We review the state of the art of knowledge regarding the Notch signaling pathway to shed light on the role that this pathway has in hematopoiesis and hematological neoplasia, focusing on acute myeloid leukemia.The Notch signaling pathway is fundamental to early fetal development, but its role in acute myeloid leukemia is still unclear. It is important to elucidate the function that contains Notch, not only in acute myeloid leukemia, but in leukemic stem cells (LSCs). LSCs seem to be the principal cause of patient relapse. This population is in a quiescent state. Signaling pathways that govern this process must be understood to increase the chemosensitivity of this compartment. In this review, we focus on the conserved Notch signaling pathway, and its repercussions in hematopoiesis and hematological neoplasia. We found in the literature both visions regarding Notch activity in acute myeloid leukemia. On one hand, the activation of Notch leads to cell proliferation, on the other hand, the activation of Notch leads to cell cycle arrest. This dilemma requires further experiments to be answered, in order to understand the role of Notch not only in acute myeloid leukemia, but especially in LSCs. Notch has an important role in several functions related to embryogenesis and cell fate in adult tissues. Indeed, Notch signaling has been extensively linked to both processes of normal and malignant stem cell (SC) self-renewal ,2. For tFor this purpose, we employ the PubMed database by using the following terms: Notch, quiescence, LSCs, leukemic stem cell, acute myeloid leukemia, crosstalk, Hedgehog and Wnt.Drosophila melanogaster [Caenorhabditis elegans to Homo sapiens [Two important works described the Notch signaling pathway at the beginning of 20th century. In 1914, Dexter analyzed variations in Notch-mutated phenotypes of the fly wing of nogaster . Some yenogaster . To date sapiens ,7,8.The ligand receptors in Notch are a single-pass type 1 transmembrane protein called Delta and Serrate, in D. melanogaster; otherwise, other species have only one receptor called LAG1, as in C. elegans. Nevertheless, the collective term for these proteins is DSL. On the other hand, in mammals, Notch ligands are classified into two groups: Delta-like  and Serrate (Jagged)-like  ,10.If we focus on the receptors of Notch, mammals, such as mice and humans, present four Notch receptors (NOTCH1–4). Although these proteins are similar among them, they have some differences; NOTCH1 and NOTCH2 receptors have 36 EGF-like repeats in their ectodomain. NOTCH1 contains a strong transactivation domain (TAD), whereas NOTCH2 has a weak TAD. On the contrary, NOTCH3 and NOTCH4 have no TAD in the endodomain and they are formed by 34 and 29 EGF-like repeats, respectively . The comFinally, the last player in this pathway is the transcription factor that is continuously bound to the DNA called CSL. CSL is composed of CBF1/RBPJ-κ, suppressor of Hairless and LAG1  Figure .MYCC [CCND1 [CDKN1A [BCL2 [The activation of the pathway is provoked by the ligand–receptor interaction between two proximal cells. When ligands interact with receptors, the NECD is cleaved by disintegrin and metalloproteinase (ADAM). The NICD of NOTCH is released by a γ-secretase/presenilins complex. This action allows NICD to enter the nucleus and displace co-repressors (CoRs) by binding itself to the transcription factor CSL, recruiting, at the same time, co-activators (CoAs). These events start the transcriptional activation of the target genes ,13,14. SMYCC , CCND1 [C [CCND1 , CDKN1A  [CDKN1A , and BCL1A [BCL2 ,19; embr1A [BCL2 ; or gene1A [BCL2 .Notch1 mutation in mouse embryos. They showed that a mutation in this gene entails deficiencies in intra-embryonic hematopoiesis [Notch1 has a major role in adult hematopoiesis by controlling events, such as lymphoid vs. myeloid differentiation [Notch1 [The important role of Notch in embryos was described by Robert-Moreno et al. when they studied opoiesis . In adulopoiesis . Indeed,ntiation , T vs. Bntiation ,26,27,28ntiation , and posntiation ,31. Furt [Notch1 , is need [Notch1 .Rbpj gene, through which canonical signaling converges from the different Notch receptors. The group examined the role of Notch in the HSC function in normal mouse bone marrow (BM) and in chimeras with an HSC transplant after radiotherapy. They observed a stable long-term reconstitution and a normal HSC engraftment either in a normal BM or a stressed BM environment. Therefore, they concluded that Notch signaling is not essential for HSC self-renewal. Nevertheless, they did not discard the fact that increased Notch expression could stimulate HSC self-renewal under other stress conditions different from radiotherapy [Rbpj-κ by a Vav-Cre and Mx1-Cre system in adult BM [On the other hand, other authors proved that Notch could be dispensable for adult HSCs. Maillard et al. inhibited the complete cascade of Notch signaling by two methods: firstly, by using dominant negative Mastermind-like1 (DNMAML), a potent and specific pan-inhibitor of Notch-mediated transcriptional activation; secondly, by silencing the otherapy . Additiootherapy . Similaradult BM .In this section, we briefly discuss the role of Notch in lymphoid malignancies, since this signaling pathway has been broadly study in this field; then, we examine Notch implications in myeloid neoplasia to finally focus on the alteration of this conserved pathway in AML.NOTCH1 [NOTCH3 promotes JAG1, a phenomena that is caused by a NOTCH3/JAG1 auto-sustaining loop [NOTCH1 mutations as a negative predictor factor of CLL patients [NOTCH1 mutated in 11% of CLL patients and found that mutations in this gene are in 90% of the cases mutually exclusive with TP53 disruptions, and confer a similarly dismal prognosis with a reduction in the overall survival (OS) [Almost 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) have mutations of NOTCH1 , but theNOTCH1 ,37,38. Ting loop . This meing loop . Moreovepatients ,41,42. INCSTN, APH1, MAML1, and NOTCH2, which, to date, have not been described [In the myeloid linage, the forced expression of Notch increased progenitor differentiation toward the T lymphoid and megakaryocyte–erythroid progenitor (MEP) lineages. Consequently, the inhibition of Notch increased the granulocyte–monocyte progenitor (GMP) and produced a phenotype compatible with chronic myelomonocytic leukemia (CMML) in mice. Interestingly, in human CMML samples, there were some mutations that inactivated Notch-related genes, such as escribed .NOTCH1 is mutated in 12% of AML patients and its mutation is an adverse independent predictor factor for OS; nevertheless, the cohort analyzed was small (50 patients) and the results were not replicated, at least in part, due to the non-inclusion of Notch pathway genes in targeted NGS panels employed in myeloid malignancies [NOTCH1, JAG1 and DLL1 compared to normal donors, and a greater expression of these genes was an adverse predictor factor in the multivariate analysis [HES1 and DTX1, showed a significant reduced expression compared to healthy controls, which suggest reduced Notch signaling in AML samples [NOTCH1, NOTCH2, and HES1 led to reduced AML growth in vivo [There are plenty of studies that seek to resolve the role of Notch in AML. Interestingly, gnancies . In addianalysis . A large samples . Additio in vivo . Likewis in vivo . More st in vivo . Further in vivo .NOTCH2 and JAG1 are shown. The inhibition of the Notch pathway, by using GSIs, blocked the cell-cycle progression during the G0/G1 phase and induced apoptosis [However, not all the studies defend the fact that the inhibition of Notch leads to an increase in the proliferation in AML. Different results were obtained by Li et al. when they studied the repercussion of the combination of tyrosine kinase inhibitors (TKIs) and the γ-secretase inhibitor (GSI) on FLT3-ITD + AML in vitro and in vivo. They found that the combination of these drugs reduced cell proliferation and induced apoptosis . Similarpoptosis . These rFinally, other studies do not focus on the role of Notch in the cell cycle of AML, but they study whether or not Notch has an anti-leukemic effect on xenograft models. The work of Kamga shows that mice treated with the γ-secretase inhibitor in combination with chemotherapy increased the overall survival, compared to the mice in which only chemotherapy was employed. For this reason, the authors suggested that Notch can be useful as a prognostic marker and therapeutic target in AML . TherefoMore than 50 clinical trials with drugs targeting Notch signaling in different tumors  were carDespite the efforts made to inhibit this pathway, most of the trials stayed in phase I with no results available (RO4929097), or with results but no further research (MK0752 and BMS-906024). Indeed, in the study of MK-0752, only a limited number of patients tolerated the proposed dose, while in the study of BMS-906024, 32% of patients showed at least a 50% reduction in bone marrow blasts ,54. On tOnly one trial reached the third phase of clinical development, the GSI called Nirogacestat (PF-03084014). Nevertheless, this approval is for desmoids tumors .Even if we manage to properly inhibit Notch, the function of this pathway in AML is not only likely to be dependent on other genetic mutations, but also on the crosstalk with other signaling pathways.There are multiples pathways that are related to Notch. Among them, we focus principally on Wnt and Hh, which seem to have a high grade of interdependence with Notch not only during embryo development, but also in stem cell regulation and the differentiation of different tissues.NOTCH2 induces a strong activation of Wnt signaling in chronic lymphocytic leukemia (CLL) tumoral cells [Some authors explain how the accumulation of active β-catenin levels are negatively regulated by Notch at a post-translational level in stem and progenitor cells . Other aal cells . Furtheral cells . Similaral cells . In furtal cells . These rShh and Hes3 [Hes1 by Hh, independent of canonical Notch signaling in stem-like cells [Another important conserved pathway is Hh, as we explained in a previous work . Nicolasand Hes3 . Certainke cells . The stuke cells . The bonke cells . This kiNOTCH1 was overexpressed when GLI1 and CTNNB1 were inhibited by a knockdown [More proof of the relationship between Hh, Wnt and Notch was obtained by Okuhashi et al. These authors found in in vitro experiments that nockdown . The intnockdown .In this section, we summarized the important crosstalk between two signaling pathways, Hh and Wnt, which are also key players in embryo development. Nevertheless, there are several signaling pathways that can interact with Notch. To further explore the different crosstalk between Notch and other signaling pathways, we encourage the reader to read a specialized review about the topic. For example, Guo et al. described in their review HER/ErbB, PDGF/PDGFR, TGF-β, VEGF/VEGFR-2, IL-1, and IL-6 in breast cancer , but theMYCC, CCND1, CDKN1A, BCL2, and embryogenesis and stemness-related genes, such as the Hes family [Since 1914, researchers tried to understand the function of Notch using different approaches. Nowadays, we know that Notch is one of the pathways in charge of the promotion of cell-cycle genes, such as s family ,18,19,20s family ,7,8. In s family ,34. In ss family ,29,30,31NOTCH1 found in AML patients and the adverse outcome conferred either by mutations in this gene or the overexpression of different elements of this pathway [Notch has been widely studied in lymphoid malignancies, where there is a broader body of evidence for its dysregulation, given the mutational frequency observed in these neoplasms. Nonetheless, we tried to focus on the role of Notch in AML. An interesting finding is the mutational frequency  of  pathway . TherefoWe sought to gather the available information regarding Notch in LSCs of AML and, more precisely, about its cell-cycle regulation. Information about this topic is contradictory and limited. Should we inhibit Notch to decrease the quiescent population of LSCs? Or is it contrariwise? To date, some studies demonstrated that the inhibition of Notch increased cell proliferation ,47. LobrSince Notch could be dispensable in the maintenance of adult HSCs, it may be possible to inhibit this signaling, as previously suggested. Indeed, multiple clinical trials have been conducted, which aimed to inhibit Notch, mostly by using a γ-secretase inhibitor. Unfortunately, only one of those studies reached the phase III clinical trial in desmoids tumors. Nevertheless, the studies in hematological neoplasia did not progress from phase I and only LY3039478 reached phase II. We should not forget that most of the studies inhibit the γ-secretase protein, which is part of the canonical activation of the pathway. Therefore, although the full inhibition would be achieved, non-canonical activation caused by other pathways is possible. There is a large list of other players, but we focused on the two other pathways, Hh and Wnt, essential in embryogenesis. The evidence sustains the relationship between these pathways from in vitro, in vivo, and even clinical studies. We believe that the combined targeting of Notch, and the Hh and Wnt pathways can show synergistic effects in the quiescence reduction in LSCs and improve the clinical benefits over the targeting of a single pathway.Notch may be dispensable for the maintenance of adult HSCs, which leaves an open door to target this signaling pathway. It is necessary to understand the role of Notch in leukemic stem cells to answer a fundamental question: does the inhibition of Notch reduce the quiescence state of LSCs by forcing cells into a cell cycle or enhance that quiescence state?Current leukemic and oncologic treatments focus on the prevention of cell division to reduce the quantity of malignant cells in patients, but this approach does not preclude the possibility of a relapse. One of the most feasible methods to reduce this possibility would be by eradicating the cells that cause the relapse: the LSCs. The LSCs used to be in quiescence; therefore, conventional chemotherapy does not affect them.In order to exploit the therapeutic potential of the Notch pathway, firstly, we believe it is essential to decipher the role of Notch in the regulation of the quiescence of the LSC population. Single cell technologies might help us in this difficult task. In a further step, targeted therapies could be employed, either directed only towards Notch or also directed towards Hh and/or Wnt, alongside conventional chemotherapy. This therapeutic strategy could decrease the quiescence of LSCs, increase their chemosensibility and achieve the eradication of LSCs and AML curation. Our group is currently working on this line of research and would be delighted to collaborate with other groups interested in this promising field.


To identify predictors of coronary artery disease in survivors of cardiac arrest, to define the best timing for coronary angiography and to establish the relationship between coronary artery disease and mortality.This was a single-center retrospective study including consecutive patients who underwent coronary angiography after cardiac arrest.A total of 117 patients  were included. Most cardiac arrest incidents occurred with shockable rhythms (70.1%), and the median duration until the return of spontaneous circulation was 10 minutes. Significant coronary artery disease was found in 68.4% of patients, of whom 75% underwent percutaneous coronary intervention. ST-segment elevation , the presence of wall motion abnormalities , an left ventricular ejection fraction ≤ 40%  and elevated high sensitivity troponin T  were predictors of coronary artery disease; the latter had poor accuracy , with an optimal cutoff of 170ng/L. Only ST-segment elevation and the presence of wall motion abnormalities were independent predictors of coronary artery disease. The duration of cardiac arrest  was an independent predictor of death, and shockable rhythm  was an independent predictor of survival. The presence of coronary artery disease and the performance of percutaneous coronary intervention had no impact on survival; it was not possible to establish the best cutoff for coronary angiography timing.In patients with cardiac arrest, ST-segment elevation, wall motion abnormalities, left ventricular dysfunction and elevated high sensitivity troponin T were predictive of coronary artery disease. Neither coronary artery disease nor percutaneous coronary intervention significantly impacted survival. Evidence suggests that coronary artery disease (CAD) is a main cause of CA, with significant CAD documented by coronary angiography or autopsy in more than 70% of patients with resuscitated CA,,7 Considering this evidence, international guidelines recommend coronary angiography and PCI when indicated in patients following CA with ST-segment elevation  and when there is a high index of suspicion of ongoing infarction .,9If acute coronary occlusion is the cause of CA, percutaneous coronary intervention (PCI) might reduce infarct size, improve hemodynamic status and reduce the recurrence of life-threatening arrhythmias and recurrent CA.However, since patients after CA have been excluded from the main randomized trials that demonstrated the benefits of primary PCI in acute coronary syndrome (ACS) with or without ST-segment elevation, the real benefit of systematic coronary angiography in these patients is still under debate.,11 this remains controversial, and some authors highlight that routine emergency coronary angiography is probably not cost-effective or entirely risk-free.,13 Indeed, the recently published COACT trial found that immediate angiography was not better than delayed angiography with respect to overall survival in these patients.Although some studies support immediate coronary angiography and possible PCI in the setting of out-of-hospital CA, as it appears to be associated with better survival at discharge,The main objective of this study was to identify independent predictors of CAD in CA survivors, with the ultimate goal of helping to determine which patients should undergo coronary angiography. The secondary objectives were to establish the best timing for coronary angiography in these patients and to determine the relationship of the presence of CAD, PCI, and mortality.Hospital Universitário de Santa Maria, Lisboa, Portugal).A retrospective single-center observational study was performed in a tertiary university hospital  from January 2015 to July 2018.Demographic, clinical, laboratory, electrocardiographic, echocardiographic and angiographic data were reviewed based on clinical records, which were fully electronic.Comorbidities were acknowledged by being referenced in the patient clinical file or by laboratory results available prior to the index hospital admission .The assumed cause for CA was adjudicated by reviewing the electronic clinical records of the patient. Other signs of ischemia  were also recorded.All electrocardiograms (ECGs) available in the electronic clinical records were reviewed by two authors to identify criteria of ST-segment elevation or equivalents according to the current recommendations.Left ventricular (LV) systolic dysfunction, based on the LV ejection fraction (LVEF), was classified according to the current European recommendations.Significant CAD was defined by a lesion resulting in a lumen reduction of at least 50% in the left main artery and of at least 70% in the remaining vessels. The first available laboratory results after ROSC were considered.The statistical analysis was performed using IBM Statistical Package for Social Disease , version 23. Categorical variables are reported in the absolute number and percentage, and continuous variables are reported as the mean and standard deviation or median and interquartile range (IQR), according to the normality of the distribution, which was tested with the Shapiro-Wilk test. Factors associated with CAD were identified by the chi-square test or Fisher’s exact test for categorical variables and Student’s t test or a nonparametric test for continuous variables with normal and nonnormal distribution, respectively. Independent predictors of CAD were identified by logistic regression analysis. Independent predictors of death were identified with Cox regression analysis. Receiver operating characteristic (ROC) curve analysis was used to determine the best cutoff of troponin level to predict CAD and the best timing for coronary angiography. A p-value below 0.05 was considered significant.All data were analyzed anonymously. Informed Consent was waived because no change was applied to the patients’ management. The present study conformed to the ethical guidelines of the Declaration of Helsinki.The patient demographics are summarized in Cardiovascular risk factors were common: 63% of the patients had arterial hypertension, 43% dyslipidemia, 27% diabetes and 32% a history of smoking. Thirty-seven patients had a previous history of CAD, and 13% had cerebrovascular disease.In 24.8% of the patients, there was no information regarding the presumed cause of CA before coronary angiography. In the remainder, based on clinical judgment, type 1 myocardial infarction was the presumed cause in 79.5%, mainly with ST-segment elevation (60%). The presumed causes of CA are detailed in Most cases (58.2%) were out-of-hospital CA, which usually occurred in shockable rhythm (70.1%). The median time until ROSC was 10 minutes (IQR = 4 - 20). Seventy-six percent of the patients underwent coronary angiography in the first 24 hours after ROSC and 38% in the first 2 hours, with a median time of ROSC to coronary angiography of 3 hours. In coronary angiography, significant CAD was found in 68.4% (n = 80) of the patients, mainly involving the left anterior descending artery , and 14% (n = 16) had left main artery involvement. Considering patients with significant CAD, 75.0% (n = 60) underwent PCI, 12.5% (n = 10) were considered for surgical intervention, and the remaining 12.5% (n = 10) did not undergo revascularization. Among these, four patients were considered to have chronic CAD, since all of these patients had previously known lesions or occluded bypass grafts; three patients had disease in vessels of small diameter only; and the remaining three patients had chronic total occlusions. Considering patients without significant CAD, eight underwent left ventriculography, of whom 71% (n = 5) had wall motion abnormalities (WMAs); one patient had WMAs compatible with Takotsubo syndrome, and 50% (n = 4) had a severely reduced LVEF.The results of the post-CA ECG were available in 86 patients. Of these, most did not have ST-segment elevation , and only 12.3% (n = 10) had left bundle branch block. However, only 7% of the patients (n = 6) had an unremarkable ECG. In the subgroup of patients without significant CAD, 17.9% (n = 5) had ST-segment elevation, 44.4% (n = 12) had other electrocardiographic signs suggestive of myocardial ischemia and 13.8% (n = 4) had an unremarkable ECG. Of the 79 patients who had available immediate post-ROSC echocardiogram results, 51% (n = 40) had WMAs, and 52% (n = 41) had at least moderate LV systolic dysfunction (LVEF ≤ 40%). In the subgroup without significant CAD, 10.7% (n = 3) had WMAs, and 37.9% (n = 11) had moderate LV systolic dysfunction.Considering the laboratory results, only 7% of the patients (n = 7) had troponin in the normal range. In the subgroup without significant CAD, only 3.0% (n = 1) had troponin levels in the normal range. During follow-up, 52% of the patients died (n = 61); 14.5% (n = 17) died in the first 24 hours after CA, 27.4% (n = 32) died between 24 hours and 30 days, and 10.3% (n = 12) died after 30 days. The mean follow-up was 24.8 ± 11.9 months, and the mean time until death was 3.6 ± 9.1 months.versus 0.52ng/mL, IQR = 0.15 - 4.68; p = 0.586, for the 4th generation troponin I - TnI - assay and 283pg/mL, IQR = 60.3 - 969 versus 96pg/mL, IQR = 32 - 549.5), p = 0.048 for high-sensitivity troponin T - hs-TnT). The ROC analysis showed that hs-TnT had a significant but poor accuracy in predicting CAD  with a best cutoff of 170pg/mL (sensitivity = 60.4% and specificity = 69.2%). A hs-TnT value above 170pg/mL was associated with the presence of significant CAD . None of the other laboratory values assessed was associated with significant CAD.Patients with significant CAD had a numerically higher troponin levels , as were the presence of WMAs in echocardiograms  and an LVEF ≤ 40% . None of the other ECG or echocardiogram characteristics were associated with significant CAD.ST-segment elevation was also associated with CAD .Considering the comorbidities, only arterial hypertension was found to be marginally associated with significant CAD , and 69% of patients with an LVEF ≤ 40% had significant CAD versus 21% of patients with an LVEF > 40% (p = 0.017). Both parameters were predictors of CAD .In the subgroup of patients without ST-segment elevation, 24 (51.1%) had significant CAD, and 54.2% (n = 13) underwent primary PCI. In this subgroup, the presence of WMAs and an LVEF ≤ 40% were also associated with significant CAD: 88% of patients with WMAs had significant CAD versus those without significant CAD .Among patients with significant CAD, 56.3% (n = 45) died, and 43.8% (n = 35) survived. The presence of significant CAD was not associated with mortality (p = 0.19). Additionally, in the subgroup of patients with significant CAD who underwent PCI, 53.3% died (n = 32), and 46.7% (n = 28) survived. Percutaneous coronary intervention in patients with significant CAD was not associated with reduced mortality (p = 0.79). Additionally, there was no significant increase in mean survival time in patients with Patients with indications for surgical revascularization had higher 30-day and overall mortality .versus 2.9 hours, IQR = 1.9 - 21.5). Performing coronary angiography in the first 24 hours after ROSC versus more than 24 hours was not associated with reduced mortality , and the same was true for the 2-hour cutoff suggested in the guidelines . ROC curve analysis further strengthened these results .There was no significant difference in the time from ROSC to coronary angiography between patients who survived and those who died .The time until ROSC was associated with mortality. Those who did not survive to 30 days had a longer time until ROSC  was similar to previous reports, where rates ranged from 53% to 95%.,15,17-21 This wide range of PCIs might be justified by the different definitions of significant CAD between studies. Importantly, our study also reported a sizeable proportion of patients considered for surgical intervention (13%), an outcome seldom reported in the literature.We evaluated the role of previous medical history, laboratory values, and electrocardiographic and echocardiographic data, usually available in the emergency department, to predict the presence of CAD in survivors of CA. More than 100 patients were included, and similar to previous studies, almost 70% had significant CAD.Herein, we found a significant difference in time from CA to coronary angiography between patients with CAD and those without CAD , which m Therefore, the indications and best timing for coronary angiography in survivors of CA are very much a matter of debate.,11The etiology of CA is often difficult to ascertain in an emergency setting, as medical history is often unavailable and complementary exams results are difficult to interpret.In our study, only direct evidence of cardiac abnormalities - ST-segment elevation, WMAs, at least moderate LV systolic dysfunction and an elevation of hs-TnT above 170pg/mL - were predictors of significant CAD.,19 ST-segment deviation might be caused by electrolyte abnormalities, defibrillation, ischemia-reperfusion injury and intravenous drugs administered during resuscitation,,17,19 rendering the ECG difficult to interpret, with poor predictive values reported.,11,17,19 However, some studies support our findings. Anyfantakis et al. studied 72 consecutive out-of-hospital CA survivors who underwent systematic emergency coronary angiography and found that ST-segment elevation after ROSC was independently correlated with the presence of CAD and strongly correlated with a final diagnosis of acute myocardial infarction. Additionally, Lee et al. found that ST-segment elevation and new-onset left bundle branch block are highly associated with acute coronary lesions.The ECG role in the post-CA setting as a predictor of ACS is still debatable. Notwithstanding, our findings suggest that emergency echocardiography might be of use in selecting patients for coronary angiography, especially if there are WMAs or an LVEF ≤ 40%.Emergency echocardiography in the post-CA setting may have limited value, as regional WMAs are not specific for acute myocardial ischemia and may be related to stunning caused by hypoperfusion or by resuscitation maneuvers.,17 We found that an elevated hs-TnT value, even with a cutoff value of 170pg/mL , while predictive of CAD, has poor accuracy in CA survivors, in keeping with previous results. These findings render the usefulness of troponin level elevations limited from a clinical perspective.In our study, 93% of the patients had elevated troponin levels irrespective of the presence of CAD. This finding was also reported in previous studies and reflects the very high sensitivity of the assay, suggesting that myocardial injury biomarkers are not reliable for the diagnosis of ACS in this setting because of their low specificity. where patients who survived to CA and present with ST-segment elevation or with high suspicion of ACS  should undergo coronary angiography. However, emergency doctors and interventional cardiologists must be aware of the limitations of these findings in this particular setting, which might lead to false-positive and false-negative results.Overall, our results support the current recommendations, Interestingly, we did not find an association between CA with shockable rhythm and CAD, which might have been the result of selection bias; the occurrence of ventricular arrhythmias rendered patients more likely to undergo coronary angiography. This finding is evidenced by the high prevalence of shockable rhythm in our cohort.When managing these patients, one should consider that the treatment of the underlying cause of CA is paramount and that ACS is the most common cause of malignant ventricular arrhythmias leading to sudden cardiac death.,13,16,23 Here, as previously described,,21,24 the only predictor of mortality was the time to ROSC, with shockable rhythm being predictive of survival. We found no association between the presence of significant CAD and PCI with survival. Our results are in keeping with those of Garot et al., who studied predictors of survival in CA complicated acute myocardial infarction in 186 patients and found that the rates of PCI success had no impact on survival. The authors concluded that although successful PCI could have played an important role in improving survival, their data indicated that it was not sufficient in itself to show any differences in both 30-day and 6-month mortality rates. Similarly, Anyfantakis et al. found that although PCI was warranted in one-third of patients, it was not associated with hospital survival. These findings might be the result of the small sample size of these studies, including ours, but also because in post-CA survival patients, other therapeutic interventions probably influence the prognosis.The results in the literature on the role of coronary angiography in this setting, performed as soon as possible after ROSC, with PCI as needed, are controversial. Several studies document the survival benefit of early coronary angiography and PCI in CA survivors.As a result of these findings, we were unable to find a threshold for the best timing for coronary angiography in our patients, nor did the time until coronary angiography have any influence on survival. These findings are in line with the results of the COACT trial, a multicentric prospective study with 522 patients without ST-segment elevation after CA, which found that immediate angiography was not better than delayed angiography with respect to overall survival at 90 days. Some authors even report that most patients die from neurological complications.According to the literature, following successful resuscitation, only one-third of patients die because of cardiovascular causes; approximately one-third of deaths are secondary to central nervous system injury, while the remaining deaths are due to a variety of other reasons. which can contribute to further mortality. Furthermore, even in patients who have genuine ischemic heart disease-related CA, some may have chronic ischemic heart disease that provides an arrhythmogenic substrate for CA. Although these patients are expected to have significant CAD, it is unclear how angiography and PCI would improve their outcomes.Therefore, the absence of survival impact of early coronary angiography and PCI might be justified by the fact that immediate angiography leads to a delay in the implementation of interventions that might contribute to brain injury prevention, since the current practice in the center is to implement these measures only when the patient is admitted to the intensive care unit. In addition, performing coronary angiography in this setting requires the mobilization of an unstable patient, exposure to contrast agents, and a risk of vascular and bleeding complications,Therefore, while the identification of an acute culprit coronary lesion amenable to urgent PCI may be important for the prognosis of CA survivors, this is not the only factor involved, and careful, individual evaluation is critical. Attempts to reduce the time to ROSC and to improve post-ROSC care are probably at least as important as coronary angiography. Further randomized studies with more patients and a prospective design are essential to define the appropriate pathway for therapeutic interventions in survivors of CA - especially for selecting who should undergo coronary angiography and when.This study has several limitations. It is a single-center retrospective study based on the information available in the electronic clinical records, and as a result, missing data are inevitable; specifically, there was a high number of patients without information regarding the post-ROSC ECG, since many of these patients are referred from other hospitals or the ECG is performed in the prehospital setting. Additionally, there was selection bias, as all patients included underwent coronary angiography based on the clinical decision of the emergency physician and the interventional cardiologist, rendering a high pretest probability, so the prevalence of significant CAD might be overrepresented. As in previous studies, the sample size was small. Another limitation was the use of two different troponin assays (TnI and hs-TnT) because we included the period of transition to a high sensitivity assay in our institution; thus, only a few patients had TnI values available. Finally, CAD was classified as significant based on the visual appreciation of the interventional cardiologist and not a core laboratory; therefore, operator bias cannot be ruled out.Our study suggests that in survivors of cardiac arrest, only direct evidence of cardiac abnormalities is strongly associated with coronary artery disease. ST-segment elevation in the post-return of spontaneous circulation electrocardiogram, the presence of wall motion abnormalities, an left ventricular ejection fraction ≤ 40% in transthoracic echocardiography, and an high sensitivity troponin T level above 170pg/mL were predictors of significant coronary artery disease. These results may help in the selection of patients for coronary angiography, thereby reducing unnecessary procedures, and may support current guidelines, which recommend performing coronary angiography in patients with a high index of suspicion of ongoing infarction.However, in our population, the time until coronary angiography, the presence of significant coronary artery disease and the performance of percutaneous coronary intervention did not influence survival, and it was not possible to establish the best cutoff for coronary angiography timing. This outcome was likely the result of the small sample size and confounding factors, especially noncardiovascular issues affecting prognosis.


Klebsiella pneumonia (Kp) strains. The aim of this study was the phenotypic and genotypic analysis of eight K. pneumoniae NDM (Kp NDM) isolates, recovered in Poland during the years 2016 and 2018 from seven patients with urinary tract infections (UTIs), asymptomatic bacteriuria (ABU), or colonization of the gut. PCR melting profile genotyping indicated a close relationship between the strains derived from 2018, which were not related to the strain isolated in 2016. WGS results were analyzed in relation to international Kp isolates. Clonal and phylogenetic analyses were performed based on multilocus sequence typing (MLST) and single nucleotide polymorphisms (SNPs) of the core genome. The metallo-β-lactamase was assigned to the NDM-1 type and the sequence was identified as ST11. Eleven antimicrobial resistance genes were detected, mostly from plasmid contigs. Unprecedented profiles of plasmid replicons were described with the IncFII/pKPX-1 dominant replicon. In terms of the KL24 and O2v1 capsular antigen profiles, these isolates corresponded to Greek strains. Strains isolated from UTI, ABU, and colonization GI tract patients were not carrying environment-specific virulence genes. Based on the assessment of strain relationships at the genome level and their direction of evolution, the international character of the sublines was demonstrated, with a documented epidemic potential in Poland and Greece. In conclusion, some groups of patients, e.g., renal transplant recipients or those with complicated UTIs, who are frequently hospitalized and undergoing antibiotic therapy, should be monitored not only for the risk of UTI, but also for colonization by Kp NDM strains.In recent years, there has been an observed increase in infections caused by carbapenem-resistant  K. pneumoniae (Kp) strains, both extended spectrum β-lactamase (ESBL) strains and Kp carbapenemase (KPC)- and metallo-β-lactamase (MBL)-producing strains [In recent years, there has been a substantial increase in the number of reported cases and outbreaks caused by  strains ,2. The p strains . Europea strains .blaNDM gene, which is capable of hydrolyzing almost all β-lactam antibiotics [The greatest epidemic threat to the hospital environment is now the strains of Kp, which produce New Delhi metallo-β-lactamases (NDMs) ,6. The ribiotics ,7. The gibiotics ,9,10.blaNDM gene [The metallo-β-lactamase NDM is a lipoprotein anchored to the bacterial outer membrane. This increases the stability of the enzyme, and its secretion in outer membrane vesicles protects against the action of β-lactam antibiotics . It is aNDM gene .rmpA and rmpA2 genes and not the capsule type [Fuursted et al. have described the virulence potential of Kp carrying NDM-1 using mouse and nematode models, and suggested a role for this virulence in rapid global spread . Virulenule type ,18. Straule type .mdh, infB, tonB, gapA, phoE, pgi, and rpoB), we classified MDR Kp strains by sequence type (ST). There are 29 recorded NDM variants across several bacterial species [Based on seven loci . UTI is the most common infectious complication after renal transplantation (RTx) [Klebsiella spp. [on (RTx) ,29. Mostlla spp. ,31,32. HA total of eight Kp NDM isolates were tested, seven of which had been isolated in either 2016 or 2018, from six patients (one patient with two isolates) hospitalized in the Department of Nephrology, Transplantology, and Internal Medicine, the Medical University of Gdańsk, Poland. An eighth isolate was sent for diagnostic purposes to the University Clinical Laboratory in Gdańsk from another hospital in Poland (Toruń) and it was also used as a reference strain for genotyping. The list of strains and their origin is given in ® 2 Compact . Susceptibility assays were interpreted according to EUCAST v 10.0 (2020) recommendations [® strips, bioMérieux, Marcy l’Étoile, France) and carbapenemase production was confirmed with a β-CARBA® test . The presence of the blaNDM-1 gene was detected by the PCR technique, as described [Identification and antibiotic susceptibility were determined using the VITEKndations . ESBL prescribed .This study was approved by the Local Bioethics Committee at the Medical University of Gdańsk  (decision no. NKBBN/120/2019). The medical personnel responsible for the taking of samples were not involved in authoring the study, and information about patients was not available to the authors of the publication.−1.Genomic DNA was extracted from 3–4 colonies on an LA agar plate using the Genomic DNA Kit , as detailed in the manufacturer’s instructions. The DNA concentrations were measured using a NanoDrop ND-100  and were at the level of 10–40 ng μLTM software, BioRad, Ver. 4.5) and UPGMA (unweighted pair-group method with arithmetic averages). The Pi cut-off for genotype definition was 96%.A PCR MP method was used according to the procedure described by Stojowska et al. , with thGenomes of all isolates were sequenced with the Illumina MiSeq platform . Read cleaning , filtration, and trimming according to quality and length criteria were performed using Trimmomatic (Ver. 0.36) . The genThe draft genomes were annotated with Prokka (Ver. 1.11)  using deA diagram showing the groups of the analyzed strains in this work is presented in An SNP pairwise difference matrix from the core gene was obtained for ST11 and other STs using snp-dists (Ver. 0.6.3). The SNP analysis of the core genome alignment was completed by aligning the short-reads data from each isolate against the reference, the first outbreak isolate from Poland from 2012 (BioSample accession number SAMN14314560), for a comparison in high resolution among ST11 using Snippy (Ver. 3.1) . The anaK. pneumoniae BIGSdb database at Institut Pasteur [The ABRicate tool was used to identify antimicrobial resistance and virulence genes by running local assemblies. Antimicrobial resistance genes were detected using ResFinder 3.1 , with a  Pasteur , with a Capsule (K) and LPS (O) antigen types were differentiated among the isolates using the Kaptive tool . PlasmidThe RFplasmid tool  was usedContig sequences for each genome were deposited in the NCBI’s database, with BioProject and BioSamples accession numbers of PRJNA688074 and SAMN17168596–603, respectively.The seven included Kp NDM cultures were isolated in 2016 and 2018 in the Department of Nephrology, Transplantology, and Internal Medicine, the Medical University of Gdańsk, from six patients with a medical history of kidney disease . The eig® test. By obtaining a 621 bp PCR product, we also confirmed the presence of the blaNDM-1 gene in all eight isolates. Most of the tested Kp NDM strains showed similar patterns of resistance. All NDM strains were resistant to penicillins, penicillins with β-lactamase inhibitors, all generations of cephalosporins, fluoroquinolones, ertapenem, meropenem, and cefoperazone/sulbactam. In the case of the remaining carbapenems (imipenem), the degree of resistance of the strains varied. Resistance to imipenem was found in four strains, and four strains were also characterized by moderate susceptibility to this drug. We observed large differences in sensitivity to aminoglycosides. None of the strains were resistant to gentamicin while only one strain was resistant, and two strains were moderately sensitive to amikacin. One strain of NDM showed resistance to tobramycin. Most strains remained resistant to trimethoprim/sulfamethoxazole, except for strain number ND32, isolated in 2016. In the analyzed material, one strain was also found to be moderately sensitive to tigecycline. All strains remained sensitive to colistin.Antibiotic resistance profiles were prepared for each of the Kp NDM isolates . ESBL prThe main aim of this task was to determine whether the strains isolated from kidney disease patients and kidney recipients were from the community or were hospital-acquired. Owing to the emergence of NDM-producing Kp in January 2018 at the Department of Nephrology, Transplantology, and Internal Diseases of the Medical University of Gdańsk, the patients had been monitored for infection and colonization during the period from January to April 2018.Genotyping of seven Kp strains isolated from six patients, and a reference Kp strain , revealed three distinct genotypes, A–C . The DicThe strain from 2016 (ND32) and the first strain from 2018 (ND24) had unique genotypes, A and B, respectively, indicating a new strain being present in the clinic’s renal ward in 2018. The other strains from 2018 were assigned a third, ‘C’, genotype, and were not consistent with the first strain isolated in 2018. For one patient (P4) from which two isolates were taken (ND88 and ND34), two distinct subgenotypes (C1 and C2) were described. One isolate came from the urine and the other from the patient’s GI tract . The strains from the local outbreak (ND33) and the reference strain (ND27/Ref) were characterized by a high degree of similarity (98%), and were assigned to a subgenotype, C3.The seven studied isolates were subjected to phylogenetic analysis against the non-outbreak reference isolate sequenced by us, and 71 publicly available Polish Kp genomes, 67 of which were NDM-1 Tn125A isolates; 4 other known Polish carbapenemase-positive Kp ST11 strains, as described previously ; 90 randblaNDM-1 gene. These 21 isolates were collected in 2013–2016 in Greece , Slovakia (n = 5), the Czech Republic (n = 3), India (n = 2), Spain (n = 1), and USA  (mainly from the EuSCAPE study). Five Kp NDM-1 sequences from the public database were not included in this clade. These isolates were collected in 2012–2013 in Pakistan (n = 2), Turkey (n = 1), Norway (n = 1), and Romania (n = 1). Two Kp NDM-1 sequences from Izdebski et al. [Phylogenetic analysis with concatenated alignment core genes showed that all seven examined isolates from our hospital represented the ST11 sequence type, with high homogeneity. The seven studied isolates were included in one clade, with 68 Polish isolates  and 21 isolates from different countries around the world, 10 of which had the i et al.  were alsAnalysis of the cgSNP (core genome SNP) in relation to the reference strain (BioSample accession number SAMN14314560) revealed 7–88 SNPs between the individual Polish isolates sampled in this study. The timing of individual isolates from the outbreak was reflected by a gradual reduction in the number of SNP differences between temporally closer epidemic isolates, from 88 SNP differences between the January 2018 isolate and the April 2018 isolate, to 16 SNP differences between the two April 2018 isolates, showing the process of population diversification.The isolates collected during the study showed the fewest differences to strains from Poland and Greece; differing from Greece isolates by 46–79 SNPs. Isolate no. ND32 (SAMN17168599), the first isolate from the ward, showed the fewest differences compared with strains from Greece, differing from them by 46–56 SNPs. The analysis showed that isolate no. ND27 (SAMN17168596), isolated at the same time (May 2018) as the outbreak strains, but from a different hospital, differed from the outbreak strains by 16–86 SNPs. Similarly, in the SNPs’ pairwise distance from core gene analysis, a Polish isolate taken from the public database  was the closest to the studied isolates, differing from the outbreak strains by 43–64 SNPs and from strains originating in Greece by 25–36 SNPs . The SNPEleven antimicrobial resistance genes in total were detected in the seven isolates from the hospital in Gdańsk, which could be used to split the strains into seven different gene profiles . The resblaNDM-1, blaCTX-M-15, blaOXA-1, and blaTEM-1 genes. All four genes were present in the ND34 (GpC2)- and ND88 (GpC1)-related isolates; three genes  in ND32 (GpA), ND111 (GpC), ND96 (GpC), and ND27 (ref) (GpC3); and only two  in ND24 (GpB) and ND33 (GpC3). The aac (6′)-Ib gene, which is responsible for aminoglycoside resistance, was detected in seven isolates. The aadA1 gene was only detected in isolate ND33. Resistance to phenicols was encoded by the cat1 and cat4 genes for ND111 and ND88, and for other isolates by the catA1gene only. The ND24 isolate lacked resistance genes for this group of antibiotics. The sul1 gene, responsible for sulfonamide resistance, was included in all isolates. One or both of the dfrA1/dfrA14 genes, which contribute to resistance to trimethoprim, were detected in all isolates. Most of the genes were determined to be plasmidic. Overall, these genes enabled us to specify a perfect phenotype to genotype correlation for antibiotic susceptibility data.A phenotype of resistance to β-lactams was generated from the The previous Polish outbreak study revealed a prevailing presence of the IncFII/pKPX-1 and IncR replicons, and high differentiation of the species. This study provides new data on the plasmid content of the epidemic genotypes resulting from a local outbreak of a nationally distributed clone. Eight replicons in total were detected across the seven isolates, which could be split into four profiles of one to six replicons each , again sThe frequency of integrative conjugation elements (ICEs) ICEKp1 and ICEKp2 in clinical Kp isolates was determined. ICEKp1 was identified in 113 (70.2%) international strains and all eight strains tested in this study. ICEKp2 was present in 159 out of 161 (98.8%) international genomes, and in all genomes sequenced by us. Its presence was not correlated with ICEKp1. Among the Kp NDM-1 strains, both ICEKp1 and ICEKp were identified in 89 (96.7%) strains. The presence of ICEKp2 correlated with the presence of ICEKp1 for Kp NDM-1 strains. Additionally, the presence of two closely related sequence types, as demonstrated by the genome analysis, suggests a stable inheritance of ICEKp2. ICE contributes to the spread of antimicrobial multi-resistance genes and pathogenicity .rmpA and rmpA2 genes encoding the so-called ‘mucus factor’, which are associated with the overexpression of proteins in strains with the K1 or K2 capsule type, and increased virulence [rmpA and rmpA2 genes always had ICEKp2 and a partial (n = 5) or complete (n = 2) ICEKp1 element , respectively, with no differences between the Polish ST11 NDM-1 strains and some of the international ST11 NDM-1 strains, especially from Greece . The witfimA-K operon, responsible for the type 1 fimbriae involved in adhesion, and the chromosomal mrkABCDF and mrkHIJ operons, responsible for the type 3 fimbriae, involved in adhesion and biofilm formation. Additional groups of virulence genes considered included those for iron secretion systems: the fyuA, irp1/2, and ybtAEPQSTUX genes specifying synthesis and secretion of yersiniabactin; iroE specifying synthesis of salmochelin; and the ybdA, entABCEF, fes, and fepABCDG genes specifying synthesis and secretion of enterobactin. The same virulence gene profile was found in close relatives of the outbreak isolates from Poland, Greece, Romania, Norway, and Turkey.Virulence-associated gene analysis  showed aKlebsiella pneumoniae (Kp) is responsible for many nosocomial infections, especially in immunocompromised patients [patients ,57. Hosppatients . The Kp In some patients, colonization is associated with carrier status for multi-drug-resistant strains. UTIs, ABU, and colonization/carriage are a serious problem as they are mainly responsible for the global expansion of carbapenemase-producing Kp clones .Among our patients were RTx recipients with symptomatic UTI, patients with asymptomatic bacteriuria, and patients with GI tract colonization. All these patients had a history of either chronic or kidney failure or acute kidney injury and were hospitalized in one hospital, and from each of them, Kp NDM-1 was isolated.In patients with kidney failure, including RTx recipients, the choice of antibiotics can be limited by impaired renal function and the possibility of interaction with other drugs; it is very difficult to determine an optimal prophylaxis . Many anIn recurrent UTIs, the probability that a carbapenem-resistant strain will be the etiological factor increases with each subsequent episode . The useThe causes of ABU can be explained in two ways. On one hand, uropathogenic strains are less virulent, or some genes are not expressed because of mutations . According to Zdziarski et al., asymptomatic bacteriuria is a result of genome reduction and inactivation of genes encoding virulence-associated factors by the accumulation of point mutations or deletions . AttenuaK. pneumoniae strains [In recent years, there have been numerous reports of epidemic outbreaks caused by MDR  strains ,66,67. TAll strains showed an identical or similar resistance profile. Only for imipenem, amikacin, and tigecycline were differences in antibiotic resistance noticeable. These differences were also reflected in the genotypes and sub-genotypes of these isolates. These slight differences in genotypes and resistance profiles are associated with the progression of the isolation timeline of the Kp NDM-1 strains. To answer the question of how much variability exists at the genomic level among Kp NDM strains, we used WGS to determine the level of genetic relatedness among our isolates and between these and other strains from worldwide sources.We have shown that our carbapenem-resistant isolates belong to the NDM-1 variant. The NDM-1 variant is considered a pandemic and has been recorded in as many as 86 countries around the world, including in Poland ,69,70.Phylogenetic analysis with cgMLST indicated high homogeneity with respect to sequence type (ST). All of our isolates belong to the lineage ST11, whose role in the hospital environment is significant. It has been shown that elements of ST11, together with ST340, participated in the evolution of the clonal complex CC258, which has global dissemination ,72. Kp cAnalysis of the cgSNP profiles indicated 7–88 SNP differences between the individual Polish isolates. The first isolate that appeared in 2018 on the ward was the least different from Greek strains, and the isolates reveal the phenomenon of an increase in SNP differences from Greek strains over the course of the epidemic situation, in line with the diversification of the population. Although related Kp ST11 strains belonging to the same basal clade occurred globally , the anaThe rapid spread of Kp NDM strains to all continents is largely related to the global development of tourism . TherefoThe virulence properties of Kp have been documented in many reports ,74,75. CWGS analysis showed the same virulence profile of genes for all isolates, regardless of each sample’s origin (ABU/UTI/GI tract). Based on the type of K24 capsule, LPS o2v1 antigen, and type 1 and 3 fimbriae were detected for our isolates. The K24 capsule type is found in Polish ST11 NDM-1 strains and some strains from Greece . Many obKp acquire iron through a system based on genes encoding siderophores such as yersiniabactin, salmochelin, and enterobactin, enabling the high virulence of multi-drug-resistant Kp NDM strains. The presence of the yersiniabactin high-pathogenicity island contributes to high virulence in our isolates. This type of siderophore is mainly characteristic of Kp strains responsible for the colonization of the respiratory system ,80,81, bBased on WGS, we found no differences in virulence profile across the strains responsible for UTI or ABU, and for isolates colonizing the gastrointestinal tract. The virulence of Kp probably does not determine the appearance or absence of symptoms of the disease. However, further research is required to examine strains from a larger population.blaNDM-1, blaKPC-2, blaVIM, blaIMP, and blaOXA-48 genes are responsible for resistance to carbapenems, and are transferred to other microorganisms via horizontal gene transfer [blaNDM-1gene and other genes encoding resistance to carbapenems, e.g., blaOXA-48, has been reported in the same bacterial cell [Besides clonal transmission, mobile genetic elements, including transposons and plasmids, play a role in the dissemination of multidrug-resistant clones of Kp. The transfer . The co-ial cell . Additioial cell ,86.blaNDN-1gene were also carriers of genes related to resistance to antibiotics belonging to different classes: β-lactams , aminoglycosides (Aac (6′)-Ib, aadA1), phenicols , sulfonamides (sul1), and trimethoprim (dfrA1/dfrA14). Various types of plasmids such as IncR, IncF, IncA/C, and IncNIncL/M are involved in the carriage of the blaNDM-1 gene [In our research, Kp NDM-1 isolates carrying the M-1 gene . ConcernrmpA and rmpA2 genes, which is typically associated with Kp (hvKp) strains with a hypermucoviscous phenotype [Mobile genetic elements, e.g., integrative conjugation elements (ICEs), are widely recognized as contributing to the spread of antimicrobial resistance and virulence genes included in islands of pathogenicity. ICEKp1 and ICEKp2 can co-exist in Kp, and were documented during our global analysis of Kp NDM-1. ICEKp2 is often detected in the presence of a plasmid carrying the henotype ,56. Desphenotype .In the case of renal transplant recipients, UTI is common because long-term immunosuppression is associated with many side effects, particularly an increased risk of infection. Organ recipients are subjected to preventive antibiotic therapy before and after transplant surgery, in order to minimize the risk of complications. Antibiotic therapy may alter the urinary microbiome composition and enrich for uropathogenic species . For chrIn addition, this group of patients is particularly exposed to hospital infections; therefore, it was necessary to analyze the epidemic situation in the hospital ward.Asymptomatic bacteriuria is also a significant problem. We ask ourselves whether or not to treat asymptomatic bacteriuria, especially considering that treatment of asymptomatic bacteriuria with targeted or empiric antibiotic therapy leads to the formation of multi-drug-resistant strains, e.g., producing carbapenemase.Researchers are constantly looking for differences in strains that cause symptoms of UTI and those associated only with the carrier and ABU. Hence our interest in Kp NDM strains isolated not only from UTI, but also from ABU and GI tract colonization.The aim of the study was the phenotypic and genotypic characterization of Kp strains producing metallo-β lactamase NDM, isolated from patients with kidney diseases and RTx recipients. We used WGS analysis to assess the genome diversity of local isolates in relation to national and global strains of Kp NDM-1 with the sequence type ST11. The correlation between strains was determined using PCR MP genotyping, confirming the presence of local outbreak and strains unrelated to the epidemic situation. Mobile genetic elements, in particular, integrative conjugative elements, are responsible for the dissemination of multi-drug resistance and virulence. Meta-analysis of genomes did not show signatures of UTI- and ABU-associated strains with high antimicrobial resistance. Colonization and immunosuppression can be additional risk factors, although this is seldom reported. In immunocompromised patients or those treated with antibiotics for prevention, the gut or ABU can act as reservoirs for the transmission of Kp NDM in the hospital setting.


Human parvovirus B19 (B19) can cause acute hepatitis and is attributed to the high mortality of alcoholic hepatitis (AH). B19 infection is generally self-healing in previously healthy people, but it can cause fatal effects in some high-risk groups and increase its virulence and infectivity. Disseminated B19 infection-induced multiple organ dysfunction syndrome (MODS) in patients with AH has not been reported yet. Here, we described B19 viremia in an adult patient with AH accompanied by hemolytic anemia (HA), leading to disseminated infection and secondary MODS, as well as self-limiting B19 infections in seven nurses caring for him. Meanwhile, we reviewed the literature on AH and B19 infection.B19 infection. After methylprednisolone, intravenous immunoglobulin (IVIG), empirical anti-infection, and supportive treatment, the lung infection improved, but hematopoietic and liver abnormalities aggravated, and systemic B19 infection occurred. Finally, the patient developed a refractory arrhythmia, heart failure, and shock and was referred to a local hospital by his family on May 8, 2021. Unfortunately, he died the next day. Fourteen days after he was transferred to MICU, seven nurses caring for him in his first two days in the MICU developed self-limiting erythema infectiosum (EI).A 43-year-old male patient with AH accompanied by HA was transferred to the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, on March 31, 2021. After supportive treatment, his transaminase and bilirubin levels were reduced, but his anemia worsened. He received a red blood cell (RBC) infusion on April 9 for hemoglobin (Hb) lower than 6 g/dl. On April 13, he suddenly had a high fever. Under empirical anti-infection, his high fever dropped and maintained at a low fever level; however, his anemia worsened. On April 25, he was transferred to the medical intensive care unit (MICU) due to severe pneumonia, acute respiratory distress syndrome (ARDS), acute aplastic crisis (AAC), and hemophagocytic syndrome (HPS), which were subsequently confirmed to be related to B19 infection is self-limiting in healthy people, with low virulence and infectivity; however, in AH patients with HA, it can lead to fatal consequences and high contagion. B19 infection has been well reported to be associated with acute hepatitis and fulminant liver failure and may underlie secondary HA, which is an important reason for the increased short-term risk of death from AH  . Approxi from AH –5.B19 is a small, non-enveloped, single-stranded DNA virus of the Erythroparvovirus genus within the Parvoviridae family (B19 is that it has a significant tropism to primary human erythrocyte progenitor cells (EPCs) in the bone marrow after respiratory transmission  in the general population ranges from 2% to 21% in children (1–5 years), 30%–40% in adolescents (15 years), and 40%–60% in young adults (20–40 years) and reaches a maximum in the elderly, with a prevalence of over 90%  in adults, and hydrops in pregnant women , acute pure red cell aplasia (PRCA), and/or aplastic crisis (AC) , respiratory diseases , kidney diseases (acute glomerulonephritis and nephropathy syndrome), heart diseases [myocarditis or heart failure (HF)], and nervous system involvement, especially in patients with abnormal hematological parameters or immune status  presented to our hospital. He complained of fatigue, bad appetite, dark urine, and yellowish sclerae for 1 month. He neither had fever nor any other disease history, including hereditary or familial clustered infectious diseases.2) under room air 98%, clear consciousness, dull face, palpebral conjunctival pallor, moderate scleral icterus, normal breath sounds in both chest regions, normal heart sounds, mild liver and spleen enlargement, abdominal distension with shifting dullness, and liver palms . Laboratory studies revealed progressive thrombocytopenia, anemia with elevated reticulocytes (Ret), and liver damage compared with those at local hospitals  104/68 mmHg, heart rate (HR) 80 beats/min, oxyhemoglobin saturation by pulse oximetry . From this day to the 25th day of admission, he received meropenem instead of cefoperazone/sulbactam sodium as an empirical antibacterial treatment, and his highest body temperature dropped from 39.6°C to 38.2°C after 2 days, and then his fever was maintained at a low level  showed arterial oxygen partial pressure (PaO2) of 91.9 mmHg and arterial partial pressure of carbon dioxide (PaCO2) of 32.6 mmHg under low-flow nasal cannula therapy. Blood laboratory tests found that WBC, CRP, PCT, and galactomannan did not change much, and LDH and ferritin increased to 3,038 mg/dl and 1123 ng/ml, respectively. Serum IL-6 and beta-1,3-glucan were much higher than before, and the Ret% decreased from the original abnormally increased level to within NR  and HPS , on the second day in MICU, we performed a bone marrow smear and pathological biopsy on the patient. The results indicated the diagnosis of AAC with HPS  of 83%, PaCO2 of 41 mmHg, alveolar-arterial oxygen gradient (A-aDO2) of 546, and lactic acid (Lac) of 8.7 mmol/L. The APACHE-II and SOFA scores increased to 32 and 28, respectively. Severe pneumonia and ARDS were newly diagnosed, and endotracheal intubation for invasive mechanical treatment was performed. Infection by B19 mixed with other pathogens in his lung was suspected. Smears, culture, and NGS of bronchoalveolar lavage fluid (BALF) were analyzed. His oxygenation did not improve under very high parameters of invasive mechanical ventilation (synchronized intermittent mandatory ventilation (SIMV): Vt 5 ml/kg, positive end-expiratory pressure (PEEP) 15 cmH2O, Ps 16 cmH2O, and FiO2 90%), so prone ventilation was administered after adequate analgesia, sedation, and muscle relaxation. After 12 h of prone ventilation, his oxygenation improved, and ventilatory parameters decreased gradually. ABG showed that the Lac reduced to a normal level of 1.7 mmol/L, oxygenation improved significantly, and ventilatory parameters were decreased to a lower level .On the third day in the MICU, the patient experienced significantly worsening shortness of breath with profuse sweating, HR of 130 beats/min, respiratory rate of 35 breaths/min, SpO2O, Ps 12 cmH2O, and FiO2 44%), and ABG showed good ventilation and oxygenation of the lungs . The previously increased transaminase, bilirubin, IL-6, and beta-1,3-glucan levels gradually decreased, but Hb, PLT, Ret count, and Ret% were still at very low levels    to maintain BP at 77–110/22–77 mmHg. His family gave up, and the patient was transferred to the local hospital, where he died the next day Figure 3B19-associated acute hepatitis should be differentially diagnosed.From his onset to the 14th day of admission to our hospital, the patient mainly manifested liver injury and blood system damage (hyperbilirubinemia manifested as indirect bilirubin (IBIL) > direct bilirubin (DBIL), aspartate aminotransferase (AST) > alanine aminotransferase (ALT), coagulation dysfunction, thrombocytopenia, anemia with Ret significant increase in count) (http://www.mayoclinic.org/girst/mayomodel10.html), the ANI score of this patient was 6.875, and the probability of ALD was 99.9%. The MELD score of this patient was 42 at admission, and 90-day mortality rate was 92%, which suggested that this patient was with severe AH.Alcoholic liver disease covers a spectrum of disorders beginning with fatty liver and progressing at times to AH; more severe cases lead to liver failure and are diagnosed predominantly on clinical history, physical examination, and laboratory testing , 25. AltB19 infection can explain the patient’s illnesses from manifestations at disease onset to later-confirmed hepatosplenomegaly, liver damage, thrombocytopenia, and HA, it cannot be concluded that B19 infected him at the beginning because of a lack of tests related to B19. However, we can be sure that B19 caused the patient’s persistent high fever, sudden respiratory distress, and hypoxemia that required his transfer to the MICU and also his subsequent deterioration. ELISA confirmed antibodies against B19 IgM and IgG in his serum specimen (specimen collected on his first day in the MICU), and NGS detected high B19 viral loads in the specimens of his blood  and tumor necrosis factor alpha (TNF-α) by recruiting striking CTLs21 that could trigger an autoimmune response, which can lead to autoimmune anemia , placental trophoblastic cells, and some megakaryocytes cells, could explain the ability of B19 to cause multiorgan diseases  N-terminus of the VP1 capsid protein. VP1u interacts with some unknown coreceptor for subsequent internalization. The VP1u region (without a capsid) is efficiently internalized by B19-permissive cells, which suggests that the primary interaction of B19 with the P antigen is required only for externalization of VP1u. Mature RBCs also express P antigen and hence show primary attachment to B19, but the virus is not internalized. It is presumed that this primary interaction may be responsible for the systemic dissemination of the virus , the patient should have been clearly diagnosed with disseminated B19 infection.There is no specific treatment for nsfusion . Under tnecrosis . The clinecrosis . MRI is necrosis . CombineB19. Due to the formation and deposition of immune complexes in the skin after B19 infection, erythema infectiosum usually appears 2 weeks after the initial infection  can be found in the article/All clinical data in this case report were either provided by the patient or collected by our team members from the patient with his consent. There was no additional invasive test performed or experimental drugs used incautiously on the patient. Written informed consent was obtained from the patient for participation in the study and the publication of this report in accordance with the Declaration of Helsinki. The case report is exempt from institutional review board approval.JL is the patient’s attending physician, responsible for the management of the patient's entire disease process in the MICU, the conception and writing of this article. JZ is patient’s resident, and is also responsible for the collation of the patient's image data and the editing of the figures in this article.WL is a doctor in the blood laboratory who was responsible for the reading and reporting of the blood and bone marrow smears of this patient. SW and LZ were the nurses in charge of the patient. In addition to clinical care of patient, they were also responsible for recording patient’ clinical data, collecting experimental data related specimens  and providing pictures of patients’ rashes. YS, JB, JH and YW were doctors involved in the diagnosis, treatment, clinical and experimental data interpretation of the patient, and provided suggestions for the conception of the article. LL was responsible for the revision and editing of this article and the PCR detection of some specimens. BW is the PI of the team and the director of the MICU. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Third Affiliated Hospital of Sun Yat-sen University for the Cultivate Special Funding Projects of 2020 National Natural Science Foundation of China (2020GZRPYQN26).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.


Helicobacter pylori infection, especially in the early phase of infection, by promoting IgA production.The stomach has been thought to host few commensal bacteria because of the existence of barriers, such as gastric acid. However, recent culture-independent, sequencing-based microbial analysis has shown that the stomach also harbors a wide diversity of microbiota. Although the stomach immune system, especially innate lymphoid cells (ILCs), has not been well elucidated, recent studies have shown that group 2 ILCs (ILC2s) are the dominant subtype in the stomach of both humans and mice. Stomach ILC2s are unique in that their existence is dependent on stomach microbiota, in sharp contrast to the lack of an impact of commensal microbiota on ILC2s in other tissues. The microbiota dependency of stomach ILC2s is partly explained by their responsiveness to interleukin (IL)-7. Stomach ILC2s express significantly higher IL-7 receptor protein levels on their surface and proliferate more in response to IL-7 stimulation in vitro than small intestinal ILC2s. Consistently, the stomach expresses much higher IL-7 protein levels than the small intestine. IL-5 secreted from stomach ILC2s promotes immunoglobulin (Ig) A production by plasma B cells. In a murine model, stomach ILC2s are important in containing  Helicobacter pylori, a bacterium found in the stomach that can lead to gastric upset and, if left untreated, ulcers or stomach cancer. Unlike the intestines, the stomach was thought to contain few bacteria owing to its high acidity, but according to new studies, the stomach harbors its own set of microbiota. Hiroshi Ohno and Naoko Satoh-Takayama at the RIKEN Center for Integrative Medical Sciences and Yokohama City University, Japan, review how gastric microbiota affects development of immune cells in the stomach. They report that the stomach’s unique microbiota influences maturation of particular immune cells, which may be crucial in fighting off H. pylori infection. These findings illuminate a new area in microbiome research.The stomach’s microbiota, and its effect on immune development, may be key factors in fending off infection with  Gut microbiota contribute to gut maturation3, host nutrition, and pathogen resistance4. By contrast, it has been thought that there are fewer commensal bacteria in the stomach due to the characteristic feature of the gastric barrier, such as the highly acidic conditions of the lumen due to acid-secreting parietal cells and the presence of proteolytic enzymes (pepsins) from pepsinogen-secreting zymogenic chief cells6. The impact of gastric microbiota on host pathophysiology is not well understood compared to the microbiota of the intestine. Compared to the intestine, where evidence has accumulated that it has evolved the unique gut-associated immune system to deal with gut microbiota and orally ingested pathogens8, studies on the stomach immune system are less common. However, recent studies have shown that wide bacterial diversity can be found in the human stomach as well9,10,11.The surfaces of an animal’s body, such as the skin and mucosa, provide a boundary between the body and the surrounding environment and are colonized by numerous commensal bacteria, called microbiota. In particular, the intestinal mucosa hosts the largest microbial community, of 40 trillion bacteria, in the bodyHelicobacter pylori infection.Here, we will review the recently identified characteristics of stomach group 2 innate lymphoid cells (ILC2s), especially their relationship with gastric microbiota and 11. In individuals who have neither of the two major gastric microbiota-perturbing events, H. pylori infection nor proton pump inhibitor (PPI) usage5, it has been shown that the microbiota in the stomach (and the duodenum) possesses a significantly lower number of bacteria than that of the lower gastrointestinal tract, which is probably correlated with the lower luminal pH in these organs6. The major phyla are Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria throughout the human gastrointestinal tract11. In contrast to the lower intestinal tract (and feces), where Firmicutes are dominant, followed by Bacteroidetes12, Firmicutes, and Proteobacteria are dominant in the stomach and the duodenum10. At the genus level, Streptococcus and Prevotella are the two major genera detected in the stomach10, which are minor genera in human feces, considered to reflect their decreased presence in the lower gastrointestinal tract12.Recent culture-independent analyses of microbial composition with 16 S rRNA sequencing have shown wide bacterial diversity in the human stomachH. pylori infection perturbs the gastric microbiota5. H. pylori is a Gram-negative bacterium categorized as a pathobiont bacteria that colonizes the gastric mucosa of half or more of the human population in the world, ranging from 20% in developed countries to >90% in developing countries14. H. pylori colonizes the gastric mucosa using virulence properties, such as vacuolating cytotoxin, VacA and cytotoxin-associated gene A, cagA, and causes acute and chronic gastritis, eventually progressing to more severe disorders, including peptic ulcer disease and gastric cancer15.As mentioned above, H. pylori needs to overcome harsh acidic conditions in the stomach lumen to colonize and establish infection in the stomach13. It has been well documented that H. pylori infection occurs in early childhood, but rarely in adults13. H. pylori infection is basically acquired in childhood and persists throughout life without eradication therapy17. One of the possible explanations is that the ontogenetic development of the stomach in early childhood life is thought to be associated with the alleviation of gastric hostility, including achlorhydria, compared to adulthood18. Gastric hostility in adults sufficiently inhibits H. pylori infection. Therefore, H. pylori infection rarely occurs in adulthood, and when it does, it likely causes acute gastritis and is then eradicated20. H. pylori possesses urase and other enzymes that produce ammonia, which aid its infection by increasing the pH19.H. pylori infection causes atrophic gastritis with a loss of parietal cells13. This leads to increased gastric pH and could modulate the gastric microbiota. The impact of H. pylori infection on the gastric microbiota has been studied. Although the majority of the gastric microbiota is occupied by H. pylori, its presence does not largely affect the composition of the gastric community9. H. pylori infection-associated gastric microbiota dysbiosis has been implicated in the pathogenesis of gastric diseases22, but the understanding of the complex interaction between the microbiota and H. pylori still needs to be further addressed in the development of gastric diseases.Chronic 5. PPIs suppress gastric acid secretion, are among the most commonly prescribed drugs and are widely used to treat gastric acid-related disorders, such as gastroesophageal reflux disease and peptic ulcers24. PPIs reduce bactericidal gastric acid secretion and lead to a significant change in the overall microbiota composition23. In the stomach, with PPI treatment, a greater diversity of bacterial species is observed, and the relative abundance of the phyla Firmicutes and Fusobacteria  is increased11.PPIs are another gastric microbiota modifier26. ILC1s used to include all ILCs producing interferon-γ; however, it is now common to say that ILC1s are distinct from NK cells, whose differentiation is regulated by E4BP4 and Eomes26. ILC3s are known to produce interleukin (IL)-17 and IL-22 upon stimulation with IL-1β or IL-23; they are subdivided into (i) lymphoid tissue-inducer cells, which express CCR6 and possess lymphoid tissue-inducing ability; (ii) NKp46-ILC3s, which express NKp46 ; and (iii) CCR6−NKp46−ILC3s, which do not belong to (i) and (ii)27. ILC3s are basically distributed in tissues facing the external environment, especially the gastrointestinal tract, and play a role in defense mechanisms against pathogens27. Although the relationship between ILCs and gut microbiota has not been well understood until recently, recent advances in ILC biology, including single-cell analysis, have unveiled the details of the relationship28.ILCs are largely classified into three groups based on the characteristic expression of transcription factors and cytokine production Fig. 25,26. IL31. ILC2s produce type 2 cytokines, such as IL-5, IL-9, and IL-13, upon stimulation with IL-25, IL-33 or TSLP, and are involved in the eradication of parasite infection31. Recent studies have attracted attention on ILC2s by showing that these cells are involved in the pathogenesis of diseases, including fibrosis, asthma, and obesity36. In these diseases, the common scheme of pathogenesis is that an alarmin, IL-33, released upon epithelial cell damage by allergens, inflammation, or stress responses, triggers the activation of ILC2s. IL-33-activated ILC2s secrete IL-4, IL-5, and IL-9, which, in turn, induce eosinophils and activate mast cells, while IL-13 from ILC2s induces fibrosis39. In addition, it has been reported that ILC2s produce not only cytokines, but also opioid methionine-enkephalin peptides to regulate adipose function and metabolic homeostasis by acting directly on adipocytes35. In contrast to ILC3s, commensal microbiota do not seem to be involved in the induction or differentiation of ILC2s41. These observations were made with ILC2s in adipose tissues, the intestine, and the lung, which is in sharp contrast to the microbiota dependency observed with stomach ILC2s as described below.ILC2s were independently identified in 2010 by three groups42. In the stomach, ILC2s are dominant, with a few ILC1s, and the stomach is almost devoid of ILC3s. This distribution is in sharp contrast to that observed in the intestine, where comparable numbers of the three ILC subsets exist. ILC2 dominance in the stomach has also been reported in humans43.Studies on ILCs have mostly been performed in the intestine and lung, but information on the distribution and function of ILCs in the stomach is still lacking. We have recently examined ILC subsets in the murine stomach42, unlike ILC2s in the other tissues/organs. In germ-free (GF) mice, the number of stomach ILC2s was significantly decreased  conditions  2 cells are the main source of IL-5, ILC2s have recently been established as another source of robust IL-5 secretion46. We have shown that microbiota-induced ILC2s are required as a major source of IL-5 for the induction of IgA+ plasma B cells, as well as efficient IgA secretion from these cells in the stomach  A coats the surface of commensal bacteria to prevent them from attaching to and invading the intestinal epitheliumS24-7 family within the order Bacteroidales are suggested to be candidate bacteria for ILC2 induction42. Our preliminary results indicate that there seems to be differential ILC2-inducing ability among several S24-7 clones, and further studies should clarify the molecular mechanisms underlying bacterial induction of stomach ILC2s.Among stomach microbiota, members of the H. pylori infection24. PPIs reduce bactericidal gastric acid secretion and lead to a significant change in the overall microbiota composition23. In the stomach, with PPI treatment, a greater diversity of bacterial species is observed, and the relative abundance of the phyla Firmicutes and Fusobacteria  is increased11. This change in gastric microflora composition might affect the number of ILC2-inducible bacteria and, thus, the number of ILC2s in the stomach, which, in turn, can alter the composition of the gastric microbiota.PPIs suppress gastric acid secretion and are among the most commonly prescribed drugs, widely used to treat gastric acid-related disorders, such as gastroesophageal reflux disease and peptic ulcer and, sometimes, H. pylori infection, especially in the acute phase, is accompanied by a mucosal IgA response, although the T-cell response is predominantly Th1 type16,48. In a murine H. pylori infection model, we recently identified that stomach ILC2s are the source of IL-5 for IgA production in the early phase of H. pylori infection (Fig. 42. The expansion of IL-5-receptor-expressing plasmablasts is initiated by ILC2 activation, which is indispensable to restrain H. pylori infection. This is also supported by the fact that a significant reduction in plasma B cells and plasmablasts, as well as a reduction in H. pylori coated with IgA, was observed in mice with ILC2-specific deficiency. IgA-mediated H. pylori elimination has a strong correlation with the restraint of gastric inflammation, which is prominently diminished in mice lacking ILC2s42. It has also been shown that the Th2 cell-dependent IgA response is induced for mucosal protection in the late phase of infection (Fig. 42.ion Fig. 42. The eion Fig. 42.Fig. 3H. pylori infection is often acquired in childhood and persists throughout life without eradication therapy46, while H. pylori infection rarely occurs in adulthood, and when it does, it causes acute gastritis and is then eradicated48. Since early childhood is a particularly vulnerable period as the immune system is still developing, one bold hypothesis is that not enough ILC2 development occurs to eradicate H. pylori infection in childhood, although there is no evidence to support this notion at this time; further studies are awaited to clarify whether this hypothesis is true.As mentioned above, H. pylori infection. H. pylori can evade or disturb innate and adaptive immune responses via multiple mechanisms to establish chronic infection, leading to clinical gastroduodenal disorders, including chronic gastritis, peptic ulcers, and two malignancies—gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Therefore, it is important to understand the stomach immune system, including ILC2s, in more detail to develop novel treatment strategies to contain its infection.It is now clear that the stomach is not a totally sterile and immunologically silent organ, but instead harbors a wide diversity of commensal microbiota with unique composition and a unique immune system within the gastrointestinal tract. In particular, it possesses ILC2s with characteristics that are distinct from their counterparts in other organs/tissues. The stomach ILC2s are dependent on the presence of stomach microbiota, and they can modulate the composition of the microbiota by promoting IgA production. Stomach ILC2s are also important in