease severity. Our results come in agreement with Malik et al., showing that the increase in the CRP level is coupled with a more severe disease . AlthougOur study is limited by its retrospective nature and limited sample size so predicting factors affecting severity and outcome is not possible due to our limited number of observations.COVID-19 in PAH and CTEPH patients is challenging, higher COVID-19 infection rate is present in those patients and is associated with increased disease severity and higher mortality.


This survey study assesses well-being among US adults by age group. Surveys were conducted online and via telephone using a 15-minute questionnaire. Data were collected from January 10 to 28, 2022. Based on US census data, post hoc weighting was performed to ensure the sample was representative of US adults 18 years or older within all 50 states and the District of Columbia. Participants responded to a validated 12-item measure of flourishing5 that assesses 6 domains of well-being, including happiness, health, meaning, character, relationships, and financial stability. Items were self-reported on a scale of 0 to 10. Domain scores were derived by calculating the mean of the 2 indicators under each domain; the overall well-being score was the mean indicator responses across domains. Ethical approval was granted by the NORC institutional review board. Participants provided written informed consent. This study followed AAPOR and STROBE reporting guidelines. Analysis of variance and the t test (2-sided) were used to examine group differences by age, race and ethnicity, and gender. Analyses were conducted using SAS, version 9.4. The eMethods in the The sample for this survey study was drawn from the NORC AmeriSpeak panel, a probability-based panel designed to be representative of the US household population.Of 8618 individuals contacted, 2598 (30.1%) provided complete responses . Well-being increased monotonically cross-sectionally with age for overall well-being, the domains, and across all individual items . Differe6 Our findings support evidence of a mental health crisis and increase in loneliness in the US that has disproportionally affected young adults2 and extend evidence of age gradients to multiple additional facets of well-being beyond mental health. Younger adults reported lower well-being even on the self-rated physical health item.This study found that mean well-being scores across multiple domains increased cross-sectionally with age, with a substantial age gradient. This finding contrasts with evidence from the early 2000s that showed U-shaped curves for some well-being domains , with well-being scores being higher in earlier adulthood and older age than in midlife.6 These findings suggest that the well-being of young people has declined compared with older age groups. Protecting the mental health of young people is regarded as a national emergency1; this study suggests that other facets of their well-being also need attention. Comprehensive strategies for expanding mental health services, supporting education and meaningful employment, and strengthening social fabric for young people are needed to enhance the well-being of this population.A limitation is that we could not disentangle age and cohort effects. Further studies are needed to replicate the findings. Although indicators in the study were obtained by self-report, this was also the case with earlier research that documented U-shaped patterns with age.


Treatment of the coronavirus disease (COVID-19) is still challenging due to the lack of evidence-based treatment protocols and continuously changing epidemiological situations and vaccinations. Remdesivir (RDV) is among the few antiviral medications with confirmed efficacy for specific patient groups. However, real-world data on long-term outcomes for a short treatment course are scarce.This retrospective observational cohort study included real-life data collected during the second and third wave of the COVID-19 pandemic in Hungary  from inpatients at a University Center (n = 947). Participants consisted of two propensity score-matched cohorts (370/370 cases): Group RDV including patients receiving RDV and supplementary oxygen and Group standard of care (SOC) as control. The primary outcome was the effect of 5-day RDV treatment on 30- and 60-day all-cause mortality. Multivariate analyses were performed to assess the effect of RDV by different covariates.Group RDV included significantly more patients from the alpha variant wave, with greater frequency of comorbidities diabetes and anemia, and larger degree of parenchymal involvement. All-cause mortality at 30- and 60-day were significantly lower in Group RDV compared to Group SOC. Significant risk reduction of 60-day all-cause mortality was observed for RDV treatment in men and patients with COPD or multiple comorbidities.Hospitalized COVID-19 patients with 5-day RDV treatment had significantly lower 30- and 60-day all-cause mortality, despite their more severe clinical condition. Men and patients with multiple comorbidities, including COPD, profited the most from RDV treatment in the long term. Due to the ongoing COVID-19 pandemic, effective treatment regimens are needed for hospitalized patients. Numerous antiviral agents have been tested for clinical effectiveness against the SARS-CoV-2 virus. Based on the ACTT-1 trial . Other sIt is often difficult to evaluate the real-world effect of a given medication due to constantly changing factors, including virus type, emerging clinical trial data of different treatments, vaccination availability, and others. Similarly, it is challenging to evaluate the efficacy of antiviral treatment, especially on the long term. Because of these factors, more real-world data based on clinical experience are essential to provide a base for the meta-analyses. Our aim was to perform a retrospective analysis regarding the clinical efficacy of a 5-day use of RDV treatment during the second (wild variant) and third wave  of the pandemic, and to identify factors influencing favorable long-term outcome of antiviral treatment.This retrospective study included PCR and/or antigen confirmed COVID-19 patients (ICD U07.10) admitted to Semmelweis University’s Department of Pulmonology between September 1, 2020, and April 30, 2021. This period covers most of the second and third wave of the pandemic in Hungary, when the predominant types of SARS-CoV2 were the wild type and alpha (B.1.1.7) variants of the virus, respectively ) were assigned to the cases, as well as daily maximum of oxygen dependency, to calculate the WHO’s Ordinal Scale for Clinical Improvement (WHOS) . The NEWAs part of the local protocol, initial low-dose chest CT was performed on the day of admission ± two days. Extent of pneumonia was classified according CO-RADS , includiaO2)<60 mm Hg or oxygen saturation measured by pulse oximetry (SpO2)≤90% at room air. Beginning oxygen flow was 2 L/min via nasal cannula and was titrated up to 15 L/min with reservoir facemask to ensure SpO2>90% [Based on the department’s SOC protocol, indication for oxygen supplementation was partial pressure of arterial oxygen  orientation of discharge forming three distinct categories by severity: death, admittance to higher intensity care unit, and end of departmental stay (lower intensity care or home); and b) proportion of patients with clinical improvement on days 7, 10, and 14 compared to the baseline date. Algorithm for defining clinical improvement or worsening is shown in 2 test for qualitative variables. Continuous variables were tested for normality  or a Mann-Whitney U test. Survival analysis was performed using Kaplan-Meier estimates. Participants were censored at day 61 or on the day of death. As an exploratory univariate analysis, relative risk (RR) was calculated for 60-day all-cause mortality and represented with 95% confidence intervals (CI) comparing the two treatment groups by subgroups. To measure the effect of RDV treatment on survival we calculated hazard ratios (HR) by performing a multivariate analysis using COX regression. The variables for COX regression were all parameters used in the exploratory univariate analysis. Age and Charlson score were included as ordinal variables. The interaction between age and Charlson score was included to compensate for the use of age in the calculation of Charlson score. Cox regression model was applied to those subgroups, which presented significant benefit in the univariate analysis, to calculate HR for RDV treatment. For each model, Harrell’s concordance index and Somers’ D statistic were calculated and displayed under the corresponding HRs, which are presented with 95% CIs.Categorical variables are presented as absolute (n) and relative (%) frequencies, while quantitative variables are presented as mean ± standard deviation (SD). Two PSM cohorts were created by calculating propensity scores by logistic regression using the presence or absence of RDV therapy as the dependent variable, and four variables as covariates: sex, age, baseline NEWS2 score, and Charlson comorbidity score. The two cohorts were compared using the Pearson χr et al. ) and comThe propensity score matching was performed using the SAS 9.4  software package, while the SPSS software ver. 27.0.1.0.  was used for all other analyses.In the observation period, 974 cases were admitted to COVID-19 wards at the Department of Pulmonology of Semmelweis University. Patient characteristics for all patients and Group RDV and Group SOC are summarized in Outcome data are summarized in Treatment modalities used during the observation period (RDV and SOC only) are summarized in Figure S1 in the Defining optimal treatment for COVID-19 patients, especially those with advanced age and multiple comorbidities, is challenging. Our data confirmed significant beneficial long-term outcome of 5-day RDV treatment in patients hospitalized for COVID-19 pneumonia in need of supplemental oxygen. Our most important finding was that antiviral RDV therapy significantly benefited men and patient with most comorbid conditions.At the time of manuscript preparation, RDV is the only known antiviral drug of confirmed benefits in COVID-19. RDV received first conditional recommendation for 10-day treatment of hospitalized patients who require supplemental oxygen, but not mechanical ventilation . The 5-dRDV was first approved for the treatment of COVID-19 patients needing supplementary oxygen due to pulmonary involvement; in our centre, all patients received RDV accordingly. According to the ACTT-1 trial, 10-day RDV treatment reduced the time to recovery in patients hospitalized with COVID-19 who had evidence of lower respiratory tract infection . SimilarRapid improvement of patients using a given treatment is always an important clinical outcome parameter. Meta-analysis of randomized controlled clinical trials of RDV treatment in 2020 showed major benefit for oxygen treated COVD-19 patients receiving RDV and defining favorable outcome as percentage of patients improving . This diIn our real-world observational study men, patients over the age of 75 years and multimorbid patients benefited the most from RDV treatment. This is in sync with the results shown in the ACTT-1 clinical trial [Our primary outcome did confirm significant benefit in view the reduction of 60-day mortality in RDV treated COVID-19 patients. Mortality is difficult to assess especially in multimorbid elderly patients. A large observational cohort study analyzing patients during the first wave did not confirm 30-day survival benefit for experimental 5-day RDV treatment . SimilarMultimorbidity is a significant risk factor for death and the Charlson comorbidity score is widely used as a prognostic factor for prediction of all-cause mortality . A studyAn additional novelty of our analysis is that in COPD, an important and prevalent comorbid condition, RDV treatment was significantly advantageous as compared to SOC therapy. Similarly, men did significantly profit over long-term from antiviral treatment. These variables were independently associated with decreased mortality following RDV treatment, defining a well-defined subgroup of patients.Age is an important predictor of mortality . HoweverThe strengths of this study are the long-term outcome analysis in a real-world setting, including several aspects of the rapidly changing pandemic, such as differences in discharge criteria, treatment protocols, virus subtypes, and vaccination of health care professionals, and high number of standardized patient care, making propensity score matching possible.The study limitations are the protocol change during third wave as all eligible patients got RDV, which could have distorted the results even after propensity score matching; the change of discharge criteria in November 2020, as patients with clinical and radiological improvement could go to home isolation without the previously needed two negative consecutive PCR results within 48 hours, making length of stay calculation inconsistent during our study; the inclusion of patients from only one hospital, as different protocols can influence the outcome; the use of all-cause mortality as an outcome, as remdesivir supposedly only decrease COVID-19 associated mortality. Lastly a strong limiting factor for our subgroups analysis is the low number of patients presenting with specific comorbid conditions.The analysis of suggested beneficial groups should be examined in later studies. A possible next step could be a retrospective meta-analysis focusing on the use of RDV in COPD patients as well as a study investigating if the proposed benefit of RDV use in men could be observed in other real-world populations.Our data confirmed a 30- and 60-day mortality benefit following 5-day RDV treatment in severe COVID-19 pneumonia. According to our data, more severe cases, especially men, COPD patients and patients with multiple comorbidities in the need of oxygen support, should receive 5-day RDV as better long-term outcome can be assumed. The study did not confirm short-term beneficial effects of RDV, as improvement at days 7, 10, and 14 were not different according to treatment protocols.


Background: The purpose of this study was to investigate the association between third-generation cephalosporin resistance and urinary tract infection (UTI) recurrence in patients who underwent voiding cystourethrogram (VCUG). Methods: In this retrospective study, data were obtained from hospitalized pediatric patients who had a first febrile UTI episode and subsequently underwent VCUG. Information based on VCUG was mandatory to identify the presence of vesicoureteral reflux (VUR). A multivariable logistic model was used to identify the risk factors for recurrence. Recurrence was divided into early (90-day) and late (1-year), and sensitivity analyses were performed according to each definition. The estimates of all the statistical models were internally validated using bootstrap samples. Results: A total of 210 patients were included, and the overall recurrence rate of UTI was 26.2% (55 of 210). Third-generation cephalosporin resistance was a significant risk factor for early recurrence (odds ratio: 2.79 [1.08–7.20]) but not for late recurrence. Sensitivity analyses showed that third-generation cephalosporin resistance was a significant risk factor for 60-day recurrence but not for 180-day recurrence. A VUR grade ≥ 3 was identified as a consistent risk factor for both early and late recurrence. Conclusions: Third-generation cephalosporin resistance was a significant risk factor for the early recurrence of pediatric UTI in patients who underwent VCUG. Enterobacteriaceae have been increasing in children ) and 89% (95% CI [83–93]) in patients with and without a VUR grade ≥ 3, respectively, and the intergroup differences were significant , the recurrence-free survival was 44% (95% CI [30–57]) and 83% (95% CI [76–88]) in patients with and without VUR grade ≥ 3, respectively, and the intergroup difference was statistically significant  and VUR grade ≥ 3 on VCUG  remained significant risk factors for 60-day recurrence. However, when an early recurrence was defined as a recurrence within 180 days after the diagnosis, third-generation cephalosporin resistance was not a significant risk factor.Sensitivity analysis was performed to assess the multivariable model for early recurrence, using shorter (60-day) and longer (180-day) time points for the definition of early recurrence . When anEnterobacteriaceae who had completed radiologic studies including the renal US, 99Tc DMSA renal scan, and VCUG, we found that third-generation cephalosporin resistance of the causative organism at the initial episode did not affect the overall recurrence rate at the 1-year follow-up. However, the recurrence-free survival analysis showed that, despite similar rates of 1-year recurrence, patients with third-generation cephalosporin resistance in our cohort had a higher rate of early recurrence than those without cephalosporin resistance  and VUR , other unidentified confounding factors could have affected recurrence rates. Treatment with non-susceptible antibiotics in pediatric UTIs caused by resistant organisms have shown favorable clinical effectiveness in previous studies [We found that in our cohort, the early recurrence rate (at 90 days) was higher in patients with third-generation cephalosporin resistance than in those without resistance. The reasons for this must be inferred. First, it is possible that patients with antibiotic resistance have higher baseline risk factors for UTI. Although the two groups had similar predisposing risks for subsequent recurrent UTIs, such as urinary tract anomalies . Susceptible antibiotics, such as carbapenems, can be cautiously suggested in patients with recurrent UTI who have these risk factors. On the contrary, for patients without these risk factors, the current practice of maintaining the initial regimen would be reasonable if the patient showed clinical and microbiological responses while on empiric antibiotic therapy to which the causative bacterium is not susceptible in vitro.n = 61, Several limitations affect the interpretation of this study. First, a small number of patients were included in the study. To overcome this limitation, we internally validated our multivariable model using bootstrapped samples, and the relative bias of our multivariable model calculated by the bootstrap method was low. In addition, we also performed a sensitivity analysis according to different time points for early recurrence and confirmed an increased rate of early recurrence in patients with third-generation cephalosporin resistance from recurrence-free survival curves. Second, although the choice of our empiric antibiotic regimen followed the previous guidelines, variability in the regimen may have influenced the clinical outcomes, including recurrence ,31. HoweIn conclusion, third-generation cephalosporin resistance was a significant risk factor for early recurrence in pediatric patients with UTI who had undergone VCUG. Although the precise cause could not be clarified in this study, further treatment strategies, including aggressive antibiotic therapy or close follow-up, in selected high-risk patients can be suggested based on these results. Further studies with larger cohorts are required to validate our results and evaluate the different treatment strategies for high-risk patients.


Background: Cardiac Magnetic Resonance (CMR) has a key role in subjects presenting with acute myocarditis, independent from left ventricular ejection fraction; it is widely used as a non-invasive imaging test for both diagnostic and prognostic purposes. However, poor data is available about the CMR-derived prognostic parameters of acute myocarditis with preserved ejection fraction (AMpEF). The aim of this study was to investigate the role of CMR in predicting outcomes in patients followed up for AMpEF, using a composite endpoint of all-cause mortality and hospitalization for heart failure (HF). Methods: We retrospectively enrolled 61 patients with diagnosed AMpEF. All patients underwent biohumoral, echocardiographic and CMR evaluation in the acute phase. Myocarditis was confirmed by Lake–Louis criteria assessed on CMR images. Mean follow-up was 4.8 ± 0.6 years during which a composite endpoint of all-cause mortality and hospitalization for HF was investigated. Results: The population was fairly homogeneous regarding baseline clinical features. In particular, no significant differences in age and main cardiovascular risk factors were found between patients with and without events at follow-up. Seven patients met the endpoint. They had significantly higher levels of circulating neutrophils in the acute phase  and a higher amount of left ventricular mass with delayed enhancement  vs. 12 (8–16) g, p = 0.028). At Cox univariate analysis, DE-LVM was the only significant predictor of endpoint, regardless of the site of inflammation. Conclusions: DE-LVM can predict the composite endpoint of all-cause mortality and hospitalization for HF in a population of patients with AMpEF, representing a new added tool for prognostic stratification. According to the World Health Organization and the International Society and Federation of Cardiology, myocarditis is an inflammatory disease of the heart muscle, whose diagnosis is based on histological, immunological and immunohistochemical criteria .Despite its clear anatomopathological findings, myocarditis is often underdiagnosed, especially in the context of a preserved left ventricular ejection fraction. Myocarditis is clinically evident in 8–10 persons out of 100,000 subjects, but its prevalence on autopsy reaches 44 persons out of every 100,000 . This suThe classification of myocarditis is controversial and still an object of debate. It can present with a wide clinical spectrum, from asymptomatic cases to cardiogenic shock, with variable symptoms according to the extension and the etiology . EtiologAccording to echocardiographic parameters, a classification based on left ventricular ejection fraction is often used because of its simple determination, with consequent impact on the therapeutic strategy. Acute myocarditis with preserved ejection fraction (AMpEF) is still a nebulous clinical entity with scarce and often conflicting evidence in the literature, without evidence-based therapeutic tools. Recently, Georgiopoulos et al. also showed that the presence of late gadolinium enhancement carries independent prognostic value, irrespective of LVEF . Other dIn particular, to our knowledge, no study has evaluated the amount of left ventricular mass with delayed enhancement (DE-LVM) as a tool for outcome stratification specifically in AMpEF patients . The ideWe retrospectively analyzed data of patients referred to our Cardiological Department for acute myocarditis from January 2011 to December 2018. In the acute phase, the diagnosis of myocarditis was suspected on the basis of the combination of personal history, symptoms, physical examination, transthoracic echocardiography and bio-humoral assays, and then confirmed by Lake–Louis criteria on CMR images. Ejection fraction was assessed both by transthoracic echocardiography and CMR in each subject, but only patients with preserved LVEF (≥50%) at CMR were included. Exclusion criteria were: LVEF at CMR < 50%, patients aged less than 18 years old, patients who underwent previous heart transplantation/left ventricular assist device implantation, patients with transvenous pacemaker/defibrillator, or acute HF at presentation.We considered a composite endpoint of all-cause mortality and hospitalization for HF. The study population was then divided into two groups according to occurrence of composite endpoint at follow-up. Informed consent regarding the anonymous use of personal data was obtained from each patient. The study was conducted in accordance with the Declaration of Helsinki .Blood samples were drawn after an overnight fasting period the day CMR was performed. Samples were stored at −80 °C. All patients underwent a thorough biohumoral investigation consisting of complete blood count, serum creatinine, C-reactive protein, hepatic function and high-sensitivity troponin. All the assays were run according to manufacturer instructions. Information about each biomarker was available for all patients.Echocardiographic examination was performed at admission, according to the American Society of Echocardiography/European Association of Cardiovascular Imaging recommendations for chamber quantification , using tLVEF, left ventricular and left atrial volume were assessed using the biplane modified Simpson method from the apical 4- and 2-chamber views. Dimensions of left ventricle and left atrium were indexed to body surface area. From the 4-chamber view, tricuspid annulus plane systolic excursion (TAPSE) was measured by M-mode . Systoli2-weighted imaging, and LGE at 10 min after gadolinium injection in the short-axis (9 to 13 images covering the entire LV), 2-chamber and 4-chamber planes [CMR was performed within three days from hospital admission, using a 1.5 T whole-body scanner . An eight-channel cardiac phased-array receiver surface coil was used for signal reception. In each patient, a set of contiguous short-axis views was acquired from the tricuspid plane to the apex with a minimum of 30 cineframes for each slice with the following parameters: slice thickness 8 mm, no gap, eight views per segment, NEX 1, field of view 40 cm, phase field of view 1, matrix 224 × 224, reconstruction matrix 256 × 256, flip angle 458, TR/TE 3.5/1.5 and bandwidth 125 kHz. According to the protocols recommended by the Society for Cardiovascular Magnetic Resonance, we acquired cine steady-state free precession (cine-SSFP) images, Tr planes . In order planes . On T2-wSemi-automated quantification was performed as follows: epicardial and endocardial left ventricular (LV) contours were carefully placed manually on all LGE images. The remote non-LGE reference region of each LGE slice was placed adjacent to the region of LGE so that the reference region was at approximately equal distance from the anterior receiver coils. We believe this method minimizes any modifying effect from LGE location on the robustness of the LGE quantification. LGE mass was then quantified by semi-automatic methods using a signal intensity threshold of > 2-SD above a reference region of remote myocardium  in the same slice, and using regions defined as above 50% of maximal signal intensity of the enhanced area for the full width at half maximum approach. Artifacts were manually erased. In all methods, LGE mass (in grams) was then expressed as a percentage of total LV mass determined by balanced steady-state free precession cine images .After inpatient baseline evaluation, patients were followed up at our outpatient clinic. Independent interviewers obtained data from the patients, relatives or general practitioners. Independent physicians performed follow-up resting echocardiography. The follow-up period lasted until May 2020. Information about the time and cause of death was retrieved from death certificates and post mortem reports.Statistical analysis was performed using IBM SPSS 25.0 program . The type of distribution of variables was assessed using the Kolmogorov–Smirnov test. Continuous variables with normal distribution are presented as mean ± standard deviation, whereas continuous variables with non-normal distribution as median and interquartile range. Non-continuous variables are presented as frequency and percentage.t-test for independent samples for what concerns normally-distributed continuous variables; to assess differences in non-normally-distributed continuous variables, a non-parametric Mann–Whitney test was performed; non-continuous variables were assessed using a Chi-square test or Fischer test as appropriate. In each of the above-mentioned cases, two tailed p values < 0.05 were considered significant. Survival was assessed with Kaplan–Meier analysis, after dividing our population into patients with left ventricular mass with delayed enhancement (DE-LVM) above and below median value. A Kaplan–Meier curve is presented at 66-month outcome. Differences in survival between groups were tested with a log-rank test (Mantel–Cox).Differences among the two groups (patients who met the primary endpoint and those who did not) were assessed using aStudent’s Univariate Cox analysis was performed in order to identify predictors of the composite endpoint.p = 0.506). Both groups were homogeneous as regards age, sex, body mass index and rates of cardiovascular risk factors (p > 0.05). All patients were in New York Heart Association (NYHA) Class I, as HF at admission represented an exclusion criterion.A thorough descriptive analysis of baseline clinical, biohumoral and instrumental features is presented in p = 0.775). This cohort of patients with events at follow-up also showed a higher relative neutrophilia , although the total count of white blood cells was homogeneous between groups. Similarly, no differences in serum creatinine levels or in troponin has emerged.A moderate increase in median C-reactive protein levels was observed  without significant difference between groups ((p = 0.459). Only two patients showed a mild left ventricular dilation, while the mean LV end-diastolic diameter was 50 ± 4 mm; eight patients showed an increase of right ventricular end-diastolic diameter (population mean 32 ± 3 mm). Both indices were comparable in patients with and without events, as well as in the estimation of systolic pulmonary artery pressure. A total of 23 patients showed LV wall motion abnormalities, but the wall motion score index did not show statistically significant differences between the groups. No patient in the event group presented anterior wall hypokinesia.Mean LVEF at admission was 57 ± 3%, with similar values between the two groups 2), and only one had an event at follow-up. Among them, only one patient had a slight reduction in LVEF at admission echocardiography that was not confirmed once CMR was performed. That patient did not meet the endpoint at follow-up. Only five males presented a slight decrease of LVEF at CMR (LVEF 50–55%), while mean CMR LVEF was 62 ± 5% with no difference between groups (p = 0.666). Interestingly, 15 patients showed a mild decrease in right ventricular EF, and four of them had events at follow-up. All patients presented normal RV dimensions.CMR confirmed a mild LV dilation in only three patients, all males (indexed LV end-diastolic volume ≥ 105 mL/mp > 0.05). Myocardial edema was found in 30 patients (49%) and LGE in 57 (93%). Despite similar distribution, the total amount of DE-LVM was statistically higher in patients with events at follow-up (18 (14–29.5) vs. 12 (8–16) g, p = 0.028; see p = 0.047).Regional LV wall motion abnormalities were comparable between those with and without events, as were regional distribution of edema and LGE . Furthermore, after splitting the population according to DE-LVM median value  helps in addressing clinical goals and investments towards a better comprehension of disease, especially as regards mid-to-long term management of myocarditis both with reduced and with preserved ejection fraction. The main result of the present study is that the amount—and not merely the presence or absence—of DE-LVM can predict the composite endpoint of all-cause mortality and hospitalization for HF in subjects presenting with AMpEF.Although preserved systolic function is a good predictor of survival in many cases of heart disease, the presence of myocardial scar is generally associated with increased risk for adverse cardiovascular events , even in patients with preserved systolic function . WhetherThere are contrasting data in the literature about the importance of CMR findings in patients with AMpEF. In 2014, Schumm et al.  evaluateMore recently, Aquaro et al.  in the Ip = 0.022). This was further confirmed when evaluating the percentage of DE-LVM to the LVM (p = 0.018). It might mean that also for relatively small fibrotic involvement that does not impair LVEF, even small differences in the percentage of left ventricular myocardium with LGE may affect outcome. In our population of AMpEF, no specific localization could predict the composite endpoint in our cohort. This finding is actually in discordance with results from the ITAMY study, but the small number of patients enrolled may explain this bias. Data from larger populations are awaited in order to clarify these different results.Despite the small sample size and being a single-center analysis, with our study we added a new datum in the comprehension of AMpEF: not only the presence of LGE, but also its amount is predictive of a worse outcome (Both groups of our study (patients with and without events at follow-up) were homogenous regarding clinical features, but AMpEF patients with events had higher levels of circulating neutrophils at admission. This may reflect a higher degree of inflammation, even if C-reactive protein levels were not significantly different between groups and routine assay of interleukins was not performed. In the ITAMY study , patientThese findings might have several consequences, although derived from a single-center, retrospective analysis. First of all, CMR should be performed in all patients with AMpEF for different purposes: making diagnosis, confirming preliminary echocardiographic data of preserved LVEF and quantifying the extent of myocardial fibrosis. Our results may also reflect the need for a closer follow-up in order to re-assess myocardial function and arrhythmic burden. Moreover, a higher degree of fibrosis would require an early therapeutic approach that is still not available for this population. Randomized clinical trials, investigating the antagonism of both neurohormonal activity and directly fibrosis, are strongly required. As in HF, fibrosis is emerging not only as merely the consequence of myocardial insult, but also as a primary therapeutic target, and AMpEF should be no exception, especially if a large fibrotic process is detected.This study has some limitations. First of all, this is a retrospective, single-center analysis on a small population of patients with AMpEF. We did not perform endomyocardial biopsy and the diagnosis was made by the summation of clinical and CMR findings; nonetheless, in clinical practice endomyocardial biopsy is rarely performed in patients with AMpEF because of disproportional risk-to-benefit ratio . FurtherAMpEF is a subpopulation of patients currently having no evidence-based management. In our study, DE-LVM and the percentage of DE-LVM to LVM were the only predictors at long-term follow-up of the composite endpoint of all-cause mortality and hospitalization for HF in subjects presenting with AMpEF at baseline, according to a univariate analysis. These data confirm that not only the presence of LGE, but also the amount of DE-LVM, may have important prognostic roles also in AMpEF, as already demonstrated in other cardiomyopathies.


BackgroundThe presence of metabolic syndrome (MS) is associated with increased disease severity in patients with coronavirus disease 2019 (COVID-19). Non-alcoholic fatty liver disease (NAFLD) associated with or without MS may be related to increased morbidity and mortality in COVID-19, but large Indian studies are lacking. The present study was carried out to assess the impact of NAFLD on the clinical outcomes in patients with COVID-19 infection.MethodsAll patients with COVID-19 hospitalized at a tertiary care hospital in eastern India from April 4 to December 31, 2020, were included in the study. Patients who underwent non-contrast CT (NCCT) chest were evaluated for the presence of hepatic steatosis based on a validated criterion liver attenuation (HU) value lower than the spleen, absolute liver attenuation lower than 40 HU, and liver to spleen attenuation ratio less than 1. Patients were divided into two groups, those with or without fatty liver. Baseline characteristics including age, sex, liver function tests, and outcomes including duration of hospital stay and mortality were compared.ResultsA total of 6003 COVID-19-positive patients were admitted during the study period. Of these patients, 214 children (<18 years) with COVID-19 infection were excluded. One hundred and eight patients with a history of significant ethanol abuse were excluded from the analysis. NCCT scan was not done in 1698 patients. Finally, 3983 patients were included in the study. They were divided into two groups depending on the presence or absence of NAFLD. Of the 3983 patients, 814 (20.4%) had NAFLD. Overall in-hospital mortality among the study group was 6.4%. The mortality rate among patients with NAFLD was 6.7% while that in patients without fatty liver was 6% (P=0.381). Similarly, the mean duration of hospital stay was also comparable between both the groups . Prevalence of NAFLD was similar in survivors and non-survivors; 759 of 2981 patients (25.4%) and 55 of 188 patients 29.2% (P=0.381), respectively. On univariate analysis, male sex, older age, elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT) along with low serum albumin and low absolute eosinophil counts (AEC) were associated with higher mortality. However, on multivariate analysis, only older age, male sex, and low albumin levels were associated with higher mortality. Surprisingly, a sub-group analysis showed that females without NAFLD were at a higher risk of mortality than those with fatty liver . Similarly, patients with lower AST levels had higher mortality compared to patients with significantly elevated AST levels ), irrespective of the presence of fatty liver.ConclusionsThe prevalence of fatty liver in severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infected patients is similar to the general population in India, the presence of which is not a predictor of severe disease. However, mortality is higher in males and elderly patients. This preliminary results of the study was previously presented as poster at the 28th INASL Annual Scientific Meeting on August 06, 2021.Coronavirus disease 2019 (COVID-19) infection was first reported in Wuhan city of China in December 2019 and later affected individuals all over the world . Though This was a prospective observational study conducted at the Kalinga Institute of Medical Sciences (KIMS) and Pradyumna Bal Memorial (PBM) Hospital, Bhubaneswar, Odisha, India, from April 4 to December 31, 2020. The study protocol was approved by the Institutional Ethics Committee (IEC), Kalinga Institute of Medical Sciences (KIMS), Kalinga Institute of Industrial Technology (KIIT) deemed to be university, Bhubaneswar, India (KIIT/KIMS/IEC/523/2020). The primary outcome measure of the study was mortality in COVID-19 infected patients with or without fatty liver while the secondary outcome measure was the total duration of hospital stay.Inclusion and exclusion criteriaAll adult patients (age≥ 18 years) who were diagnosed as COVID-19 positive, admitted into our COVID-19 hospital from April 4 to December 31, 2020, and underwent a non-contrast CT (NCCT) scan of the chest were included in the study. All these images were acquired in a craniocaudal direction (from the thoracic inlet to the mid-portion of kidneys) at shallow inspiration. This enabled us to acquire images of both the spleen and liver.All patients less than 18 years of age, patients with prior history of cirrhosis (compensated or decompensated), and/or significant ethanol abuse were excluded. Patients who didn’t undergo an NCCT scan were also excluded.Study definitionsThe diagnosis of COVID-19 infection was based on either a positive reverse transcriptase-polymerase chain reaction (RT-PCR) or rapid antigen test (RAT). Fatty liver was diagnosed on the basis of the following radiological parameters in the NCCT scan of the chest performed as part of a workup done in sick patients with COVID-19 infection: Liver attenuation value lower than the spleen, absolute liver attenuation of less than 40 HU and liver to spleen attenuation ratio less than 1 . Liver aData collectionThe individual medical records of all the patients were analyzed. Information regarding demographic data, hemoglobin, total leukocyte count (TLC), platelet count (PC), coagulation parameters and biochemical parameters , duration of hospital stay, and final outcome (death or discharge) were populated in the master chart. All the CT scans were analyzed for the presence of fatty liver by an experienced hepato-radiologist. The imaging of the liver and spleen in the NCCT chest from the Picture Archiving and Communication System (PACS) of KIMS and PBM Hospital was used to detect fatty liver in these patients.Statistical analysisCategorical data were presented as ; Continuous data were written in form of mean ± SD or median± interquartile range (IQR) as appropriate. The Chi-square test was performed for categorical variables while the student-t-test was performed for continuous variables. For skewed data, a non-parametric Mann-Whitney U- test was used for the analysis of the two groups. Multivariate regression analysis was performed to estimate the odds ratio for variables on the severity of COVID-19. All statistical tests were two-sided and were performed at a significance level of α=0.05. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 22.0 .A total of 6003 patients were admitted to the COVID-19 hospital of KIMS, PBM hospital, between April 4 and December 31, 2020. Of these, 218 patients belonging to the pediatric age group (age <18 years) were excluded; 108 patients had a history of excessive alcohol intake while an NCCT scan of the chest was not done in 1698 patients. Finally, a total of 3983 patients with COVID-19 were included in the study  were diagnosed to have NAFLD while 3169 patients didn’t have NAFLD. Thus the prevalence of NAFLD in the COVID-19 population was at par with the Indian studies reported previously . In bothWith regard to the laboratory findings, TLC, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and absolute eosinophil count (AEC) were comparable in both groups. Inflammatory markers like C-reactive protein (CRP), ferritin, and D-dimer levels were also similar. Liver lab parameters including serum bilirubin, albumin, and Alkaline Phosphatase (ALP) levels were comparable across both groups. In contrast, serum aspartate transaminase (AST), alanine transaminase (ALT), and gamma-glutamyl transpeptidase (GGT) were higher in the NAFLD group compared to the non-NAFLD group. A detailed comparison of these baseline characteristics has been provided in Table With respect to final clinical outcomes, there was no significant difference between NAFLD and non-NAFLD patients. A total of 243 (6.4%) patients died during a hospital stay. The mortality rate was comparable between the two groups (54 (6.7%) patients in the NAFLD group and 188 (6%) in the non-NAFLD group; p=0.381). Similarly, the mean duration of hospital stay was also comparable . Due to logistics reasons, the mean duration of ICU stay between the two groups could not be compared. Based on final outcome, patients were divided into two groups and were compared as per baseline clinical and laboratory parameters  males and 12 (4.9%), females had NAFLD. In contrast, 158 (65%) males and 30 (12.3%) females had no fatty liver. Females without fatty liver were found to have a significantly higher mortality risk than females with fatty liver (p=0.006). Surprisingly, these patients were younger and had lower GGT levels.Next, the mean duration of hospital stay was compared between patients with NAFLD and non-NAFLD with respect to age, sex, and presence of diabetes mellitus. However, no significant difference in the mean duration of hospital stay was noted with respect to age, sex, or presence of DM. Deranged liver functions were also analyzed as the predictor of mortality Table  and meanPatients who died during hospitalization were first divided on the basis of the presence or absence of NAFLD. Afterward, patients in both groups were divided into subgroups based on levels of AST, ALT, and GGT. No statistically significant difference in mortality was noted between NAFLD and non-NAFLD groups with respect to deranged liver functions. Similarly, no difference in the mean duration of hospital stay was noted between both groups.However, significantly lower mortality was noted in patients with NAFLD who had significantly elevated AST levels  compared to patients with AST levels less than 2ULN ). A similar observation was also noted in patients without NAFLD. Patients without NAFLD who had AST levels less than 2ULN had higher mortality compared to the patients with AST levels more than 2ULN  . Patients with lower baseline AST levels (<2ULN) had higher mortality compared to patients with significantly elevated AST (>2 ULN) levels irrespective of the presence of fatty liver. These findings are in stark contrast with two of the previous studies, which included patients with COVID-19 and NAFLD and found a positive correlation between the presence of fatty liver and severe illness -15. HoweThe retrospective study by Wang et al. analyzed the association of NAFLD with susceptibility and outcomes of COVID-19 infected patients. They found out that 39.4% of patients with COVID-19 had NAFLD, which was higher than the prevalence in the Chinese population . It can Similar to previous studies, we found significantly elevated AST and ALT levels in patients with NAFLD ,23. In aIn the study by Mahamid et al., patients with NAFLD had higher lymphopenia at admission, and the presence of lymphopenia correlated with the severity of the illness . The preAnother interesting finding in our study was that females without NAFLD were at a higher risk of mortality compared to those with NAFLD. At present, we are not able to explain this finding. However, data regarding the severity of illness, ICU stay, ventilator requirement, and presence of other comorbidities were not available to us, which might have contributed to these results. Future prospective studies are needed to confirm this finding.The main limitation of this study is that data collection was dependent on the medical officers looking after the patients as gastroenterologists/hepatologists were not managing the patients on daily basis. Some of the laboratory data were not recorded due to the pressure of workload when admission rates were high. This could have caused a few inaccurate data entries. However, the large number of patients included in the study would have neutralized some of these limitations. To conclude, the presence of NAFLD per se may not be associated with higher mortality or longer hospital stay in COVID-19-infected patients. The etiology of elevated liver enzymes in COVID-19-infected patients is multifactorial and may not be associated with severe disease in COVID-19-infected patients with fatty liver as these patients might have elevated baseline liver enzyme levels. Surprisingly, females without NAFLD may be at higher risk of mortality, although future prospective studies are needed to validate this finding further. Moreover, older age and male sex may be the two most important predictors of mortality.