Cancer is one of the most serious human diseases, causing millions of deaths worldwide annually, and, therefore, it is one of the most investigated research disciplines. Developing efficient anticancer tools includes studying the effects of different natural enzymes of plant and microbial origin on tumor cells. The development of various smart delivery systems based on enzyme drugs has been conducted for more than two decades. Some of these delivery systems have been developed to the point that they have reached clinical stages, and a few have even found application in selected cancer treatments. Various biological, chemical, and physical approaches have been utilized to enhance their efficiencies by improving their delivery and targeting. In this paper, we review advanced delivery systems for enzyme drugs for use in cancer therapy. Their structure-based functions, mechanisms of action, fused forms with other peptides in terms of targeting and penetration, and other main results from in vivo and clinical studies of these advanced delivery systems are highlighted. Enzymes leading to cell damage or death have garnered significant attention since they play key roles in cell metabolism. Cytotoxic enzymes appear to be efficient tools to combat cancer. Ideally, one molecule of a protein toxin would be enough to induce the death of a single cell. However, a number of reasons hamper the utilization of cytotoxic enzymes. Modern approaches to drug delivery and genetic engineering have led to the current stage of their development. The fundamental action of potential cytotoxic enzymes has been highly scrutinized, and studies on these have yielded significant results. Unfortunately, only few of these enzymes have reached clinical stages. Here, we introduce a few mechanisms of action of enzyme drugs currently used in cancer therapies .Many of the most efficient toxic enzymes play a significant role in key processes such as protein synthesis; they transduce damage in cell metabolism and amplify it from a single point. Ribosome-inactivating proteins (RIPs) are of these key proteins of plant origin that function to inhibit ribosomal function . Well-kn2O2. A change in the pH of culture medium or the quality and quantity of substrates derived from their activities can cause oxidative stress and cytotoxicity, thus leading to a disruption in cell metabolism . O. O3O4/GO+3 ions and tannic acid (TA) (MPN)  has been efficiently used to target mesothelin, a 40-kD tumor differentiation antigen  Table 5Table 5. A recombinant immunotoxin, termed RFB4(dsFv)-PE38 (BL22), reached the second phase of clinical trials for hairy cell leukemia (HCL) by 2008 . The potCD22-targeting is a widely used strategy that has provided exotoxin with enhanced efficacy . Both inThe fusion of the interleukin-4 (IL-4) sequence with truncated PE38 represents another immunotoxin that has reached clinical stages. It was used against IL-4-expressing glioblastoma  as an inThe overexpression of ErbB2 in cancer cells was utilized to develop a PE-fused immunotoxin with the Fv fragment portion of the e23 monoclonal antibody . ResultsEGFR binding ligand is another useful ligand that has been used in fusion to PE 38 exotoxin . This stP. aeruginosa exotoxin discussed the PE-based immunotoxins that have reached the second and third phases of clinical trials [The most recent review paper on l trials . Some ofl trials . The maxl trials . Thus, tThe fusion of PE with scFv, which is an anti-epithelial cell adhesion molecule, has been developed by Viventia Biotech Inc. and has passed phase II, III clinical trials in patients with head and neck squamous cell carcinoma and bladder cell carcinoma .Corynebacterium diphtheriae [DT is a 58-kD protein secreted by htheriae . In the htheriae . The trahtheriae . Studieshtheriae . A low pImproving strategies for the use of DT includes both obtaining its conjugates with antibodies and fusing it with other peptides or fragments of proteins . One of In a number of cases, DT has been expressed in targeted cells by delivering genes using different approaches. For example, Adeno-associated virus vector expressing DT A chain was found to efficiently inhibit pancreatic carcinoma cells in vitro . Toxic eThe delivery of DT A genes has reached clinical stages in a few cases. DNA plasmid for a gene regulated by the H19 promoter sequence (BC-819) has made it to phase II clinical trials for use in cases with invasive bladder tumor . The resThe next stage of the development of DT-based prodrug forms involved fusing it with targeting sequences. In one work, T22-DITOX-H6 and T22-PE24-H6 sequences were developed by Geneart (Thermofisher), and recombinant proteins were obtained on this basis to target CXCR4-expressing cells . T22 is The cell viability levels of Panc-1 human pancreatic cancer cells and HeLa cervical cancer cells treated with T22-DITOX-H6 and T22-PE24-H6 recombinant fusions reduced by more than half compared to controls, while exotoxin fusions showed higher efficiency. The administration of T22-DITOX-H6 inhibited tumor growth in mice bearing colorectal cancer cells by more than 5-fold compared to buffer-treated controls. T22-PE24-H6 administration led to a 2.3-fold reduction in colorectal cancer cells. Furthermore, T22-DITOX-H6 has been shown to induce apoptosis in CXCR4-expressing leukemia cells . In an aThe fusion of DT with VEGF was created to target the vasculature associated with pancreatic cancer  Table 6Table 6. Protein toxins have been shown to be efficient anticancer drug tools. Ideally, a small number of molecules have been shown to be enough to cause the death of cancer cells. This feature provides protein toxins advantages over lower molecular weight drugs. Their application, however, is hampered by a number of obstacles such as being attacked by immune cells, their heavy weights and bulky volumes, aggregation during storage, and low productivity. Therefore, different approaches have been used to improve their efficiencies, including fusion with peptides that provide targeting and penetrating functions, encapsulation in liposomes and nanoparticles, different releasing options that activate at lower pH, linking  with transport proteins such as albumin or lactoferrin, providing their expression in targeted cells by delivering appropriate genes, and utilizing physical-stimuli responsive systems. These different modifications have resulted in some of these toxins reaching the market. Ongoing studies should focus on the application of these protein toxins with other drug forms that might provide synergistic effects.


Vaccination against SARS-CoV-2 has been conducted frequently to limit the pandemic but may rarely be associated with postvaccinal autoimmune reactions or disorders.We present a 35-year-old woman who developed fever, skin rash, and headache 2 days after the second SARS-CoV-2 vaccination with BNT162b2 (Pfizer/Biontech). Eight days later, she developed behavioral changes and severe recurrent seizures that led to sedation and intubation. Cerebral magnetic resonance imaging showed swelling in the (para-) hippocampal region predominantly on the left hemisphere and bilateral subcortical subinsular FLAIR hyperintensities. Cerebrospinal fluid analysis revealed a lymphocytic pleocytosis of 7 cells/μl and normal protein and immunoglobulin parameters. Common causes of encephalitis or encephalopathy such as viral infections, autoimmune encephalitis with well-characterized autoantibodies, paraneoplastic diseases, and intoxications were ruled out. We made a diagnosis of new-onset refractory status epilepticus (NORSE) due to seronegative autoimmune encephalitis. The neurological deficits improved after combined antiepileptic therapy and immunomodulatory treatment including high-dose methylprednisolone and plasma exchange.Although a causal relationship cannot be established, the onset of symptoms shortly after receiving the SARS-CoV-2 vaccine suggests a potential association between the vaccination and NORSE due to antibody-negative autoimmune encephalitis. After ruling out other etiologies, early immunomodulatory treatment may be considered in such cases. Adverse effects of SARS-CoV-2 vaccination with mRNA vaccines are rare and mostly limited to local reactions or slight systemic symptoms  includinA heterogeneous group of sporadic autoimmune encephalitis cases associated with SARS-CoV-2 infection has also been reported . AdditioHere, we describe a case of new-onset refractory status epilepticus (NORSE) due to autoantibody-negative autoimmune encephalitis in close temporal association with the second SARS-CoV-2 vaccination dose of BNT162b2 (Pfizer/Biontech).A 35-year old woman was referred to our center with recurrent focal to bilateral convulsive seizures after an episode of fever, headache, and skin rash 6 days prior. Two days before the onset of fever, she received the second dose of an mRNA-based SARS-CoV-2 vaccine . The first dose was well-tolerated. The patient's medical history revealed only sporadic but not recent cocaine consumption and mild SARS-CoV-2 infection a year before the vaccination.The clinical, laboratory, and imaging findings are summarized in The electroencephalogram (EEG) initially showed a generalized rhythmic delta activity with superimposed multifocal interictal epileptic discharges mainly over the frontal and right-sided leads. Frequent brief electrographic seizures were also detected . InitialDue to recurrent convulsive seizures without regaining consciousness, a diagnosis of new-onset refractory status epilepticus (NORSE) was made. Levetiracetam up to 4 g/d and lacosamide up to 200 mg/d were started. Under continuous EEG monitoring, phenytoin up to 750 mg/d, midazolam up to 0.57 mg/kg/h, and ketamine up to 4 mg/kg/h were added. Phenytoin was later replaced by phenobarbital. Initially, acyclovir 3 × 10 mg/kg body weight/day and ceftriaxone were given until the CSF and serum testing for herpes viral and bacterial CNS infections turned negative. Immunomodulatory treatment with high-dose methylprednisolone  was started 2 days after hospital admission with subsequent slow tapering over 8 weeks. Because of continued seizures, plasma exchange for over 10 days starting on day 7 after the admission was performed.Subsequently, the patient's condition rapidly improved beginning on day 12 after the admission; she regained full consciousness and had only infrequent seizures. The follow-up MRI 1 month after the hospital admission showed edema reduction in the hippocampal, amygdala, and external capsule. On discharge, she had persisting moderate mnestic deficits and infrequent seizures (2–3/month). However, after 2 months, the patient was readmitted to our hospital because of deterioration in seizure frequency  and neuropsychological deficits. She again received high-dose methylprednisolone for 5 days, which led to significant reduction in seizure frequency.We report the first case, to our knowledge, of NORSE due to seronegative autoimmune encephalitis 8 days after vaccination with the mRNA SARS-CoV-2 vaccine. A causal relationship between vaccination and autoimmune encephalitis with NORSE is not established with certainty. Nevertheless, the onset of neurological symptoms 8 days following the vaccination suggests a potential association as well as the hyperintensities of the claustrum as a common finding in NORSE and autoimmune encephalitis , 10.Given the assumed low incidence of autoimmune reactions and their favorable outcome, benefits of vaccination far outweigh the risk of side effects. Importantly, SARS-CoV-2 infection itself may trigger a heterogeneous group of autoimmune encephalitis. Hence, the observation of SARS-CoV-2 vaccine-associated NORSE does not argue against the broad use of SARS-CoV-2 vaccines.Whether the patient's history of SARS-CoV-2 infection approximately 1 year before the encephalitis may have played a role remains unknown. Our findings should encourage clinicians to consider SARS-CoV-2 vaccination or infection as a potential trigger for autoimmune encephalitis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JW collected and interpreted the clinical and radiological data and was a major contributor in the writing and revision of the manuscript. IJ collected and interpreted the clinical and laboratory data and revised the manuscript. GB interpreted the clinical findings and revised the manuscript. MG interpreted the clinical and electroencephalographic findings and revised the manuscript. All authors read and approved the final version of the manuscript.This study was partly supported by the SNF  and the Loop Zurich, COVID-19 project (Title: SARS-CoV-2-Induced Immune Alterations and their Role in Post-COVID Syndrome).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.


Apart from the direct killing of cancer cells, cyclophosphamide-based chemotherapy has been shown to induce an antitumor immune response, and is being used in combination with immunotherapies in cancer care. We assessed the interaction of chemotherapy with immune biomarkers expressed on primary tumor tissue from a randomized phase III clinical trial, and confirmed that the presence of tumor-infiltrating lymphocytes is linked to improved survival in premenopausal women with high-risk breast cancer, regardless of their treatment allocation. However, immune biomarkers including tumor-infiltrating lymphocytes do not predict extra benefit from cyclophosphamide chemotherapy. This finding applies across the major molecular subgroups, including non-luminal and basal breast cancers that tend to be more immunogenic, and are often considered the most suitable subsets for receiving immunotherapy.p-value = 0.03), consistent with previous studies. However, none of the immune biomarkers predicted benefit from chemotherapy in the full study set nor within major breast cancer subtypes. Our study indicates that primary tumors with higher immune infiltration do not derive extra benefit from cyclophosphamide-based cytotoxic chemotherapy.Recent studies have shown that immune infiltrates in the tumor microenvironment play a role in response to therapy, with some suggesting that patients with immunogenic tumors may receive increased benefit from chemotherapies. We evaluated this hypothesis in early breast cancer by testing the interaction between immune biomarkers and chemotherapy using materials from DBCG77B, a phase III clinical trial where high-risk premenopausal women were randomized to receive chemotherapy or no chemotherapy. Tissue microarrays were evaluated for tumor-infiltrating lymphocytes (TILs) assessed morphologically on hematoxylin and eosin-stained slides, and by immunohistochemistry for CD8, FOXP3, LAG-3, PD-1 and PD-L1. Following REMARK reporting guidelines, data analyses were performed according to a prespecified statistical plan, using 10-year invasive disease-free survival as the endpoint. Differences in survival probabilities between biomarker groups were evaluated by Kaplan–Meier and Cox proportional hazard ratio analyses and prediction for treatment benefit by an interaction test. Our results showed that stromal TILs were associated with an improved prognosis (HR = 0.93;  Breast cancer is the most common malignancy in women worldwide . AdjuvanWith the advent of immunotherapy, the immune-modulating features of conventional cytotoxic chemotherapies have received considerable attention in recent years. Breast cancer is not a particularly immunogenic malignancy and therefore combining immune checkpoint inhibitors with immune-augmenting modalities such as chemotherapy is a strategy under active investigation; for example, pembrolizumab has been granted approval for use with chemotherapy in high-risk, early-stage triple-negative breast cancer . Other cSeveral studies highlight the important role of the immune microenvironment in response to therapies . Tumor-iTaken together, these studies suggest that pre-existing immune infiltrates in the primary tumor microenvironment might predict additional benefit from immune modulating chemotherapies such as cyclophosphamide. To test this hypothesis, we used archived excisional tumor materials from the Danish Breast Cancer Cooperative Group (DBCG)77B phase III clinical trial. This randomized trial has both chemotherapy and no chemotherapy arms in high-risk early breast cancer patients, thus providing an ideal setting to test our hypothesis . The priThe DBCG77B clinical trial included premenopausal women diagnosed with high risk, non-metastatic invasive breast cancer  during 1977–1983. Patients were randomized into four treatment arms receiving either intravenous cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), the single agent oral cyclophosphamide (C), levamisole (a non-chemotherapy antiparasitic drug), or no systemic therapy. All patients underwent mastectomy with axillary dissection (level I or II) and none of the patients received endocrine therapy as per the trial protocol. However, radiation to the chest wall and axilla was administered concomitantly with chemotherapy in the adjuvant setting to all enrolled patients . The levThis study received ethics approval from the Biomedical Research Ethics Committee of the Capital Region of Denmark and the University of British Columbia BC Cancer Research Ethics Board . Biomarkers were immunostained on existing tissue microarrays, previously constructed from surgically excised primary tumors. Each case was represented by 2–3 cores, measuring 2 mm in diameter, with the full trial cohort spread over 24 tissue microarray blocks. Tissue microarray slides were created by sectioning at 4 µm and staining with hematoxylin and eosin (H&E) or by immunohistochemistry (IHC) for CD8 , FOXP3 , LAG-3 , PD-1 , and PD-L1  as previously described [All biomarkers except for PD-L1 were stained at the Genetic Pathology Evaluation Centre  on the Ventana Discovery Ultra autostainer. PD-L1 was stained at the Deeley Research Centre  . StainedCD8, FOXP3, LAG-3, and PD-1 were scored by DG, and PD-L1 and stromal TILs on H&E-stained sections were assessed by ESS as trained pathologists blinded to the clinical data. The absolute number of positive cells per tissue core was reported for CD8, FOXP3, LAG-3, and PD-1 as intraepithelial TILs (iTIL) when they are located inside of carcinoma nests in close contact with the malignant cells, and stromal TILs (sTIL) as those present in the stromal tissue between epithelial nests. PD-L1 was reported as per the manufacturer’s protocol, as the percentage of the tumor area occupied by the immune cells expressing PD-L1 . MorpholFor each case with duplicate or triplicate cores, an average score was calculated. For IHC-assessed biomarkers, predefined cutoff points were locked down prior to data analyses based on those used in previous studies ,31,37,39The tissue microarrays from this trial have been previously stained and scored for other biomarkers including ER , PR , HER2 , CK5 , EGFR , and Ki67  for defining IHC-based breast cancer intrinsic subtypes, as previously published . Brieflyp-values are 2-tailed and are reported without adjusting for the multiple comparisons and a value of less than 0.05 was considered statistically significant. DBCG Statistical Office performed the statistical analyses using the SAS Enterprise Guide Version 7.15 software program package . Statistical analyses were independently performed by the DBCG Data Center using a hypothesis driven, prespecified statistical plan. The Chi-squared test was used to analyze the associations between biomarkers and clinicopathological features as well as differences between included and excluded patients (excluding unknowns). IDFS was defined as the end point indicating the probability of survival without invasive breast cancer-related events at contralateral, locoregional or distant sites, non-breast primary malignancies or death irrespective of the cause, and was estimated using the Kaplan–Meier method. Univariate and multivariate analyses were performed using Cox proportional hazards regression models to calculate the unadjusted and adjusted hazard ratios (HR) and their corresponding confidence intervals (CI). The following factors were considered in the multivariate modeling: age (<40 years vs. ≥40 years), tumor size , the number of involved axillary lymph nodes , histological type and grade , treatment regimen (chemotherapy (C + CMF arms) vs. no chemotherapy (control + levamisole arms)) and biomarkers in separate models. Invasion of the deep fascia was of no significance and was not included. No multivariate analysis was performed for the basal subset due to the limited number of events, and for the non-luminal subset the model was reduced. H&E sTIL were included as a continuous as well as a categorical variable, and other biomarkers were categorical. Functional form and proportional hazards assumptions were assessed by residuals and by including a time-dependent component in the model for each covariate. The hazard ratios for histological type and tumor grade were not proportional; therefore, stratification was used. For PD-L1, separate estimates were included according to the time since randomization. The Wald test for interaction was used to assess heterogeneity. All n = 681) in the current study set or excluded according to the original trial per protocol analysis can be seen in 709 of the patients included in per protocol analysis also had tissue available for this study. Cores with insufficient tissue or less than 50 invasive carcinoma cells were considered unevaluable for biomarker assessment and therefore excluded from the analyses. Differences in the baseline clinicopathological characteristics between the patients included  as defined by predetermined cutoff points . p-value = 0.009). Lower levels of CD8+ iTILs and LAG-3+ sTILs were more prevalent in smaller tumors (<20 mm). However, higher levels of FOXP3+ regulatory sTILs were seen in tumors smaller than 50 mm . Grade III tumors showed higher frequencies of TILs  but lower frequencies of PD-L1 negative immune cells when compared to grade I and II. By histological type, medullary breast cancers not surprisingly exhibited higher levels of CD8 iTILs and LAG-3 sTILs, whereas lobular carcinomas had lower levels of these immune cells compared to the other subtypes . ER-negative tumors displayed significantly higher levels of H&E sTIL, CD8 iTIL, LAG-3 sTIL, PD-1 sTIL and PD-L1 immune cells . Moreover, lower levels of H&E sTIL, and LAG-3 sTIL, and PD-1 immune cells were observed in patients with HER2-negative tumors . Other clinicopathological factors and their associations with immune biomarkers are highlighted in A significant association was detected between the level of immune infiltrates and age, tumor size, histological type, and tumor grade . PD-1+ sp-value = 0.03) and multivariate analyses  . Furthermore, the presence of high levels of CD8+ iTILs was not associated with a significant survival advantage in the full study set nor in the non-luminal A subgroup. Likewise, H&E sTILs when evaluated as a categorical variable in an exploratory analysis were not prognostic  is associated with a significant improvement in breast cancer survival (10 years IDFS), with a 10-point difference in the continuous values in univariate  . Howeverognostic . n = 681) in the multivariate analysis . The primary prespecified hypothesis in the statistical plan was to assess the capacity of H&E sTIL (as a continuous variable) and CD8+ iTILs  to predict the benefit of chemotherapy . The multivariate analyses did not reveal a significant predictive value for H&E-assessed sTILs in any breast cancer subgroup  in the non-luminal group, these results should be interpreted with caution due to the small number of the events in the no-chemotherapy group with low CD8+iTILs. The predictive exploratory analysis in the basal subset was not performed due to the limited number of events. When compared to the no chemotherapy group, the chemotherapy arm was associated with significantly improved IDFS in the full study set  when analyzed for the full study set. However, these significant results were not maintained when the analysis was restricted to the non-luminal A subgroup. None of the other secondary immune biomarkers expressed on intraepithelial or stromal lymphocytes were predictive of an enhanced benefit from chemotherapy in the full study set nor in the non-luminal A subgroup  immune infiltrates . The resp-value in the context of an exploratory finding, and the different behavior of the HER2-positive patients with CMF chemotherapy in DGCG77B [Although a significant predictive interaction was seen for the CD8 iTIL in the non-luminal subset , this exploratory finding needs to be interpreted with caution due to the limited number of cases and events occurring for this analysis, its borderline  DGCG77B . A possible explanation for the negative result of the primary tested hypothesis may be that antitumor immunity induced by cyclophosphamide-based chemotherapy is dependent on dose and chemotherapy schedules . GhiringThere are ongoing investigations on different combinations of immunotherapy and chemotherapy, particularly in the neoadjuvant setting in triple-negative breast cancers . EncouraStrengths of our study include using material from a randomized phase III clinical trial and long-term clinical follow up data. Most importantly, this older trial included a no-chemotherapy arm in relatively high-risk patients, something no longer ethically possible to assess in newer trials, that allowed for the assessment of the predictive value of immune biomarkers for chemotherapy with cyclophosphamide, a major backbone drug in most breast cancer regimens to this day. Another factor that provided strength to this study was compiling a pre-specified statistical plan with predetermined cutoff points and independently executed statistical analyses, following the REMARK guidelines . It is plausible that study limitations may have affected our conclusions. Firstly, we used tissue microarrays for evaluating biomarkers. Although tissue microarrays will not completely capture the heterogeneity of the tumor microenvironment, they do facilitate assessing a large number of cases that would otherwise require enormous resources . For thiAnother limitation that may have contributed to the negative result in this paper is our approach to capture the immune biomarkers. Conventional yet widely used techniques such as staining the slides with the single marker IHC or H&E provides basic information about a complex tumor microenvironment. Although these approaches are affordable and accessible, utilizing novel high plex techniques to study the spatial relationships between malignant and immune compartments can potentially uncover new findings that we were unable to assess with these conventional methods . DiffereImmune infiltrates in the tumor microenvironment assessed by H&E are prognostic of improved IDFS in the full study set. However, no significant interaction was observed between any of the assessed immune biomarkers and chemotherapy benefit, including among major breast cancer subgroups, indicative of their lack of predictive capacity for cyclophosphamide-based adjuvant chemotherapy.


Fusarium oxysporum remains one of the leading causes of economic losses and poor crop yields; its detection is strained due to its presentation in various morphological and physiological forms. This research work sought to identify novel biomarkers for the detection of Fusarium oxysporum using in silico approaches. Experimentally validated anti-Fusarium oxysporum antimicrobial peptides (AMPs) were used to construct a profile against Fusarium oxysporum. The performance and physicochemical parameters of these peptides were predicted. The gene for the Fusarium oxysporum receptor protein PR-1-like Protein, Fpr1, was identified and translated. The resulting protein model from the translation was then validated. The anti-Fusarium oxysporum AMPs and Fusarium oxysporum receptor protein 3-D structures were characterized, and their docking interaction analyses were carried out. The HMMER in silico tool identified novel anti-Fusarium oxysporum antimicrobial peptides with good performance in terms of accuracy, sensitivity, and specificity. These AMPs also displayed good physicochemical properties and bound with greater affinity to Fusarium oxysporum protein receptor PR-1-like Protein. The tendency of these AMPs to precisely detect Fusarium oxysporum PR-1-like Protein, Fpr1, would justify their use for the identification of the fungus. This study would enhance and facilitate the identification of Fusarium oxysporum to reduce problems associated with poor crop yield, economic losses, and decreased nutritional values of plants to keep up with the growing population. Fusarium oxysporum is a significant threat to agricultural production. Due to its considerable variation of morphological and physiological makeup resulting from an anamorphic species complex, it tends to escape detection [etection . This fuetection . The patetection . The conetection . Hence, Fusarium oxysporum may cause infection such as severe vascular wilts and root rot diseases to not only Phaseolus vulgaris but also other plant hosts such as tomato, banana, cotton and legumes [Fusarium oxysporum strains only infect very few plant species during inoculation [The pathogenic strains of  legumes . It is a legumes . Despiteculation . This inFusarium oxysporum to recognize and penetrate a host and subdue the innate defenses and nutrient components of the host. The overall effect of the virulence factors produced determines both the infectious potential and severity of the disease caused [Fusarium oxysporum has been functionally characterized and its function is required for full virulence [Fusarium oxysporum as a virulence factor, its biochemical function and biological importance remain elusive.Several mechanisms have been described that allow e caused . Plants e caused . Of all e caused . A secreirulence . DespiteFusarium oxysporum strains that are less accurate due to their similarity to other species. One detection method is based on the characteristic shape and size of macro-and microconidia with the presence and absence of chlamydospores [Fusarium species [Fusarium oxysporum and has proven to be highly dependable for the differentiation of strains at the intra-specific level [Fusarium oxysporum has a cell wall that impedes its efficient lysis and liberation of DNA. However, this can lead to false negative PCR results, creating demand for more sensitive and accurate diagnostics [Several methods are available to detect dospores . There adospores . These m species . Polymeric level . Howevergnostics .Fusarium oxysporum in Phaseolus vulgaris.Antimicrobial peptides (AMPs) are produced as part of the innate immune response, and they exhibit a broad spectrum of activities against pathogens . They haFusarium oxysporum AMPs were retrieved from antimicrobial peptide databases such as Antimicrobial Peptides Database (APD3) [The anti-e (APD3) ,20 and Ce (APD3) . Thereafe (APD3) .The screened, experimentally validated AMPs were randomly partitioned into two subsets: 75% of the data were utilized as the training partition (to build each profile), while the remaining 25% was used for testing .The Hidden Markov Models (HMMER) algorithm version 3.8  was utilFor the initial step, the training datasets of each target class were arranged by utilizing the Clustalo alignment tool .The autonomous query of the profiles was performed in a step called “Query profiles”. The testing data were queried against each target profile utilizing the command line as stated above in the flow chart (iii)  with an The statistical measures were carried out utilizing sensitivity, specificity, accuracy, and Matthews Correlation Coefficient as parameters. The parameters utilized are as described below where TP indicates true positive, TN indicates true negative, FP indicates false positive, and FN indicates false negative:Fusarium oxysporum AMPs against a specific pathogen (testing sets) effectively predicted as anti-Fusarium oxysporum AMPs (positive). The equation of the sensitivity is written below as (1):Percentage sensitivity of the anti-Fusarium oxysporum AMPs (negative sets) effectively predicted as non-anti-Fusarium oxysporum AMPs (negative). The equation of the specificity is written below as (2):Percentage specificity of the non-anti-Fusarium oxysporum AMPs and non-anti-Fusarium oxysporum AMPs). The equation of the accuracy is written below as (3):Percentage accuracy of the effectively predicted peptides (anti-Matthew’s correlation coefficient (MCC) measures the sensitivity and specificity. MCC = 0 is an indication of absolutely random prediction, while MCC = 1 means perfect prediction. See the Equation (4) as below:http://www.ensembl.org/index.html, accessed on 22 December 2019) [http://www.uniprot.org/, accessed on 23 December 2019) [Fusarium oxysporum AMPs. The accomplishment of this task was done using “hmmsearch” module of the HMMER software with the command line employed stated in the flow chart above (iv)   and the er 2019) . An E-vaove (iv) .Fusarium oxysporum (FO) profile.Specific FOTrainings.hmm in the profile, target class query.txt representing the species scanned against the profile and resultfile.txt is the output file acquired after testing the species against the constructed Fusarium oxysporum (isolate 4287 PR-1-like protein) and collected from the National Center for Biotechnology Information (NCBI)  [Fusarium oxysporum gene was complete. Thereafter, the translate tool of ExPAsy  [https://www.uniprot.org/help/uniprotkb, accessed on 23 January 2020) [Fusarium oxysporum.The gene for the receptor, PR-1-like protein, Fpr1, was identified for er 2019) , throughhttps://www.3ds.com/products-services/biovia/, accessed on 27 January 2020) [https://salilab.org/modeller, accessed on 27 January 2020) [The quality of the receptor PR-1-like protein, Fpr1, was analyzed using BIOVIA (ry 2020)  online sry 2020)  by compuFusarium oxysporum AMPs were calculated using the prediction interface of BACTIBASE  [https://dbaasp.org/, accessed on 31 January 2020) [https://wangapd3.com/main.php, accessed on 28 February 2020) [http://web.expasy.org/protparam/, accessed on 28 March 2020) from the ExPAsy server [Physicochemical properties of the anti-ry 2020) , DBAASP ry 2020) , and APDry 2020) ,34 and ty server  using thFusarium oxysporum AMPs as well as the Fusarium oxysporum PR-1-like protein, Fpr1, structures [The I-TASSER (Iterative Threading ASSembly Refinement) server, which is an example of a de novo method of peptide or protein structure prediction, was used to generate the putative anti-ructures . In brieructures . PyMOL (ructures .http://bioinfo3d.cs.tau.ac.il/PatchDock/ (accessed on 31 March 2020) was used for the docking of the anti-Fusarium oxysporum AMPs to the Fusarium oxysporum PR-1-like protein, Fpr1 [Fusarium oxysporum putative AMPs and the Fusarium oxysporum protein receptor were uploaded onto the PatchDock server. The complex formation with the interaction analysis between the anti-Fusarium oxysporum putative AMPs and the PR-1-like protein receptor was achieved using RasMol 2.7.5 Software  [http://hdock.phys.hust.edu.cn/, accessed on 3 March 2021) [The PatchDock 1.3 web-server that enables the docking of the protein-small ligand molecule, available at in, Fpr1 . In brieark, UK) . Subsequch 2021) .Fusarium oxyporum AMPs were collected from different databases—literature mining revealed that CAMP, APD3, DBAASP, and BACTIBASE had 2, 32, and 6 experimentally validated anti-Fusarium oxysporum antimicrobial peptides, respectively. After duplicate removal, a final list of 32 anti-Fusarium oxysporum AMPs was generated.Experimentally validated anti-Fusarium oxysporum.The first step in the profile creation was the random partitioning of the experimentally validated AMPs . HMMER wFusarium oxysporum activity  was identified for Fusarium oxysporum from the National Centre for Bioinformatics Institute (NCBI) database. It was translated using the ExPAsy translate tool using the coding unit of the gene. It was projected that this PR-1-like protein is potentially relevant in detecting Fusarium oxysporum because of its compensatory advantages ranging from production in very high concentration to accessibility for detection [etection .BIOVIA, an online tool for verification of structure using modeler Ramachandran plot , was useFusarium oxysporum AMPs. From Physicochemical features such as molecular weight amino acid composition, hydrophobicity, Boman index, net charge, isoelectric potential, and half-life were used to evaluate the anti-In Fusarium oxysporum AMPs was predicted using certain parameters such as C score, TM score, and RSMD as indicators  and the protein receptors are indicated in Representative output images from the I-TASSER server after predicting the 3-D structures of the anti-Fusarium oxysporum Frp1. Using binding energy scores from the HDock server, it was observed that all the anti-Fusarium oxysporum displayed high binding energy with BOMK-7 having the highest binding energy, followed by BOMK-3 and -5. The complex formation occurred at the Fusarium oxysporum PR-1-like protein, Fpr1, the most favored regions.The docking results of the complex between the putative AMPs and PR-1-like protein, Fpr1, is displayed in Fusarium oxysporum, and anti-Fusarium oxysporum AMPs downloaded as PDB files and visualized using RasMol software is shown in Fusarium oxysporum AMPs are shown in red. BOMK-1 and -2 bound at the same position, BOMK-3 and -9 bound at the same position, BOMK-4 and -7 bound at the same position, BOMK-5 and -8 bound at the same position, while BOMK-6 bound at the different orientation of the PR-1-like protein from others.The structural complex of the docking results between the PR-1-like protein, Fpr1, of Fusarium oxysporum AMPs that have been experimentally approved were recovered from different databases since they have been demonstrated to possess activities against Fusarium oxysporum by utilizing the agar dilution or broth microdilution techniques with the minimum inhibitory concentration (MIC) assay [Fusarium oxysporum AMPs from the databases was recovered after eliminating duplicates to take into account specific species/pathogen profile creation.Anti-C) assay . The runThe training dataset was made up of ¾ of the retrieved peptides required to prepare the algorithm to test whether the functionally critical amino acid consensus is preserved. After this, multiple alignments were created utilizing HMMER, which keeps the profile from being sensitive to little misalignments and report significant E-values. This allows the tendency to capture sequence diversity since the AMPs were obtained from various life forms . ClusterFusarium oxysporum activity. The utilization of a negative dataset (neuropeptides) was done to affirm whether the prepared profiles would discriminate non-anti-Fusarium oxysporum peptides. The utilization of random sequences as a negative dataset is a regularly utilized method [The profile constructed utilizing the training dataset was applied against the held out, positive testing dataset to assess the trained model’s ability to recognize and distinguish this subset of AMPs. Since experimentally confirmed AMPs were utilized, the assumption will be that the profiles developed should have the ability to identify different sequences with similar action and reject those that have no anti-d method .Fusarium oxysporum AMP and false negative anti-Fusarium oxysporum AMPs. The FO (against Fusarium oxysporum) profile had six of its eight positive datasets as true positive, bringing about high sensitivity. The MCC is considered to give the best performance estimation of profiles since it provides the best connection between sensitivity, specificity, and accuracy [The assessment of the autonomous profile testing was accomplished utilizing the TP, FP, TN, and FN measures as inputs to the sensitivity, specificity, accuracy, and MCC descriptive statistics. The HMMER E-value cut-off was set to 0.05 to improve the discovery capacity of the profile between the true positive anti-accuracy . The higFusarium oxysporum profile yielded true positives with E-values lower than 0.05 showing that there was just a 5% possibility that the hit was false or arbitrary. This outcome concurs with the work of Bhadra, Yan [HMMER utilized a default E-value of 0.05 for each hit viewed as a true positive. The anti-dra, Yan  where peFusarium oxysporum in infected plant tissues. This was carried out to discover the AMPs with the same signature/motif as the input sequences. A final list of twelve AMPs was identified, and the AMPs were categorized according to their E-values, with those having the smallest E-values considered the most probable putative anti-Fusarium oxysporum AMPs. There was a very small likelihood that these peptides were incorrectly predicted to be anti-Fusarium oxysporum AMPs.The discovery stage was to identify novel AMPs with the ability to detect anti-Fusarium oxysporum used for proteolytic processing and activation of secreted effectors by fungal and plant host proteases (Avr4). It is a well-characterized type of PR-1 like protein in humans that has been associated with rudimentary biological processes such as cancer, reproduction, and immune response, which are inferred indirectly based on gene expression, localization in specific cell types (glioma or sperm cells), or in response to certain stimuli (pathogen attack) rather than by firm genetic evidence [Fusarium oxysporum detection.PR-1-like protein, Fpr1, is a protein of evidence . The higFusarium oxysporum is a moderately stable protein. The 3-D model structure validation using BIOVIA also supports its use because of its high quality in terms of the distribution of amino acid residues  with a net charge of −5 has an anti-bacterial action, and maximin H5 with a charge between −1 and −7 has a bacterial growth restraint action against Listeriamonocytogenes [The physicochemical properties of the putative AMPs were resolved by utilizing APD and BACTIBASE to guarantee that the distinguished sequences adjust to other AMPs dependent on the qualities estimated. The hydrophobicity result, which was lower than 30% is not an ideal physicochemical parameter . Peptideytogenes . The ranytogenes . The isoytogenes . In any ytogenes .Fusarium oxysporum in terms of stability (as indicated by the instability index), with alanine, valine, isoleucine, and leucine being the most abundant contributors to the aliphatic side chains resulting in an increased thermo-stability .The physicochemical parameters of the PR-1-like protein, Fpr1  indicateFusarium oxysporum protein receptor was analyzed in Fusarium oxysporum AMPs and the Fusarium oxysporum Fpr1 had high confidence in terms of the templates used for their prediction.The structure prediction of the AMPs and the On the other hand, TM-score is a proposed scale for estimating the basic convergence/similarity between two structures . A TM-scFusarium oxysporum peptides because of the absence of experimental proof for these molecules at present.Even though there is certifiably not a characterized RMSD value for 3-D structure prediction, an RMSD estimation of 2–4 Å is viewed as acceptable, and an RMSD of ≤1 Å is considered to be ideal. All models had ideal qualities for RMSD. The results similarly showed that all the AMPs different secondary structures, including α-helices, parallel β-sheet, anti-parallel β-sheet, extended, and loop conformational structures. The outcomes observed associate with the various structural conformations displayed by known AMPs. Examples of known AMPs and their structures include tachyplesin from horseshoe crabs and bovine lactoferricin, which have beta-sheet structures ; magainiFusarium oxysporum. The AMPs also displayed high binding energy scores with PR-1-like protein, Fpr1, with the AMPs having the most noteworthy inclination to identify the fungus. PatchDock and HDock servers use the scoring function as provided in this research to sample ligands’ conformations on the protein receptor [Fusarium oxysporum in plants by utilizing PR-1-like protein, Fpr1, as a target under high sensitivity, specificity, and accuracy.Utilizing the binding geometry scores in receptor ,41. The Fusarium oxysporum using HMMER in silico technology, where 12 anti-Fusarium oxysporum AMPs were generated. The putative anti-Fusarium oxysporum AMPs showed conformity to other known AMPs in terms of their physicochemical characteristics. This diagnostic system’s primary goal is to ease the search and identify a standard reference for a biomarker for early detection of the fungus to solve the current problem, which leads to the reduction of crop yield, market value, and nutritional value of crop plants, including Phaseolus vulgaris. AMPs have demonstrated incredible promise in evading the downsides related to the current diagnostic systems of this fungus. This research work could be pursued for molecular validation through the binding of these AMPs with the PR-1-like protein, Fpr1, using an “on/off” binding experiment in an LFD setting to develop a prototype with these specific AMPs conjugated to gold nanoparticles (AuNPs) to accurately and sensitively detect the fungal pathogen within plant samples.This research identified novel AMPs for the potential diagnosis of


SARS-CoV-2 is the etiological agent COVID-19, one of the most impactful health crises afflicting humanity in recent decades. While research advances have yielded several treatment and prevention options, the pandemic is slow to abate, necessitating an expansion of our treatment arsenal. As a member of the coronaviridae, SARS-CoV-2 contains several ion channels, of which E and 3a are the best characterized. Since ion channels as a family are excellent drug targets, we sought to inhibit both viroporins as a means to curb infectivity. In a previous targeted study, we identified several blockers to each channel from an extensive drug repurposing library. Herein, we examined the ability of said compounds on the whole virus in cellulo. Gratifyingly, many of the blockers exhibited antiviral activity in a stringent assay examining protection from viral-driven death. In particular, darapladib and flumatinib, both 3a blockers, displayed potent antiviral activity. Furthermore, appreciable synergism between flumatinib and several E blockers was identified in a concentration regime in which the compounds are present in human plasma following oral administration. Taken together, targeting ion channels represents a promising approach to both augment and complement our antiviral arsenal against COVID-19. The COVID-19 pandemic emerged as a significant public health crisis since first detected in Wuhan, China ,3,4. TheCoronaviruses (CoVs) order Nidovirales, family Coronaviridae, subfamily Coronavirinae, are enveloped viruses with a positive sense, single-stranded RNA genome. SARS-CoV-2, the etiological agent of COVID-19 ,6, belonThe immense impact of COVID-19 has brought about the development of treatment and prevention options at a record pace. Vaccines have proven particularly efficacious in preventing severe disease . HoweverDirect-acting antiviral drugs have also been developed against SARS-CoV-2, the first receiving emergency usage approval a handful of months after the pandemic broke ,13. HoweConsidering the speed at which new viral strains arise, it is imperative to continue developing new drugs, especially those targeting other virus proteins. To that end, we have decided to focus on the virus’s viroporins since, as a family, ion channels are excellent drug targets ,18,19,20As a member of the coronaviridae, SARS-CoV-2 contains several viroporins, the best characterized of which are E and 3a. The E protein is the most conserved of all Sarbecoviruses’ proteins ,21 and hRecently, we screened an extensive repurposed drug library for inhibitors against each channel using bacteria-based assays. Employing repurposed drugs can prove beneficial since the compounds’ toxicity is known while concomitantly focusing the chemical space for investigation. Our results yielded ten blockers identified against the 3a protein  and eighThe present study reports the protective effect and antiviral activity of the aforementioned hits in a tissue culture setting. We demonstrate that flumatinib and darapladib, both 3a blockers, are particularly potent in preventing virus-induced cellular death. Additionally, several E channel blockers, such as mavorixafor and cyclen, exhibited marked synergism with the Flumatinib at low concentrations. Combinatorial regimens as such may improve efficacy and reduce the potential of the emergence of drug-resistant variants.The assay we chose to evaluate antiviral efficacy was protection from virus-induced cellular death. Precisely, the ability of every compound to enhance cellular viability was measured 48 h after infection and compared to untreated and uninfected cells.Before analyzing the ability of compounds to protect cells from a viral infection, it was imperative to evaluate the inherent toxicity of every blocker. The reason being is that any toxic drug would score poorly in our assay even if it effectively inhibits a critical viral function. Moreover, tolerability studies as such provide a safe concentration range for investigation. Therefore, we tested cellular viability after 48 h in the presence of each drug at concentrations from 0.1–30 μM . ResultsWe next assessed the potential of each drug to exert antiviral activity against SARS-CoV-2 using Vero E6 cells as an infection model . Vero E6As shown in To evaluate the stringency of the protection assay, we measured the resulting viral RNA levels as a function of different flumatinib concentrations . The resM is 26% . The exaA recent study suggested that many cationic amphiphilic drugs indirectly exert their anti-SARS-CoV-2 activity via phospholipidosis induction . Since dIn order to examine potential synergism between the drugs, we tested the ability of different combinations to enhance viability. Particular synergistic effects were found between several E channel blockers and flumatinib, a 3a channel blocker . For exaThe onset of the COVID-19 pandemic has spurred the rapid development of several prevention and treatment options. Vaccines have been particularly impactful at preventing severe disease . HoweverTwo proteins in the virus have received considerable attention as targets for pharmaceutical intervention: polymerase  and protTo that end, we have recently completed two extensive screens to identify inhibitors of the E  and 3a  channelsWe chose protection from virus-induced cell death from the several assays capable of investigating the antiviral activity. While it is a more stringent assay, protection from cellular death considers drug toxicity and does not necessitate additional controls. Furthermore, comparing protection from virus-induced death to measurements of viral progeny reduction demonstrates that the former is far more demanding: Even small changes in protection lead to considerable differences in viral RNA levels .Analysis of all inhibitors at a single 3 ar death . Daraplaectively .We recognize that both darapladib and flumatinib can be classified as cationic amphiphiles and have the potential to induce phospholipidosis. Moreover, a recent study showed that phospholipidosis might underpin the non-specific anti-SARS-CoV-2 activity of many cationic amphiphiles  Yet, botIt is important to note that the targeted screen that retrieved the compounds was conducted at a significantly higher concentration of 100 μM ,30. Theriroporin .One synergism combination, flumatinib and mavorixafor, is particularly auspicious. Pharmacokinetic analyses following oral administration in humans demonstrate that both drugs are present in the plasma at the sub-microMolar concentrations in which pronounced synergism is obtained ,34.A library of 2839 repurposed drugs was purchased from MedChem Express . The drugs for this study were selected from the previously characterized bacteria-based assays against the SARS-CoV-2 E protein channel  and 3a cSimian kidney Vero E6 (ATCC Vero C 1008) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM); , supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 10 IU/mL Penicillin, and 10 SARS-CoV-2, isolate USA-WA1/2020, NR-52281 was deposited by the Center for Disease Control and Prevention and obtained through BEI Resources, NIAID, and NIH. The virus stock was prepared by infecting Vero E6 monolayers with SARS-CoV-2 isolate followed by harvest of culture supernatants three days post infection. The virus titers of cleared infected cells’ supernatants were determined by a standard TCIDA 10 mM stock of listed compounds was prepared in DMSO and stored at −80The effects of the drugs on the metabolic activity of Vero E6 cells were assessed at 48 h post treatment using CellTiter 96 Aqueous Non-Radioactive Cell Proliferation reagent .To examine the effect of different drugs, cells were pretreated with the specific compound for one hour followed by infection with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. Each compound concentration was tested in triplicate and each assay plate contained the following controls: no cells (background control), cells treated with medium , infected/untreated cells and infected/solvent-treated cells (infection control).At two days post infection, drug efficacies in control of toxicity were assessed by the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation reagent  for 3 h at 37We have presented a targeted three-tier approach to identify anti-SARS-CoV-2 drugs. We started by identifying channels in the virus that may serve as drug targets. We continued by screening inhibitors against these viroporins utilizing a rapid bacteria-based methodology. We concluded by examining the ability of each of the hits to exhibit antiviral activity in tissue culture cells. Encouragingly, our approach was able to identify new anti-SARS-CoV-2 drugs that are attractive candidates for further development.


Intraoperative blood salvage autotransfusion(IBSA) has been widely used in a variety of surgeries, but the use of IBSA in hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) is controversial. Numerous studies have reported that IBSA used during LT for HCC is not associated with adverse oncologic outcomes. This systematic review and meta-analysis aims to estimate the clinical prognosis of IBSA for patients with H+CC undergoing LT.2 test. Publication bias was evaluated by funnel plots, Egger’s and Begg’s test.MEDLINE, Embase, Web of Science, and Cochrane Library were searched for articles describing IBSA in HCC patients undergoing LT from the date of inception until May 1, 2022, and a meta-analysis was performed. Study heterogeneity was assessed by I12 studies enrolling a total of 2253 cases (1374 IBSA and 879 non-IBSA cases) are included in this meta-analysis. The recurrence rate(RR) at 5-year and 7-year in the IBSA group is slightly lower than non-IBSA group. There are no significant differences in the 1-year RR, 3-years RR ,1-year overall survival outcome(OS) , 3-year OS, 5-year OS,1-year disease-free survival rate(DFS) , 3-year DFS, and 5-year DFS. Subgroup analysis shows a difference in the use of leukocyte depletion filters group of 5-year RR. No significant differences are found in other subgroups.IBSA provides comparable survival outcomes relative to allogeneic blood transfusion and does not increase the tumor recurrence for HCC patients after LT.https://www.crd.york.ac.uk/prospero/, identifier CRD42022295479. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancers, the sixth most common neoplasm, and the third most common cause of cancer death . Liver tBlood transfusion could be divided into autotransfusion and allogenic blood transfusion (ABT) based on the blood source. Three types of autologous transfusion exist: prestored autotransfusion, dilution autotransfusion, and intraoperative blood salvage autotransfusion(IBSA). ABT is the primary technique employed in conventional application, but it may transmit hepatitis virus and human immunodeficiency virus, as well as cause an immunological transfusion reaction , 7. NoniThe use of IBSA in HCC patients involving LT is controversial, the critical point is whether IBSA increases the risk of recurrence or metastasis due to reperfusion of tumor cells , 13. EveThis systematic review and meta-analysis adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. This syThe search strategies were created by an investigator (KY) with database search experience. We conducted database searches in the following databases: Medline (via PubMed), Web of Science databases, EMBASE and The Cochrane Library. Databases were used to identify suitable studies that were published up to 1 May 2022. Three search themes were combined with the Boolean operator ‘and’ in searching databases, and the search terms were as follows: ‘Autotransfusion’, ‘Liver Transplantation’ and ‘Hepatocellular Carcinoma’. Detailed search strategy was shown in The following parameters were extracted from the full-text article: the name of the first author, periodical titles, country, publication year, type of study, characteristics of IBSA group and non-IBSA group . Two reviewers(YJ and DL) extracted data from studies in accordance with the screening process, and any inconsistencies were resolved by a third reviewer(SL). In case of any ambiguity or insufficient information, wherever possible, authors of primary studies were contacted by either telephone, email or post to obtain missing data. We made a summary sheet containing all the data fore-mentioned. On the other hand, we assessed the quality of published literature by two independent reviewers (ZW and YJ). The risk of bias of RCTs was assessed with items in the Cochrane Collaboration’s tool . Non-RCTThe primary outcome of this meta-analysis is the tumor-related recurrence rate of use IBSA during LT for HCC. The recurrence time points will be 1-year, 3-year, 5-year and 7-year after LT. Radiological data was used to determine whether HCC had recurred . Other s2 statistic were used to assess statistical heterogeneity among the included studies   will be used to analyze the outcomes, otherwise, a fixed-effect model(the Mantei-Haenszle method) would be applied. The sources of heterogeneity will be explored by using sensitivity analyses. A subgroup analysis will be conducted to determine whether the results differed according to the use of leukocyte depletion filters (LDFs). The potential for publication bias will be assessed by the funnel plot, Egger test and Begg’s test  and corresponding 95% confidence intervals(CI) were used to measure the association for each study. We will apply mathematical operations to convert data that is presented in the literature as median and quartiles into mean and standard deviation format . Forest ctively) . I2 > 50g’s test –26.The database searches returned 123 results, 22 of which were excluded due to duplication. Further, 34 studies were excluded because they were reviews or qualitative study or were not relevant to the topic being studied. The remaining articles were fully read. Finally, 12 studies enrolling a total of 2253 cases (1374 IBSA cases and 879 non-IBSA cases) were included in the meta-analysis , 27–37. The selected studies had been published between 2005 and 2022. The sample size of studies ranged from 23 to 397. All of the studies were cohort studies. There were no randomized controlled trials. According to the Newcastle-Ottawa Scale, most  of the studies were defined as high-quality studies (score more than 7), the detailed assessments are shown in 2 = 0%), 3-year RR (I2 = 0%), 5-year RR (I2 = 0%), and 7-year RR(I2 = 0%), the fixed effect model was adopted.Twelve studies reported the recurrence rate(RR) outcomes of IBSA and non-IBSA patients. Of them, seven studies provided a specified description of criteria for determining the recurrence and follow-up methods , 35–37. 2 = 0%), 3-year OS (I2 = 29%), 5-year OS (I2 = 28%). For all included studies performed in statistics of disease-free survival(DFS), there were no significant differences at 1-year DFS, 3-year DFS, 5-year DFS (2 = 0%), 3-year DFS (I2 = 20%), and 5-year DFS (I2 = 0%).Eight studies reported the overall survival(OS) outcomes of IBSA and non-IBSA patients Figure 3A predesigned subgroup analysis was conducted according to the use of LDFs. Six studies attached LDFs to IBSA during LT , 31–33. For primary outcomes, pooled effects of ORs remained stable after removing any single study at 1-, 3-, 5-, and 7-year RR. For secondary outcomes, the removal of Kwon’s study led to a reduction in heterogeneity at 3-year and 5-year OS . Filled We used Egger’s test and Begg’s test to evaluate the publication bias for RR and OS outcomes. No indication of publication bias was observed for 1-year RR , 3-year RR , 5-year RR , and for 1-year OS , 3-year OS , 5-year OS . Funnel plots were visually examined for symmetry for all outcomes reported 3.In this comprehensive systematic review and meta-analysis, we identified 12 cohort studies investigating the clinical prognosis of IBSA during LT for HCC. The recurrence rate was used as the primary outcome, and the overall survival and disease-free survival were used as the secondary outcomes. The analyses showed that the RR at 5- and 7-year in the IBSA group was slightly lower than non-IBSA group. No significant differences were found between the IBSA and non-IBSA groups in the 1-, and 3-year RR outcomes. For secondary outcomes, the OS outcomes at 1-, 3-, and 5-year and the DFS outcomes at 1-, 3-, and 5-year were not significantly different. Sensitivity analysis was carried out to evaluate whether the result is stable and reliable, adjusted effects did not fluctuate much by omitting each study. Given the above, though no randomized studies were included, results of the meta-analysis could be considered relatively solid and trustworthy based on the current studies.The use of IBSA reduces the requirement for allogeneic blood during surgery, preventing adverse transfusion reactions without having a negative impact on other clinical outcomes. However, oncological surgery is still regarded as a relative contraindication to IBSA over concern of reinfusing tumor cells and thereby causing tumor dissemination , 38, 39.in vitro and vivo studies . But the included studies of this meta-analysis ranged from different cancer types, only three studies involved patients with hepatocellular carcinoma . In addiSeveral limitations of our study should be mentioned. First, the included studies were retrospective research and selection bias should not be ignored, since no RCT research on this question has been found after searching the databases. Well-designed, randomized, controlled, prospective trials are urgently required to clarify the existing concerns. Second, we only included English language studies due to the constraints of translating foreign language studies. Third, the included studies did not explore the use of allogeneic blood products, which may affect survival outcomes and prognosis due to their impact on immunity. Moreover, although significant heterogeneity was not found, patients’ characteristics varied across included studies. Only part of included studies use a propensity score to control for the effect of confounding and address selection bias, more detailed subgroup analyses were difficult to conduct, because of multiple outcomes and insufficient studies.These 12 studies represent the best reliable evidence to date. This meta-analysis may at least indicate that intraoperative blood salvage autotransfusion provided comparable survival outcomes relative to allogeneic blood transfusion and did not increase the tumor recurrence for hepatocellular carcinoma patients after liver transplantation. A reappraisal of the appropriate strategy for blood management during liver transplantation is warranted. High quality researches are required in the future to provide more sufficient evidence.The original contributions presented in the study are included in the article/ZW developed the initial idea for this study. SL and KY developed and revised the search strategy. MS and ML finished the study design. KY and KL carried out data extraction and assessment of risk of bias. ZW, SL and YJ contributed to the original draft. DL and KL and were responsible for the revision of the draft. All of the authors approved the final work prior to submission ZW, SL and YJ have contributed equally to this work. All authors contributed to the article and approved the submitted version.The authors thank all the authors of the original studies included in this meta-analysis.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.


Objective: Despite advances in treatment, the management of fracture non-union remains a challenging and complex problem in orthopaedics. Low-intensity pulsed ultrasound (LIPUS) treatment has been shown to be an effective, non-invasive, affordable treatment option. This treatment was evaluated in a Scottish district hospital over a nine-year period, which included the COVID-19 pandemic.Materials and methods: This submission describes a case series at Dr Gray’s Hospital in Scotland, 18 patients in whom fracture non-union was treated using LIPUS.Results: An overall healing rate of 94% was achieved. Exogen™  proved to be most successful in oligotrophic non-union. No observed patient demographic appeared predictive of outcome. LIPUS treatment failed in one case. No significant adverse effects of LIPUS were detected.Conclusion: LIPUS represents a useful, cost-effective potential alternative to revision surgery. LIPUS may therefore be the preferred treatment when surgical intervention and face-to-face interactions are to be minimised, as during the COVID-19 pandemic. Optimum treatment of non-union following fractures remains a challenging problem. The current gold standard management, open reduction and internal fixation with bone graft, carries a significant socio-economic burden  and neceThe incidence of fracture is estimated at 3.6 fractures/100 people/year in the UK . LiteratThe COVID-19 pandemic created its own unique challenges in this already complex situation, with hospital beds at a premium, delays to non-emergency surgery and mandatory avoidance of face-to-face intervention . After tTherapeutic low-intensity pulsed ultrasound (LIPUS) delivers micro-mechanical stress  to the tThe exact definition of fracture non-union varies widely within the orthopaedic community . In commIn this retrospective study, Exogen™  was used to treat a variety of cases of fracture non-union at a district hospital in a remote, rural area of Scotland, in 18 patients who underwent LIPUS during the nine-year period from 2013 to 2021.Patients were identified retrospectively from the local Exogen™ Order Registry between 2013 and 2021. The inclusion criteria were persistent pain at the fracture site and either absence of radiological union or no progression of callus on two consecutive radiographs at six months (in the case of long bone fracture) or three months  from the date of injury or initial fixation .All patients were managed by consultant orthopaedic surgeons at Dr Gray’s Hospital. The decision to offer LIPUS was individualized in this series. During the COVID-19 pandemic, remote consultations were employed to comply with restrictions. LIPUS was offered to patients with persistent pain and absence of callus progression on radiographs in accordance with the criteria. Patients gave informed consent for the treatment with LIPUS on the understanding that surgical intervention would be offered should LIPUS fail to generate adequate bone healing.2, operating frequency of 1.5 MHz, pulse width of 200 microseconds and repetition rate of 1 KHz, as described by NICE [LIPUS stimulation employed Exogen 150™ (formerly Exogen Express™) equipment. The intended treatment area was delineated in ink by the orthopaedic consultant. All patients received adequate training and were deemed competent to self-administer low-intensity pulsed ultrasound stimulation via a transducer held over the delineated area and fixed in place using a strap, using a coupling gel to aid acoustic conduction. The transducer delivered acoustic radiation with a temporal average power of 30 milliwatts/cm by NICE . TreatmeClinical and radiological progress was assessed by the consultant orthopaedic surgeon at regular intervals.The primary outcome measure was fracture healing, as defined by the complete resolution of clinical symptoms including pain, accompanied by satisfactory radiological union leading to discharge from the fracture clinic with no requirement for further follow-up.The outcome was deemed successful or unsuccessful on the basis of both clinic letters and radiographs recorded after treatment with LIPUS. The outcome was evaluated at the final outpatient visit prior to discharge from the clinic (in the case of the full resolution of symptoms and satisfactory radiographic appearance) or when a decision was made to offer subsequent operative fixation.Discharged patients were given self-awareness instructions and offered an open return to the Outpatient Department.Eighteen patients with fracture non-union were identified from the registry from 2013 to 2021, and all were included in the study.The patient group comprised six males and 12 females. The average age at the time of injury was 46.6 years (range: 14-88 years). The average age did not differ significantly between males (48.3 years) and females (45.8 years). The age distribution is shown in Figure Five patients underwent operative fixation prior to LIPUS therapy, and 13 patients were managed conservatively throughout. A wide range of fracture types was represented in the study, of which 15 developed oligotrophic non-union and three developed hypertrophic non-union, as shown in Table Available data were reviewed for the presence of comorbidities considered to be associated with failure of union post-fracture ,17, the Smoker or ex-smoker status was documented in 39% of patients and diabetes or other relevant comorbidities in 22%. Of our cases, 28% declared themselves non-teetotal; however, only patient (6%) one was identified as drinking alcohol to excess. The average BMI (for the 15 patients for whom this data was available) was 30, with 27% being in the healthy range. Overall, no risk factors were identified in six out of the 18 patients, while the remaining 67% of patients had one or more risk factors.The Exogen 150™ or Exogen Express™ machine was fitted on an average of 6.6 months after the index injury or fixation (range: 74-557 days). All patients bar one reported compliance with the planned treatment regime, which lasted between 2.1 months and 7.3 months (average duration of 4.2 months or 127 days). Patient 8 demonstrated significant initial improvement with LIPUS treatment and became symptom-free, before ceasing to attend further follow-ups. Clinical and radiological follow-up at the Outpatient Department by the consultant orthopaedic surgeon continued throughout treatment, with reviews occurring every nine weeks on average Figure . PatientTreatment with Exogen™ was successful in 17 out of 18 cases. Three patients reported initial increase in pain during the first week of Exogen™ treatment, which quickly settled. No other complications were reported. There were no late failures in successfully treated patients, and no patient required re-admission in respect of the same fractured bone. The overall healing rate was therefore 94%.2 (the highest recorded BMI in our series), this patient had no other characteristics predictive of failure of union after fracture [Exogen™ treatment was unsuccessful in one patient, who underwent ORIF with bone grafting two years after the index injury of the fourth metatarsal stress fracture. Apart from a BMI of 45 kg/mfracture , unless Our case series of 18 fracture non-unions spanned 10 different bones, including lateral malleolar fracture (five cases), fifth metatarsal fracture (five cases), clavicle fracture (two cases), fourth metatarsal stress fracture, femoral shaft fracture, femoral neck fracture, tibial fracture, combined radius and ulna fracture, and combined third and fourth metacarpal fracture (one case each).LIPUS has been used in the treatment of recalcitrant fracture non-union for many years. Several studies demonstrate the efficacy of LIPUS in this context ,15,16.A NICE meta-analysis evaluated the treatment of 1,441 cases of fracture non-union using LIPUS, suggesting an estimated healing rate of 82%. When the definition of non-union was restricted to fractures of at least eight months duration, the pooled estimate of effect size was 84% . OverallThere are a number of documented patient-specific risk factors for the development of fracture non-union ,17. ZuraOnly one fracture non-union remained unresolved after treatment with LIPUS. This was the only stress fracture and one of only three cases of hypertrophic non-union in this case series.As LIPUS can be self-administered at home, this alternative to operative fixation was particularly valuable during the COVID-19 pandemic. In the Scottish Highlands, where frequent travel to attend a face-to-face appointment is routinely inconvenient and costly, difficulties were exacerbated by limitations imposed by COVID-19 restrictions. LIPUS remained accessible and convenient both for patients and for the orthopaedic team despite the COVID-19 pandemic.No firm conclusions can be drawn from this study owing to the limitations imposed by its retrospective nature and the small number of participants. However, this study encompasses a wide variety of fracture types, and its results suggest that oligotrophic non-union may be particularly amenable to LIPUS treatment.The healing rate of LIPUS treatment of fracture non-union in this study was 94%, concurring with larger published trials and comparable to outcomes after surgery. While the current waiting time for elective non-union surgery is excessive, LIPUS treatment is readily available and can be initiated soon after non-union has been diagnosed.Although circumstances dictate minimal face-to-face contact with patients (as was the case during the COVID-19 pandemic), this study demonstrates that LIPUS presents a viable alternative to surgical fixation, which is acceptable to patients and may permit significant savings to be made in both direct and indirect costs.


Febrile neutropenia (FN) is a medical emergency with significant morbidity and mortality for oncology patients, requiring comprehensive workup and timely antibiotic administration. We evaluated concordance with locally developed FN guidelines and outcomes of cancer patients admitted to general internal medicine at an academic teaching hospital.We conducted a retrospective observational cohort study of patients admitted between July 1, 2016, and June 30, 2017, for FN. Patients were classified as having low-risk or high-risk FN according to their malignancy and chemotherapy. Primary outcome was the proportion of patients receiving guideline-concordant antibiotics within 48 hours of admission to general internal medicine. Secondary outcomes were the proportion of patients in whom empirical antibiotics were active against pathogens isolated, rate of antibiotic-associated adverse events, and in-hospital mortality. We used logistic regression to model relationship between FN risk and guideline-concordant antibiotics.P = .001). Empirical antibiotics were active against 17 (94%) of 18 isolated pathogens. The mortality rate was 3%, and 16% of patients experienced antibiotic-associated adverse events. Hematological malignancy and infectious diseases–trained physician involvement were associated with guideline-concordant prescribing, with adjusted odds ratios of 3.76  and 3.71 , respectively.Among 100 patients included, 34 (34%) were low-risk FN and 66 (66%) were high-risk. Proportion of guideline-concordant empirical antibiotics was significantly lower among low-risk FN patients than high-risk patients: 12 (35%) of 34 versus 47 (71%) of 66 (Guideline concordance was low compared to published reports. Factors influencing appropriate antimicrobial prescribing in patients with FN warrant further exploration. Signs and symptoms of infection are often absent or ambiguous due to diminished inflammatory responses in this patient population, but given the risk of infectious complications, timely administration of empirical antibiotics is recommended. The guideline from the Infectious Diseases Society of America (IDSA) for antimicrobial stewardship programs (ASP) has highlighted the value of ASP interventions in oncology patients to ensure appropriate selection of antibiotics tailored to local epidemiology while reducing antibiotic-associated adverse events associated with inappropriate use. The IDSA has emphasized the role of facility-specific, locally developed FN guidelines in this patient population. The implementation of institutional FN guidelines can help to optimize patient care by promoting best practices, improving patient outcomes, and reducing costs.Febrile neutropenia (FN) is a medical emergency associated with significant potential morbidity and mortality for oncology patients. One guideline is specific for patients with hematological malignancies such as acute leukemia and recipients of stem-cell transplant pre-engraftment, who are at high risk for infectious complications. The other guideline is specific for patients undergoing treatment for solid tumor, who are considered to be at low risk of infectious complications. The guideline for patients with hematological malignancies or stem-cell transplant recommends piperacillin-tazobactam plus tobramycin as empiric antimicrobial therapy. The guideline for the solid-tumor patients recommends cefazolin plus tobramycin. Empirical tobramycin is limited to a maximum of 72 hours for both guidelines while microbiological investigations are pending and diagnostic workup are underway. The inclusion of tobramycin is to ensure adequate gram-negative coverage, reflecting the epidemiology from the PM-specific antibiograms and Public Health Ontario’s provincial report. The prevalence of multidrug-resistant gram-negative rods in blood isolates is estimated to be between 20% and 25% in the local patient population. Meropenem is the empirical alternative for patients with self-reported allergy to penicillin and those with history of colonization or infection due to extended-spectrum β-lactamase–producing gram-negative bacilli.The University Health Network (UHN) is a tertiary academic teaching hospital system affiliated with the University of Toronto. It includes 3 acute-care sites: Toronto General Hospital (TG), Princess Margaret Cancer Centre (PM), and Toronto Western Hospital. The UHN ASP created 2 FN guidelines based on local susceptibility pattern, patient population, and resources.In this study, we evaluated concordance with the FN guidelines and appropriateness of antibiotic prescribing among cancer patients admitted to TG general internal medicine (GIM) service. Although PM specializes in treating complex cancer patients, those who experience infectious complications are commonly admitted to TG. We aimed to identify opportunities to optimize febrile neutropenia management in GIM through this quality improvement initiative.International Classification of Disease, Tenth Revision (ICD-10) codes, who had a current oncology diagnosis and were currently receiving chemotherapy within 6 months prior to admission. We included patients admitted from TG emergency department or from PM’s rapid assessment clinic . Patients were excluded if they were hematopoietic stem-cell transplant recipients in the postengraftment phase or if they were not undergoing chemotherapy. The study was approved by the UHN Institutional Review Board.We conducted a retrospective observational cohort study of patients admitted to GIM between July 2016 and June 2017. The GIM service consisted of between 60 and 65 beds. Patients were identified by the UHN Department of Clinical Decision Support. We included patients with a primary discharge diagnosis of first-episode FN based on the No direct antimicrobial stewardship interventions were underway in GIM during the study period. Following the launch of the FN guidelines (version 2016), presentations to introduce the guidelines were given by the ASP team to clinical teams across the UHN, including the TG GIM program. The guidelines are available on the institution’s intranet and on the ASP website.Following eligible patients identified by the clinical decision support team, baseline patient demographics, clinical parameters including neutrophil counts and documented fever, imaging studies and microbiology data were collected through manual review of the institution’s electronic health record (EHR) system. An antimicrobial stewardship report generated by the ASP was provided to record the antibiotic regimens used.9/L or an ANC that is anticipated to decrease to <0.5 × 109/L during the following 48 hours. Fever was defined as any reported or measured temperature of ≥38.3°C once or sustained temperature of ≥38°C for at least 1 hour. Oncology diagnoses were classified as follows: active solid tumor included breast, lung, colorectal, other gastrointestinal, gynecologic, genitourinary, head and neck, and low-risk lymphoma; active hematological malignancy included acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), mixed ALL and AML, high-grade lymphoma , chronic lymphoblastic leukemia (CLL), multiple myeloma, and myelodysplastic syndrome. We stratified patients as having low-risk or high-risk FN based on the IDSA guidelines, which were adopted in the UHN guidelines in 2014 and first updated in 2016. Low-risk patients were expected to have a short neutropenic period of ≤7 days; high-risk patients were anticipated to have prolonged neutropenia of >7 days; and profound neutropenia was defined as an absolute neutrophil count ≤0.1 × 109 cells/L. In practice, patients undergoing treatment for solid tumor were managed under the low-risk guideline and patients with hematological malignancies were managed under the high-risk guideline. Age was categorized into <65 years or ≥65 years.FN was defined as per the IDSA guideline, with a composite of neutropenia as an absolute neutrophil count (ANC) of ≤0.5 × 10 Adverse events reported during hospitalization were categorized by body systems: gastrointestinal, dermatologic, musculoskeletal, hematologic, hepatobiliary, renal, cardiac, and/or neurologic.Guideline concordance was defined as the prescription of an empirical antimicrobial regimen that was consistent with the UHN FN guidelines recommendation according to the risk stratification of the patient. Empirical antimicrobials were considered appropriate if the regimen was active against the pathogen isolated from a microbiological culture and/or appropriate for the source of infection  according to the indication in the medical records. Antibiotic-associated adverse events were defined according to Tamma et al.Appropriateness of antimicrobial and guideline concordance were adjudicated by a study investigator (R.L.). For quality assurance, another clinician independently audited 20 randomly selected charts for accuracy of data collection.Clostridioides difficile infection, antibiotic-associated adverse events, length of stay under the GIM service, and in-hospital all-cause mortality.The primary outcome was the proportion of patients who received guideline-concordant antimicrobials within 48 hours of admission to GIM. We excluded antimicrobials administered while patients were in the emergency department because they had not been officially under the care of the attending internists. Secondary outcomes evaluated whether empirical antibiotics were active against the isolated pathogen or appropriate for the source of infection, factors associated with guideline concordance, incidence of nosocomial 2 test for categorical variables and the Wilcoxon rank-sum test for continuous variables. Univariable and multivariable logistic regression models were used to assess the association between FN risk and receipt of guideline-concordant antibiotics, adjusting for age, sex, type of cancer , and infectious diseases (ID) physician involvement. ID involvement was defined as having received ID specialist consultation or being under the care of an attending internist who was also a qualified ID consultant. Odds ratios (OR) and adjusted odds ratios (aOR) with 95% confidence intervals (CIs) for the primary outcome were reported. Model goodness-of-fit was assessed with the Akaike information criterion and likelihood ratio test. We defined P < .05 as statistically significant. Statistical analysis was completed using STATA version 16.0 software .Baseline characteristics were reported with descriptive statistics. Outcome measures were compared using the χ9 cells/L. In the high-risk group, 73% had ANC ≤0.1 ×109 cells/L and 11% had neutropenia lasting 7 days or longer. Length of stay (LOS) was longer in the high-risk group  than the low-risk group . Duration of fever was shorter in the low-risk group, with a mean of 1.1 days , compared to 2.2 days (±1.9) in the high-risk group (P = .002).In total, 100 patients met eligibility criteria to be included in the study. Baseline characteristics are presented in Table P = .001) (Table P = .022). Factors associated with receiving guideline-concordant antimicrobials included type of cancer  and involvement by an ID physician   of 100 patients was guideline concordant. In the low-risk group, 12 35%) of 34 patients received guideline-concordant antimicrobials, and in the high-risk group 47 (71%) of 66 received guideline-concordant antimicrobials  of 34 patients in the low-risk group received piperacillin-tazobactam and tobramycin empirically, and 6 (18%) of 34 received piperacillin-tazobactam monotherapy, instead of cefazolin and tobramycin as recommended by the local guideline. In the high-risk group, 8 (12%) of 66 received piperacillin-tazobactam monotherapy, rather than piperacillin-tazobactam plus tobramycin as recommended by the local guideline.Enterococcus (VRE) and 1 extended-spectrum β-lactamase–producing (ESBL) Escherischia coli were isolated from a blood and urine source, respectively. Clinically defined infections occurred in 28 (28%) of 100 patients. Pneumonia was diagnosed in 21 patients, and 7 had skin and soft-tissue infections.Source of infection was identified in 50% of patients. The cause of fever was not identified in the other half of the study population. Of the 22 microbiologically defined infections, empiric antibiotic regimens were active against 17 (94%) of 18 bacterial pathogens isolated. Of the pathogens isolated, 8 of 22 were from blood specimens: 3 were gram-negative bacilli, 3 were gram-positive cocci, and 2 were polymicrobial. Of the 14 nonblood isolates, 7 were from urine specimens, all of which were gram-negative bacilli. Several multidrug-resistant organisms were identified. One vancomycin-resistant C. difficile infection occurred in 4 (4%) of 100. In-hospital all-cause mortality occurred in 3 (3%) of 100. Antibiotic-associated adverse drug events occurring in 16 (16%) of 100 patients with the most common being non–C. difficile associated diarrhea (n = 9), followed by dermatologic reaction (n = 3), and 1 patient each with hepatobiliary, gastrointestinal, renal, and neurological adverse events.Hospital-onset of At our institution, the overall guideline concordance among FN patients in GIM at the Toronto General Hospital was low at 59%. Concordance was higher in the high-risk group (71%) than in the low-risk group (35%). Overall, guideline-concordant empirical antimicrobials were active against the clinical syndromes and isolated pathogens in FN patients, except for 1 VRE infection. A point-prevalence audit conducted at Princess Margaret Cancer Center in 2017 found FN guideline concordance to be 73% in patients with acute leukemia. The practice model and staffing at the malignant hematology units differed from that in the GIM units at Toronto General Hospital, which are clinical teaching units and patient care is provided by learners at various stages of training under the direct supervision of the attending internists. Furthermore, the ASP team conducted twice-weekly audit-and-feedback in the malignant hematology units at Princess Margaret. However, in TG GIM units, no direct antimicrobial stewardship interventions were conducted. However, our results shared common patterns with contemporaneous reports. Guideline concordance was 85.4% in a series of point-prevalence surveys conducted between 2014 and 2018 in a combination of academic and community hospitals in Australia. In this cohort, hematology patients were more likely to receive antimicrobials concordant with local guidelines than solid-tumor patients. Baugh et al conducted a case-vignettes survey among 107 oncologists of an academic cancer center and 44 physicians from the emergency department (ED) of an affiliated teaching hospital in 2015. These researchers reported that 96% of oncologists and 100% of ED physicians selected guideline-concordant antimicrobials for high-risk patients but that only 61% of oncologists and 23% of ED physicians selected appropriate plans for low-risk patients. In a retrospective cohort study by the same authors, guideline concordance was 70% among patients who presented with FN to the ED in 2010–2014, including 44 low-risk and 129 high-risk visits. There was a significant difference in the prevalence of guideline-discordant antimicrobials between low-risk and high-risk patients . Schuttevaer et al reported guideline adherence to be 395 (43.5%) of 909 in a retrospective cohort of FN patients who presented to the ED at a tertiary-care university hospital and had bloodstream infections between 2012 and 2017. In this study, over-treatment (spectrum too broad) with empirical antibiotics was not associated with survival benefit.Our guideline-concordant antimicrobial prescribing was lower than the 79% reported in a database study of admission records between 2000 and 2010.Several factors may have contributed to poor guideline compliance in GIM. First, there were barriers to effective communication. UHN had a hybrid system of paper chart plus EHR that was not integrated with venues of communication, mainly paging and e-mail. GIM patients were located in several units, which were physical barriers of communication between prescribers and the ASP team. In addition, the GIM program is located at Toronto General Hospital—a different building from the Princess Margaret Cancer Centre—and did not receive scheduled audit-and-feedback interventions from the ASP team. No GIM pharmacist was dedicated to reviewing antimicrobial management in oncology patients or to liaising with the ASP team. in 2015, only 54% of oncologists and 26% of ED physicians were familiar with the IDSA guideline. Unfamiliarity with the guideline was associated with unnecessary empirical vancomycin in high-risk patients. In the Australian point-prevalence surveys, among hematology patients, 20.2% of meropenem and 11.3% of vancomycin were adjudicated as spectrum being too broad. In our study, the most common guideline-discordant prescribing in low-risk patients was the spectrum being too broad. Piperacillin-tazobactam monotherapy or in combination with tobramycin were prescribed instead of cefazolin and tobramycin. Because our recommended empirical therapy differed from the international guidelines, prescribers were not familiar with the rationale based on local epidemiology. Concerns regarding ototoxicity and nephrotoxicity from tobramycin exposure, albeit for ≤72 hours, may have contributed to a reluctance to prescribe.Second, there appeared to be a lack of familiarity of the FN guidelines among the GIM teams. Although the guidelines were on our institution’s intranet and the ASP website, active implementation strategies, education, and promotion efforts were not sustained in GIM units. In the survey conducted by Baugh et al Conversely, as demonstrated by Douglas et al, solid-tumor patients may have less exposure to oversight by ID specialists and ASP interventions. ID specialist involvement may promote guideline-driven practice by raising the awareness of other clinicians toward them.Lastly, cancer type and ID physician involvement  were independently associated with guideline-concordant antimicrobials. ID physicians may be more familiar with the guidelines, whereas patients with hematological malignancies were more likely to receive ID consultation service. We are upgrading our guidelines to reflect a more nuanced approach to risk stratification, accounting for cancer diagnosis and up-to-date treatment.Since the conclusion of this study, TG GIM has installed a clinical pharmacist for oncology patients, who liaises with the ASP team and ID consultants. Recently, UHN replaced the hybrid system of paper chart and EHR system with an enterprise-wide integrated EHR system. The new system has built-in communication tools, documentation, and clinical decision support. Clinicians can access information on patients’ current cancer therapy directly. We anticipate that the new system will facilitate guideline-based prescribing and will improve the efficiency of evaluating prescribing practice. Second, beyond process measures, we assessed appropriateness of guideline-recommended antimicrobials in the initial 48 hours after admission and clinical outcomes based on microbiological investigations. In comparison, others have focused on evaluating a single agent, most commonly vancomycin, route of antimicrobial administration, or admission according to disease burden score. There is increasing emphasis on auditing adherence to guidelines by standards-setting organizations such as the Center for Disease Control and the Joint Commission. In an era with growing prevalence of multidrug-resistant organisms, receiving inappropriate empirical antimicrobial treatment is associated with morbidity and mortality. Third, in our study population, patients were admitted over a 1-year period after guideline implementation. These results reflect the validity of the guideline recommendations. Previous publications have reported on data for cohorts of patients accumulated over long periods, often upwards of 5 years. The validity of those guidelines may wane over time owing to changes in the epidemiology of infectious complications, antimicrobial resistance, and advances in cancer treatment.The strengths of our study were intertwined with the quality improvement process. First, we focused on evaluating concordance and outcomes of patients admitted to GIM for FN, whereas many recent publications have concentrated on patients presented to the ED.Our study had several limitations. First, given its retrospective nature, the accuracy and validity of the data were limited by the quality of the documentation. A detailed examination of the prescriber’s clinical reasoning was precluded because the rationale and thought process behind antimicrobial prescribing was infrequently recorded. Second, there was overlap in duration and nadir of neutropenia between the high-risk and low-risk groups, suggesting that risk differentiation can be improved by incorporating details of cancer diagnosis and treatment. Third, as a single-center study, local factors contributing to guideline discordance may not apply elsewhere. Serial point-prevalence audits on guidelines concordance and appropriateness of antimicrobial prescribing using an a priori adjudication framework with prescriber-specific data may be useful. In conclusion, we demonstrated that after developing local FN guidelines and making them available to prescribers, maintaining antimicrobial stewardship efforts, giving feedback, and evaluating adherence are imperative to ensuring that prescribers use antimicrobials according to best practices.Further studies with quantitative and qualitative methods are needed to better understand why prescribers are not compliant with FN guideline recommendations and how those barriers can be removed.