It was found that both XFEM and VCCT produced better results, but XFEM had better flexural simulation. Contrarily, the CIT models failed to express the softening behavior and to capture the crack path. Furthermore, the flexural capacity was increased after reducing the (ao/D) and after decreasing the S.S/D. Additionally, using EPT increased the flexural capacity, showed the ductile flexural response, and reduced the flexural softening. Moreover, using reinforcement led to more ductile behavior, controlled damage propagation, and a dramatic increase in the flexural capacity. Furthermore, CIT showed reliable results for reinforced concrete beams, unlike plain concrete beams.The numerical simulation of concrete fracture is difficult because of the brittle, inelastic-nonlinear nature of concrete. In this study, notched plain and reinforced concrete beams were investigated numerically to study their flexural response using different crack simulation techniques in ABAQUS. The flexural response was expressed by hardening and softening regime, flexural capacity, failure ductility, damage initiation and propagation, fracture energy, crack path, and crack mouth opening displacement. The employed techniques were the contour integral technique (CIT), the extended finite element method (XFEM), and the virtual crack closure technique (VCCT). A parametric study regarding the initial notch-to-depth ratio (a Employing numerical finite element (FE) simulations for different concrete elements reduces the need for further physical testing and helps researchers undertake complex parametric studies precisely. The complex nonlinear behavior of concrete in both tension and compression makes it challenging to simulate. The complexity stems from the brittle response of concrete, leading to failure due to cracking or crushing when subjected to tension or compression, respectively. Also, it is difficult to simulate crack initiation and propagation due to the tensile damage.Various constitutive numerical models were presented to simulate the concrete behavior in tension and compression ,2,3,4,5.Many studies integrated the use of different composite materials with notched concrete beams through various experiments ,16,17,18De Domenico et al.  investigChen et al.  conducteThe previously mentioned studies focused on the incorporation of different composite materials with notched concrete beams to enhance flexural tensile capacity and to improve cracking damage resistance. However, studies scrutinizing the effects of geometrical characteristics on the flexural performance of notched concrete beams are lacking. Along this research line, this paper provides numerical investigations regarding the effects of different geometrical aspects, such as loading scheme and notch height, on flexural performance. Additionally, this paper provides further numerical investigations on methods of enhancing the flexural tensile capacity and damage control performance of notched concrete beams. Unlike the studies mentioned in the literature that addressed this problem using different composite materials, the current study implemented steel reinforcement and un-bonded post-tensioning. Moreover, the simulations conducted in the current work were achieved by employing different numerical techniques in a comparative study to determine the most appropriate technique for concrete fracture simulation. o/D), the shear span-to-depth ratio (S.S/D), and external post-tensioning (EPT). The flexural behavior, damage initiation and propagation of six notched plain concrete beams having various span-to-depth ratios (S/D) were numerically investigated. All investigated beams were subjected to a three-point bending setup. Their response was captured as a relationship between loading level (P) and crack mouth opening displacement (CMOD). Three techniques for crack modeling in ABAQUS, such as the contour integral technique (CIT), XFEM and VCCT were considered, to determine the most suitable crack modeling technique. These techniques employed different material damage criteria: concrete damage plasticity (CDP), maximum principal stress (MAXPS) and critical energy release rate (Gc). The numerical results were compared with those of the experimental investigation to find out the most suitable technique for expressing the behavior and the damage of all tested beams.The current study utilizes the experimental work conducted by Yin et al. . The curo) of 60 mm at the middle of all beams was considered. According to the experimental work done by Yin et al. , a threeThis section explains the details of developing ABAQUS FE models employing CIT, VCCT and XFEM. The contour integral technique (CIT) was used to study the commencement of damage in quasi-static problems . To use /;The uniaxial behavior of concrete in tension was defined using a relationship between the tensile stresses and their corresponding cracking strains. Defining this relationship makes it necessary to convert the tensile strain  model expresses concrete damage utilizing a damage parameter ranging between zero  and one . Thus, the operative modulus of elasticity after tensile and compressive damage can be evaluated using Equations (7) and (8), respectively.Ψ) measured in the p–q plane at high confining pressure and is necessary to evaluate the potential plastic flow, which uses the Drucker–Prager hyperbolic function. The dilation angle ranges between The dilation angle designates the rate at which the hyperbolic Drucker–Prager function reaches the linear Drucker–Prager function. A value of 0.1 was used. This value guarantees that the material has nearly the same dilation angle among a wide band of confining pressure values [Flow potential eccentricity  to the initial uniaxial compressive strength (c0f) is responsible for the evolution of yield surfaces. This ratio contributes to the evaluation of the yield function proposed by Lubliner et al. [The ratio of initial equibiaxial compressive strength  of the second stress invariant on the tensile meridian (The ratio (.5 and 1 . The def.5 and 1 . μ) helps to make the tangent stiffness of the degrading material have a positive value for small time increments. This value is achieved by allowing stresses to be outside the developed yield surfaces. Using small values of µ compared to the characteristic analysis time increment tends to enhance the rate of solution convergence in the softening regime. The default value of zero [μ has no significant effect on the analysis precision. Thus, a value of 0.0007 was utilized.The viscosity parameter  was used. This element is a second-order element type, which gives higher accuracy and is effective in bending dominated problems . As resuThe virtual crack closure technique (VCCT) employs the principles of linear elastic fracture mechanics (LEFM) ,27 and IThe VCCT technique requires a predefined crack path along which to propagate . To defiA value of 0.06 N/mm was adopted for GTC . Based oThe extended finite element method (XFEM) utilizes the principles of LEFM until the crack is initiated. This technique was first proposed by Belytschko and Black , and wasTo initiate a discontinuity among the damaged elements to represent a crack, the Heaviside function The asymptotic enrichment function ectively .(15)Fα; and 5. Kling–Gupta efficiency (KGE) [2 assesses the degree of collinearity between the numerical and experimental data. The KGE assesses the correlation, bias, and variability between the experimental and numerical data, thus providing a complete similarity estimation. The last four statistical indicators have an optimal value of one. The RMSE, NSE, md, R2, and KGE were computed as shown below:2, and KGE for CIT, VCCT, and XFEM and shows that both XFEM and VCCT have better correlation to the experimental results. The XFEM managed to achieve the closest optimal value for each statistical indicator, indicating that XFEM offered the best flexural simulation.All models were developed using CIT, VCCT, and XFEM. Based on the results deduced from the T6 simulations, the most suitable technique was determined by using five different statistical indicators that are used by researchers in different fields ,38,39. Tcy (KGE) . The RMSThe fracture energy According to the experimental results , the craIt can be concluded that both XFEM and CIT are the most suitable and least suitable techniques for plain concrete beam fracture simulation, respectively. To support this conclusion and to identify the discrepancy between the two techniques, both XFEM and CIT were considered to verify the experimental results of the other beams stated in In the current study, three parameters were investigated for plain and reinforced concrete beams to study their effect on the flexural behavior. These parameters were: 1. the initial notch-to-depth ratio; 2. the shear span-to-depth ratio; 3. the use of external post-tensioning. This investigation was carried out on beam T6. For reinforced concrete beams, the reinforcement consists of two top corner bars as stirrup hangers, two bottom corner bars as the main reinforcements, and shear stirrups. All bars had a diameter of 6 mm. The spacing between the stirrups was 100 mm. The reinforcement steel grade used was the standard ASTM (A 615M/A 615) grade 300. This steel grade has a minimum yield strength of 300 MPa. A constant concrete cover of 10 mm was maintained in the current work.o/D) values of 0.3, 0.4, and 0.5 were studied. These values imply that the corresponding values of initial notch height (ao) were 45 mm, 60 mm, and 75 mm, respectively. The FE models for plain and reinforced beams were executed. o/D for plain and reinforced concrete beam models, respectively. For plain concrete beams, it can be noted that the flexural capacity increases with reduction of the ao/D. This is because reducing the ao/D increases the ligament area of the notched beam, causing increased resistance to flexural damage leading to higher flexural capacity. Furthermore, a complete flexural softening can be noticed for all XFEM results unlike CIT. For the reinforced concrete beam, it can be noted that the behavior became more ductile as there was no sudden failure. Instead, a failure plateau took place. Also, the flexural capacity was drastically increased due to the presence of reinforcement. Moreover, the flexural capacity of the reinforced concrete beam seems to be nearly constant regardless the ao/D value. This means that even with a light amount of reinforcement, damage initiation and propagation can be controlled, causing stability. Additionally, it is noted that both the CIT and XFEM models gave very similar results, as no softening occurred in the reinforced beam. Thus, it can be concluded that CIT can give reliable results for reinforced concrete beams, unlike plain concrete beams. Initial notch-to-depth ratio (aIn order to study the effect of changing the shear span-to-depth ratio (S.S/D), the model was modified to follow the four-point loading setup. As seen in External post-tensioning (EPT) rods were used in a three-point bending loading scheme at three locations and four levels of post-tensioning stress. This was study their effects on the flexural behavior of the plain and reinforced T6 concrete beams. These rods had a yield strength of 900 MPa . The EPTEach configuration was investigated at four levels of post-tensioning stress, as follows: 1. rods are present but without post-tensioning; 2. rods are post-tensioned with 25% of their yield strength; 3. rods are post-tensioned with 50% of their yield strength; 4. rods are post-tensioned with 75% of their yield strength.Two cylindrical steel anchor pins were placed at both ends of the concrete beam to connect the post-tensioning rods at both beam sides. Surface-to-surface contact interaction was used for the mutual surfaces between the steel anchor and the concrete beam. The motion of the steel anchor pins was constrained to the motion of the prestressing rod ends to apply the prestressing effect on the beam.Placing the EPT rods towards the crack mouth and increasing the EPT stress gives a better effect. This is because increasing EPT towards the crack mouth enhances the role of post-tensioning in resisting the crack opening, resulting in improved crack control.For plain concrete beam models, For plain concrete beam models, EPT rods reduce flexural softening when they are placed closer to the crack mouth. The reduction of the flexural softening occurred because the stresses were handed over to the EPT rods earlier when they were closer to the crack mouth. As result, more softening and degradation was captured for the beams having EPT rods closer to the crack tip.For plain concrete beam models, increasing the EPT stress to reach 75% of the rod’s yield strength reduced flexural softening even with a rod location at the crack tip. This indicates that this amount of post-tensioning stress is capable of enhancing damage control even if the EPT rods are not located at crack mouth.For plain concrete beam models, a clear discrepancy can be noticed between the CIT and XFEM results. The discrepancy is attributed to the stationary nature of a CIT crack that does not grow, preventing a complete failure regime. Conversely, XFEM is capable of representing complete damage softening.For the reinforced concrete beam models, For the reinforced concrete beam models, the flexural results are close as the reinforcement managed to control the damage initiation and propagation.For the reinforced concrete beam models, both the CIT and XFEM results are in good agreement. This supports the conclusion that CIT can give trustworthy results for reinforced concrete beams, unlike plain concrete beams. Additionally, this agreement reveals that using reinforcement has more performance in controlling damage than using EPT only. This is due to the complete bond with the concrete, the bond between the reinforcement bar and the concrete is achieved along the entire bar. Conversely, EPT rods are placed outside the concrete section and are bonded to the concrete at the end anchorages only.For all EPT beam models, the following findings can be drawn:Based on all previous simulations, beneficial practical implications can be interpreted. For both plain and reinforced concrete fracture simulations, the XFEM model is favored over the CIT model. However, the CIT model still can be used effectively to simulate reinforced concrete fractures. Additionally, using a minimal bonded steel reinforcement is capable of controlling the fracture damage propagation and enhancing the flexural performance significantly. Moreover, decreasing shear spans to eliminate shear stress at notched sections can contribute to the escalation of the flexural capacity and fracture control performance. For rehabilitation and renovation purposes, using unbonded external post-tensioning at the crack mouth at higher stresses can improve flexural tensile capacity and tensile damage resistance.o/D, S.S/D, and EPT, was investigated. The XFEM and VCCT models simulated flexural response in good agreement with the experimental outcomes. The CIT model showed a discrepancy for plain concrete simulations but yielded reliable results for reinforced concrete beams. Furthermore, the XFEM model was considered the most suitable, as it had better fracture energy estimation, solution-dependent crack path, and lower RMSE. It was found that increasing the ao/D and S.S/D decreased the flexural capacity. Reinforcement implementation controlled concrete damage and increased the ductility and the flexural capacity for all studied parameters. Additionally, using EPT rods and increasing EPT stress increased the flexural capacity and ductility. Also, placing EPT rods near the crack mouth reduced flexural softening. However, for higher EPT stresses, flexural softening was reduced even if EPT rods were at the crack tip. For future research, it is recommended to extend this study by investigating the effect of using different strengthening materials to replace the bonded steel reinforcement and the unbonded steel external post-tensioning. The strengthening materials to be studied may include carbon fibers, glass fibers, and shape memory alloys.This study aimed to develop numerical models using different notch modeling techniques built in ABAQUS. Notched plain and reinforced concrete beams subjected to three-point-bending and four point-bending loading setups were simulated. These notch modeling techniques were CIT, VCCT, and XFEM. The outcomes were compared with those of a pervious experimental study. Additionally, the influence of changing some parameters, such as a


FGFR2 fusions or rearrangements, IDH1 mutations, and NTRK gene fusions. Also, several clinical studies involving molecular stratification have been performed in defined patient groups, for example, BRAF V600E and HER2. Mesenchymal epithelial transition(MET)encodes a tyrosine kinase receptor and its ligand hepatocyte growth factor is a proto-oncogene. Targeting the MET signaling pathway is an effective strategy in numerous cancer types. However, the poor efficacy of MET inhibitors has been demonstrated in several phase II studies, but currently no reports have explained the potential mechanisms of resistance to MET inhibitors in BTC. In this article, we report a case of metastatic GBC with MET amplification that exhibited a rapid response to crizotinib after the failure of two lines of chemotherapy. After the patient had progressed and discontinued crizotinib, cabozantinib was introduced. Analysis of circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) indicated a loss of MET amplification status. To our knowledge, this is the first case study demonstrating the use of NGS in ctDNA to monitor the development of acquired resistance during anti-MET treatment in GBC.Gallbladder cancer (GBC) is a highly invasive disease and the most prevalent malignancy of the biliary system. Patients with GBC are commonly diagnosed at a late stage and have an unfavorable prognosis. Palliative chemotherapy has been the standard care for recurrent or metastatic disease in the past decades. Recently, several targeted therapies have been investigated in advanced biliary tract cancer (BTC) including inhibitors of genes or pathways such as  Palliative chemotherapy is the standard of care for patients with recurrent or metastatic disease; however, outcomes remain poor, with a median overall survival (OS) of <12 months. The poor outcomes in GBC are mainly due to low responses to first-line chemotherapy and the absence of effective second- or third-line therapeutic strategies. Recently, novel targeted therapies have been developed for the management of advanced biliary tract cancer (BTC) that have used next-generation sequencing (NGS) technology for patient stratification. Several targeted agents have been approved for advanced BTC by the FDA (Food and Drug Administration) including inhibitors of particular genes or pathways such as FGFR2 fusions or rearrangements, IDH1mutations, NTRK gene fusions and MSI-H status. These alterations are present in around 0.1%–20% of patients with BTC and are less frequent in patients with GBC.3 Several precision medicine trials are ongoing, for example, BRAF V600E and HER2. These data suggest that it is very necessary to carry out accurate treatment for BTC based on molecular stratification.Gallbladder carcinomas (GBCs) are the most prevalent malignancies in the biliary tract and are highly invasive diseases with late diagnosis and an unfavorable prognosis. The five-year survival rate of GBC is 5% for patients with stage III disease and 1% for patients with stage IV disease.(MET)encodes a receptor tyrosine kinase and that is activated by binding of its ligand, hepatocyte growth factor (HGF).4 Dysregulation of the MET pathway involves amplification, fusion, overexpression, and mutation.5 Several studies have indicated MET overexpression in 11%–43% of intrahepatic cholangiocarcinoma (IHCC) and 16%–80% in extrahepatic cholangiocarcinoma (EHCC). MET amplification has been reported with a fairly low frequency ranging from 0% to 7% in IHCC and EHCC, and from 0% to 18% in GBC.6 Activation of MET signaling is involved in tumor angiogenesis, metastatic progression, and acquired resistance to anti-EGFR treatment.8 In a study by Hida and colleaguesMET was also strongly correlated with tumor location, T category, AJCC stage, and perineural invasion. These factors can lead to successive local recurrence and poor prognosis in extrahepatic BTC. Therefore, targeting the MET signaling pathway is a potential treatment strategy. Combinations of multiple kinases or selective tyrosine kinase MET inhibitors (TKIs) and anti-MET antibodies, antibody–drug conjugates have been studied. For example, selective MET TKIs including capmatinib10 and the multikinase MET inhibitor crizotinib11 have shown promising efficacy in lung cancer patients with MET amplification and MET exon 14-alterations. However, the effectiveness and safety of MET inhibitors in GBC with MET amplification or mutations remains unknown.Mesenchymal epithelial transitionMET amplification that had metastasized to the lymph nodes and liver. The patient showed a rapid response to crizotinib after the failure of two lines of chemotherapy. Moreover, the MET amplification status was negative after the failure of anti-MET therapy.Here, we report a case of terminal GBC with In August 2019, a 55-year-old woman visited our clinic with mild right upper abdominal pain on a numerical rating scale (NRS) of 2–3 with a poor appetite and weight loss Fig. . MagnetiOne month after surgery, the patient had a new palpable hard nodule in the left neck that was 2 cm in diameter and had back pain. An enhanced abdomen computed tomography (CT) scan displayed a new nodule in the liver and the level of serum CA19-9 increased to over 1000 U/ml. CEA levels had also increased Fig. . The perwas identified and the copy numbers were 5, 7, 5, and 6, respectively. NGS also revealed that the tumor harbored two mutations,ATMp.G2765S Exon57 and KEAP1 p.I145Hfs*29 Exon2. After obtaining informed consent for the use of off-label targeted therapy, the patient started treatment with crizotinib at an initial dose of 250 mg q.d. in March 2020. After 2 months of treatment, the back pain was significantly reduced (NRS 2–3) and the left supraclavicular lymph node had almost disappeared. The serum CA19-9 level was significantly reduced to 383 U/ml. A partial response (PR) was observed by abdominal CT scan on reexamination with a significant reduction in the mass located in the liver as well as the retroperitoneal lymph node  performed in a CLIA and CAP-certificated laboratory. Amplification of four genesode Fig. . HoweverMET targeted-drug (cabozantinib) was precribed. Cabozantinib was administered only for 18 days because of disease progression with bilateral supraclavicular lymph node growth. To reevaluate the dynamic change of the genetic characteristics, a peripheral blood sample was assessed for circulating tumor deoxyribonucleic acid (ctDNA) using NGS with an array of 61 genes (3D medicines Inc.) as the patient could not tolerate tissue biopsy. The ctDNA analysis was negative for MET amplification, and mutation of gene GNAS p.R201HExon8. The condition of the patient (PS 3–4) rapidly deteriorated and the best supportive care was given. The patient passed away on July 24, 2020. The OS of the patient was 9 months.Unfortunately, the patient developed rapid jaundice 10 days after discontinuation of crizotinib and underwent percutaneous transhepatic catheter drainage. One week later, bilirubin levels dropped and due to adverse effects another anti-MET amplification that confirmed a rapid PR within 2 months. The patient rapidly progressed with cabozantinib treatment potentially due to tumor heterogeneity. After the patient had progressed, crizotinib was discontinued and cabozantinib was introduced. ctDNA analysis was performed by NGS and showed that MET amplification was lost. In this case, it was indicated that MET inhibitors might be effective in harboring MET amplification advanced GBC offering a potential targeted therapy option in patients with GBC carrying this lesion. Also, the loss of MET amplification may be a mechanism that can promote resistance to subsequent anti-MET therapy, although the exact mechanism remains unclear. Mutation profiling of the patient's ctDNA by NGS revealed posttreatment dynamic changes in genomic status and may be used as a novel clinical strategy for personalized therapy in refractory BTC.In this case study, the patient was treated with crizotinib after the failure of two lines of chemotherapy. NGS genomic profiling of the operative tumor specimen revealed FGFR2 fusions or rearrangements, IDH1mutations, HER2 amplifications and NTRK gene fusions. Precision therapies in BTC are still being explored. Targeted therapy, particularly agents targeting angiogenesis, have provided encouraging results, for example, regorafenib has been approved for advanced BTC by the FDA.Traditionally, systemic chemotherapy with cisplatin plus gemcitabine has been the standard of care for patients with terminal BTC. Recently, targeted therapies have been proposed in the treatment of metastatic BTC. Several targeted agents have been approved by the FDA for advanced BTC including inhibitors of special genes or pathways such as MET is an attractive new target that is involved in the pathogenesis of some malignancies. In recent years, MET inhibitors have been increasingly investigated and evaluated for the treatment of advanced BTC. Crizotinib is a small molecule tyrosine kinase inhibitor that inhibits ALK12, ROS1,13 and MET.11 IHCC patients harboring EHBP1–MET fusions present a continuous partial response for 8 months with crizotinib.14 The first phase II clinical trial of cabozantinib in terminal BTC patients using cabozantinib as a second- or third-line treatment demonstrated limited clinical efficacy in advanced refractory BTC with a median OS of 5.2 months and a median progression-free survival of 1.8 months.15 The MET inhibitor, tivantinib, has been combined with gemcitabine in BTC patients in a phase I study and patients demonstrated a partial response at 46% and stable disease at 27%.16 A randomized phase 2 study of merestinib is being conducted in patients with terminal BTC (NCT02711553).MET amplification, yet the development of acquired resistance remains a major problem. Mechanisms of resistance to targeted drugs have been investigated in patients who initially benefited from MET inhibitors; for example, those with bypass signaling and second-site mutations, MET kinase domain mutations, and MET amplifications.17 However, the mechanism of drug resistance to MET inhibitors in advanced BTC patients with MET amplification remains unknown. In this case, the patient progressed after being treated with MET inhibitors and ctDNA analysis by NGS showed that MET amplification status was lost. Similarly, loss of MET amplification has been reported in some lung cancer patients who received first- or third-generation EGFR-TKI and crizotinib at progression.18 We hypothesize that loss of MET amplification may contribute to the occurrence of drug resistance and disease recurrence.Crizotinib has shown promise as a targeted therapy in lung cancers with 19 Previous studies have shown that positive results on ctDNA NGS are highly consistent with NGS analysis of tissues in oncogenic driver alterations. Furthermore, in the study of Ikeda et al.,20 ctDNA NGS showed higher rates of MET alterations (including MET amplification) than the tissue NGS testing. ctDNA can avoid temporal and spatial (intratumor and intertumor) heterogeneity caused by tissue biopsy. Also, ctDNA may overcome the limitations of NGS in tissue biopsies including insufficient quantities of tissue and risks associated with repeated invasive procedures.21 Liquid biopsy provides the possibility to monitor genetic changes during crizotinib treatment of terminal GBC. A similar approach has been reported in non-small cell lung, colorectal, and breast cancer. These data indicate that dynamic monitoring of ctDNA is useful in selecting the appropriate therapeutic strategies to extend patient survival.ctDNA is composed of short double-stranded DNA fragments that are released into the bloodstream by tumor cells during apoptosis or necrosis. ctDNA samples can be noninvasively and conveniently obtained in real-time during the disease and can be used to detect the genetic information of the tumor. This information can be used to predict treatment responses, monitor disease relapse, and track mechanisms of therapeutic resistance.MET amplification of GBC, there was ATM mutation of this patient. ATM mutation has been reported by Zhang et al. as a good target by olaparib in GBC,22 there may be a change of response by combining PARP inhibitors. However, this patient was in poor health and she discontinued crizotinib due to intolerable treatment-associated adverse effects despite being prescribed the relevant drugs. During the late stage of disease development, the patient could not tolerate anticancer therapy as the physical status score was 3–4. Nevertheless, this is a good choice for patients of good health; meanwhile, this combined treatment strategy deserves to be explored for its efficacy and safety in subsequent studies. This patient was treated with a reduced dose of crizotinib due to poor health status. A similar case has been reported in the study of Yang et al.,13 where an elderly female patient harboring ROS1 rearrangement non-small cell lung cancer achieved a stable disease for 14 months on a reduced dose of crizotinib treatment.In addition to detecting MET amplification. Gain and loss of gene copy numbers can be more difficult to confirm in plasma than in tissue. Nevertheless, this report provides valuable proof of concept for exploring the genomic mechanisms of resistance to MET inhibitors in the clinic.This report has several limitations. Tissue was prioritized for genetic profiling after disease progression. We used peripheral blood samples to assess ctDNA using NGS because the patient could not tolerate tissue biopsy. However, ctDNA is the best alternative for when a conventional biopsy is unavailable or when insufficient quantities of DNA are available for sequencing. Also, the difference in the panel size of the genes in two tests can influence the depth of sequencing coverage and the possibility of further exploring mechanisms of resistance other than MET treatment in advanced GBC. Our data indicate that ctDNA can be a useful tool for tracking acquired therapeutic resistance and to analyze mechanisms of resistance. In the era of precision therapy, the accurate detection and identification of therapeutic targets should be prioritized in advanced BTC cases that have failed conventional treatments. This is a successful clinical case report of a GBC patient with MET amplification who received crizotinib as a monotherapy. Our findings suggest that crizotinib may be a promising therapeutic option for GBC patients with MET amplification and warrants further clinical investigation. In this study, the patient unavoidably became resistant to MET inhibitors and the mechanisms of resistance to MET inhibitors in patients with MET amplification remain unknown and require study.In summary, this is the first report using NGS of ctDNA to monitor acquired resistance during anti-


Experiments were conducted using this concrete slurry water and supernatant water under different conditions of temperature (40 and 80 °C), pressure (100 and 150 bar), and reaction time (10 and 30 min). The results showed that reaction for 10 min was sufficient for complete carbonation at a sludge solids content of 5%. However, reaction products of supernatant water could not be identified due to the presence of Ca(HCO3)2 as an aqueous solution, warranting further research.To prevent drastic climate change due to global warming, it is necessary to transition to a carbon-neutral society by reducing greenhouse gas emissions in all industrial sectors. This study aims to prepare measures to reduce the greenhouse gas in the cement industry, which is a large source of greenhouse gas emissions. The research uses supercritical CO Concrete is the most widely used construction material, and approximately 4100 million metric tons of concrete were produced globally in 2020 . Accordi3) with other clay minerals  and sintering at a high temperature above 1450 °C. The amount of CO2 generated during sintering accounts for approximately 93% of the total emissions during cement manufacturing, where 33% originates from the combustion of fossil fuels and 60% from the decomposition of limestone. The process of limestone decomposition proceeds according to Equation (1) [Cement, the main binder material of concrete, is produced by mixing and pulverizing limestone  ,8,9.Ca2 emitted from industrial sectors and sequestering it in the Earth’s strata to maintain environmental sustainability by preventing the average global temperature from reaching 1.5 °C above pre-industrial levels. However, problems such as leakage of CO2 isolated in the stratum and groundwater pollution are recognized as concerns associated with CCS [2 as carbonate minerals and utilizes it as an effective resource [2 to carbonate is very slow and inefficient under the conditions of ambient temperature and pressure, there has recently been an increasing trend in research on maximizing the mineral carbonation reaction using supercritical CO2. Existing studies on mineral carbonation using supercritical CO2 have revealed an absence of any significant effect of temperature in the supercritical state and have also shown that the carbonation efficiency increases with increasing pressure [2 are summarized in Various other industries are also responsible for emitting a significant amount of greenhouse gases, and such increase in greenhouse gas emissions leads to intensified global warming and rapid climate change . Carbon with CCS . Thereforesource . Since tpressure ,19,20,21During concrete production, the generation of concrete slurry water is inevitable. Concrete slurry water refers to the surplus concrete returned from over orders or from failure to meet the requirements of construction sites and concrete cleaning requirements, which ensure that concrete does not adhere to truck agitators or mixers in batching plants at ready-mixed concrete factories. Since concrete slurry waste is strongly alkaline, it may contaminate the soil and water and, thus, needs to be neutralized or treated with recycling equipment, such as a filter press ,23,24.2+ derived from the non-hydrated cement component; therefore, it shows promise for application as a material for immobilizing a substantial amount of CO2 [In general, concrete slurry waste is a dehydrated cake primarily obtained from air drying or filter pressing concrete slurry water upon separation into residual aggregate and coarse aggregate in a ready-mixed concrete factory. The waste contains a large amount of Cat of CO2 ,26.2 capture.However, most reported studies on concrete slurry waste have primarily explored the use of a small amount of dry/wet concrete slurry waste as a cement substitute, and there is currently a lack of reported research on the use of concrete slurry waste for large-scale CO2+ concentration increases in the supernatant water during this induction period and that CO2 may react with the ions to be sequestered in the form of CaCO3 [In addition, for supernatant water that lies above the precipitate of concrete slurry water, hydration is delayed by 0.5–2 h or more due to the coating of cement gel formed around the non-hydrated cement. It is understood that the Caof CaCO3 .2 sequestration by concrete slurry water and supernatant water by utilizing a supercritical CO2 reaction and exploring the effects of temperature (40 and 80 °C), pressure (100 and 150 bar), and reaction time (10 and 30 min).Therefore, in this study, we investigate the possibility of large-scale CO2 reaction was initiated by diluting 2 kg of supernatant water and 100 g of dried concrete slurry waste. The chemical compositions of the supernatant water and concrete slurry waste are presented in Concrete slurry water was obtained from a ready-mixed concrete factory owned by company “Y”, located in Gyeonggi-do, South Korea. The concrete slurry water extraction is illustrated in 2 reactor, comprising a gas booster  and a reactor . The reactor is equipped with an electrical heater for temperature control, an agitator for mixing, and a thermocouple and a pressure gauge for temperature and pressure measurements, respectively. The gas booster is connected to an air compressor that maintains the CO2 in its supercritical state by pressurizing the reactor vessel with CO2 gas at high pressure.2 reactor were 80 °C and 200 bar, respectively, and the internal volume of the reactor was 4 L. In addition, the agitator was designed to enable control of the rotation speed up to 400 rpm.The maximum operating temperature and pressure of the supercritical CO2 were performed using the following algorithm:The sample diluted with supernatant water and concrete slurry waste is added to the reactor, which is then assembled. The concrete slurry water used in this study is shown in 2 is injected until the desired pressure is achieved.The electric heater is powered on. Once the reactor vessel reaches the target temperature, CO2 inside the vessel reaches the target pressure, the agitator is operated at 200 rpm to perform accelerated carbonation over the specified reaction time, while maintaining the temperature and pressure.Once the CO2 is released, the reactor is disassembled, and the sample is retrieved.After the specified reaction time has elapsed, COThe supernatant water and concrete slurry waste are separated from the sample. The concrete slurry waste is dried at 105 °C until it reaches a constant weight.SEM , XRD , and TG–DTA  characterizations and pH  measurements are conducted on the dehydrated concrete slurry waste to determine the degree of carbonation reaction.Carbonation experiments using supercritical CO2, SEM, XRD, and TG–DTA analyses and pH measurements were performed on samples before and after the carbonation reaction. The pH measurements were conducted on the eluate after mixing the sample with distilled water at a ratio of 1:5. To quantify the amount of CaCO3 produced from the reaction, TG–DTA measurements were performed at a ramp rate of 10 °C/min over a temperature range from 25 to 1000 °C.To investigate the mineralogical phase transition of the concrete slurry waste upon carbonation by supercritical CO2 carbonation are shown in 2 carbonation revealed no temperature and pressure dependence, but the pH was shown to decrease by 2% as the reaction time increased from 10 to 30 min. In general, the pH of high-purity CaCO3 is 9.4; therefore, the pH of the products of the supercritical CO2 carbonation reaction (9.0–9.3) indicate the conversion of CO2 to CaCO3 via the reaction shown in Equation (2).The pH measurement results before and after supercritical CO3 converts to Ca(HCO3)2 when CO2 is supplied continuously. In generally Ca(HCO3)2 exists as an aqueous solution. It is understood that no products were formed due to the presence of Ca(HCO3)2 in its aqueous solution state because an excessive amount of CO2 is available under supercritical CO2 conditions of supernatant water that contains a small amount of Ca2+ (800 mg/L). However, since the reaction shown in Equation (2) is reversible, Ca(HCO3)2 reverts to CaCO3 at room temperature, along with release of CO2 gas, and further research is required to clarify this point [In contrast, no products were formed from supernatant water and pH measurements could not be performed. As shown in Equation (3), CaCOis point ,29.CaCO2 carbonation. In general, in the case of Ca(OH)2, a microcrystalline layer of CaCO3 is densely formed on the surface of the particle during the carbonation reaction with supercritical CO2 and diffuses inwards to approach complete carbonation as the reaction proceeds [2 carbonation, this morphology changed to microcrystalline layers on the surface of the particle regardless of temperature, pressure, and reaction time, indicating the progression of carbonation by supercritical CO2.proceeds . In this2 carbonation are displayed in 2 and a small amount of calcite were found in the XRD pattern of concrete slurry waste before reaction; after reaction, the Ca(OH)2 peak disappeared, while peaks reflecting the presence of calcite and a small amount of aragonite were detected. Further, comparing the XRD patterns of the reaction products obtained at different reaction temperatures, pressures, and times showed that similar peaks appeared irrespective of the reaction conditions. The three crystal structures of CaCO3 are aragonite, vaterite, and calcite, among which calcite is the most stable form [2+/CO32− ≤ 1. The supercritical CO2 conditions in this study also produce a large amount of CO32− and, therefore, a decrease in Ca2+/CO32− ratio indicates that calcite is the main reaction product [XRD patterns obtained before and after supercritical COble form ,30. Acco product .2 carbonation. Generally, the major cement hydrate, Ca(OH)2, undergoes pyrolysis near 450–550 °C and reacts with CO2 to form CaCO3, which pyrolyzes at 600–800 °C. The TG–DTA measurement on the sample before supercritical CO2 carbonation revealed small weight losses for Ca(OH)2 and CaCO3. Measurements after the reaction showed no signs of Ca(OH)2 weight loss, but showed signs of CaCO3 weight loss regardless of the temperature, pressure, and reaction time. Therefore, it was confirmed that the sludge with a solids content of 5% underwent complete carbonation in only 10 min via supercritical CO2 carbonation.2 reactions were shown to reduce CO2 emissions when utilized in the developed carbon capture and utilization technology using concrete slurry water produced during concrete manufacturing as a new means for CO2 sequestration. The major findings are as follows:2 conditions, reaction products could not be confirmed due to Ca(HCO3)2 existing as an aqueous solution in the presence of an excess amount of injected CO2. However, since the reaction is reversible, CaCO3 can precipitate from aqueous Ca(HCO3)2 over time at room temperature, along with the release of CO2 gas; hence, further research is necessary.For supernatant water under supercritical CO2 demonstrated that complete carbonation can be achieved in only 10 min of reaction at a sludge solids content of 5%. However, the reaction seemed to be independent of the supercritical CO2 temperature and pressure at 5% sludge solids content. Further investigation on the change in the reaction with respect to sludge solids content is to be performed.Mineral carbonation of concrete slurry water by supercritical CO2 carbonation  and CO2 storage capacity with respect to sludge solids content will be conducted.In future studies, quantitative analyses on supercritical CO2 via supercritical CO2 carbonation, and the implementation of a new CO2 fixation source with concrete slurry water is considered viable. The implementation of supercritical CO2 mineral carbonation technology using concrete slurry water will enable carbon-neutrality to be achieved by reducing greenhouse gas emissions from not only cement industries, but all industrial sectors.Collating the results, it is possible to fix COIn this study, supercritical CO


Lung cancer is one of the most common malignant tumors and it is the leading cause of cancer-related mortality worldwide. For early-stage Non-Small Cell Lung Cancer (NSCLC), surgical resection is the treatment of choice, but the 5-year survival is still unsatisfying, ranging from 60% to 36% depending on the disease stage. Multimodality treatment with adjuvant chemotherapy did not lead to clinically relevant results, improving survival rates by only 5%. Recently, immune checkpoint inhibitors (ICIs) are being studied as neoadjuvant treatment for resectable NSCLC too, after the satisfactory results obtained in stage IV disease. Several clinical trials are evaluating the safety and feasibility of neoadjuvant immunotherapy and their early findings suggest that ICIs could be better tolerated than standard neoadjuvant chemotherapy and more effective in reducing cancer local recurrence and metastasis. The aim of this review is to retrace the most relevant results of the completed and the ongoing clinical trials, in terms of efficacy and safety, but also to face the open challenges regarding ICIs in neoadjuvant setting for resectable NSCLC. Lung cancer is one of the most common malignant tumors and it is the leading cause of cancer-related mortality worldwide, with 1.76 million victims annually . Thanks However, the 5-year survival rates of these patients are still unsatisfying, ranging from 60% in stage IIA to 36% in stage IIIA . MoreoveMoreover, it is now common knowledge that resectable NSCLC benefits from a multimodality treatment rather than surgery alone. Adjuvant chemotherapy, for example, has been shown to improve 5-year survival rates by around 5%, though it cannot be defined as a successfully achieved goal .p = 0.007) compared to surgery alone [Moreover, the neoadjuvant trials showed that neoadjuvant standard chemotherapy followed by surgery for stage I to III NSCLC improved the 5-year OS by 5% (40–45%) .At the same time, we have limited evidence regarding the efficacy of the induction chemoradiotherapy (CRT) followed by surgery. The INT0139 study, a phase 3 trial, compared the standard of CRT vs. induction CRT (45 Gy) followed by surgery for pathologically diagnosed cN2 resectable NSCLC . In an eICIs improve the prognosis of patients with stage IV NSCLC ,11,12,13Recently, the IMpower010 trial revealed that adjuvant chemotherapy followed by maintenance with atezolizumab showed the significant prolongation of disease-free survival (DFS) in patients with PD-L1 TC ≥ 1% stage II to IIIA  NSCLC .Neoadjuvant therapy may control micrometastases in the early phases and may offer an opportunity to evaluate drug sensitivity. Adjuvant therapy may or may not be performed if the patients are not fit for chemotherapy after surgery. Neoadjuvant therapy can be performed with good compliance, but may cause increased postoperative complications and treatment-related adverse events.This is why there is an urgent and unmet need to seek novel and more effective treatments for NSCLC, such as neoadjuvant therapy. In particular, neoadjuvant immunotherapy with immune checkpoint inhibitors (ICIs) is being explored in an increasing number of studies and clinical trials that are moving from metastatic disease to early-stage NSCLC looking for efficacy in resectable patients too.ICIs have been successfully used against many solid tumors such as triple-negative breast cancer, melanoma, and urothelial carcinoma ,16,17. WThe clinical data from this trial stated not only the efficacy of durvalumab in prolonging the overall survival of non-resectable NSCLC, but also suggested that immunotherapy could be applied in a multimodality treatment alongside surgery too, thanks to its control over distant metastases.The rationale of using immunotherapy against NSCLC  lies in the clinical need to inhibit the tumoral pathways that downregulate the patients’ immune T-cell response. The majority of the drugs used  modulate the interaction of programmed cell death protein 1 (PD-1)/programmed cell death protein 1 ligand (PD-L1). The antibodies lead to the blockade of this immunosuppressive interaction and allow the patient’s T cells to recognize the antigen-presenting tumor cells .Ipilimumab, on the other side, modulates the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to block its pathway and to ensure the immune T-cell response .This is why the use of immunotherapy in the neoadjuvant setting seems a reasonable and effective application, in fact, the entire tumor exhibits a high number of antigens that can be linked to the antigen-presenting cells to induce a stronger and prolonged immune T-cell response, preventing tumor recurrence.The majority of the advantages in administering ICIs as neoadjuvant therapy have been highlighted by Liu and colleagues in their preclinical study . The autThere are several more benefits that make neoadjuvant immunotherapy a suitable treatment option.First of all, one of the goals of administering neoadjuvant ICIs is to reduce the size of the primary tumor and, as a result, to increase the chance of radical surgical resection, but also to control and ideally eradicate the circulant micrometastases. In fact, activated T cells run through the lymphatic and the bloodstream both to the primary tumor and against micrometastatic sites to perform their tumor-killing effect . The ratMoreover, the interaction of these drugs with the microenvironment of the tumor induces, among other things, a devascularization of the tumor itself, that may result in adhesions and fibrotic retraction. This effect may represent the downside of neoadjuvant immunotherapy, leading to a more challenging surgical field, as well as after neoadjuvant chemotherapy.In addition, administering drugs to a patient with an intact immune system gives earlier endpoints to assess the patient’s response to therapy in terms of sensitivity or resistance to it. The data collected during this phase of the therapeutic pathway enable a more accurate choice of the most appropriate treatment regimen after surgery and ineffective agents can be stopped and substituted with alternatives.Moreover, even if immunotherapy showed some adverse effects such as pneumonia, myocarditis, and neuromuscular toxicity ,24 theirNeoadjuvant immunotherapy may also shorten clinical trials and lead to a more diffuse and widely accepted use of surrogate predictors of overall survival, such as major pathological response (MPR) and pathological complete response (pCR).These short-term efficacy indicators are increasingly used as primary endpoints in the majority of completed and ongoing clinical trials.MPR, for example, has been found to correlate with long-term survival in patients with NSCLC treated with neoadjuvant chemotherapy, and its use is accepted as a reliable endpoint in this setting ,26.MPR: The resected specimen presents ≤ 10% of viable tumor cells. It is the most commonly used in the ongoing trials;pCR: The evaluation of the specimen and the regional lymph nodes does not detect any residual invasive cancer . UnfortuThe use of these surrogates may fasten the approval of immunotherapy agents too and they are defined as follows:The aim of this review is to examine the clinical results of the most relevant studies on neoadjuvant immunotherapy in resectable NSCLC and to discuss the emerging data from the most innovative ongoing clinical trials. We will also focus on the potential problems related to this new approach.Clinical data and results were found by searching PubMed for articles only in the English language from 2018 to 2021. The keywords searched were neoadjuvant immunotherapy, early-stage NSCLC, resectable lung cancer. Moreover, clinicaltrials.gov was searched by inserting the words neoadjuvant immunotherapy in NSCLC. In this review, we decided to cite the more relevant studies after an accurate screening of the ones found.The results of the most relevant clinical trials using neoadjuvant immunotherapy alone are depicted in The CheckMate 159 by Forde and colleagues  exploredThe Lung Cancer Mutation Consortium conducted the LCMC3 trial  where twThe IONESCO trial  set as tNeoadjuvant immunotherapy has been and is being tested synergically with chemotherapy. Clinical trials evaluating neoadjuvant immunotherapy in combination with chemotherapy and with or without adjuvant therapy are reported in The NADIM trial by Provencio and colleagues  assessedNeoadjuvant immunotherapy is being tested not only in monotherapy or in combination with standard chemotherapy, but dual immunotherapy is being investigated too. The phase 2 randomized trial NEOSTAR , for exaThe majority of clinical trials investigating neoadjuvant immunotherapy are still ongoing and only partial results are being released. The current studies are focusing not only on single drug neoadjuvant immunotherapy, but also on neoadjuvant chemo-immunotherapy and on multimodality treatment with neoadjuvant and following surgery adjuvant immunotherapy. The endpoints are still heterogeneous and evaluate the efficacy of neoadjuvant immunotherapy through survival surrogates  and the safety and feasibility of ICIs.The PRINCEPS trial enrolled 30 patients with clinical stage IA-IIIA resectable NSCLC . The priMoreover, the trial proved again the safety and feasibility of neoadjuvant immunotherapy, in fact, there was only one treatment-related adverse effect, a grade 1 parietal pain.The NEOMUN trial is a single-arm monocentric study that aims to study the safety and feasibility of neoadjuvant pembrolizumab in patients with resectable NSCLC stage II-IIIA . The firGrade 2–3 treatment-related adverse events happened in five patients (33%), the overall postoperative morbidity was 7% and 30-day mortality was 0%. In conclusion, neoadjuvant pembrolizumab resulted as a feasible and safe treatment.Shu and colleagues assessed four cycles of atezolizumab plus carboplatin and nab-paclitaxel in a neoadjuvant setting in 30 patients with stage IB-IIIA NSCLC . The priCheckMate 816 (NCT02998528) is a phase 3 randomized multicenter trial whose results have been recently announced. It randomized 358 patients with resectable NSCLC to receive three cycles of neoadjuvant nivolumab and histology-based platinum doublet chemotherapy or neoadjuvant chemotherapy alone. The primary endpoints are pCR and event-free survival (EFS). The group treated with neoadjuvant nivolumab reached 24% of pCR versus only 2.2% of the group matched to platinum doublet alone. The significant improvement in pCR is the first to be demonstrated in patients with NSCLC treated in a multimodality treatment with neoadjuvant immunotherapy and it is not related to PD-L1 expression. One of the secondary endpoints is MPR, which is reached in 36.9% of patients treated with nivolumab, while patients undergoing chemotherapy alone reached an 8.9% MPR rate.Moreover, 83% of patients who received nivolumab underwent surgery and achieved a complete surgical resection (R0). Surgery-related and treatment-related adverse events were similar in both arms of the study. In conclusion, CheckMate 816 demonstrated that neoadjuvant chemo-immunotherapy does not affect the feasibility of surgery and meanwhile increases pCR.Another phase 3 trial is the Aegean trial (NCT03800134), which is a double-blind multicenter study. It is still recruiting patients and it aims to randomize approximately 800 patients with stage II and III NSCLC to receive neoadjuvant durvalumab or placebo and concurrent platinum-based chemotherapy and adjuvant chemotherapy with durvalumab or placebo.The primary endpoint is pCR, while secondary endpoints comprehend safety assessments, MPR, and overall survival. The estimated study completion date is April 2024.It is also worth citing the NeoCOAST trial (NCT03794544), which evaluates in patients with stage I-IIIA NSCLC the safety of neoadjuvant durvalumab alone or in combination with novel agents oleclumab (+MEDI9447), monalizumab (IPH2201), and danvatirsen (AZD9150). The primary endpoint is MPR and the secondary includes feasibility of tumor resection and pCR. The study ended in January 2021 and its results will be likely published soon.An increasing number of trials are demonstrating the feasibility and safety of immunotherapy in neoadjuvant settings against NSCLC. Furthermore, many trials are still recruiting and their first results are encouraging.On the other hand, the majority of the collected data use surrogate predictors of survival, while the goal is to assess the impact of neoadjuvant immunotherapy in terms of overall survival of patients. This lack of uniformity in clinical trials endpoints could lead to heterogeneous results that are difficult to compare. On the other side, there is an unmet need to discover new and more effective treatments for NSCLC, which is the leading cause of cancer-related mortality worldwide and it could be counterproductive to assess long-term endpoints of five years or more. This is why pCR and, more frequently, MPR are currently used to predict patients’ response to neoadjuvant therapy, but cannot provide long-term disease monitoring which will be necessary to definitely validate the efficacy of neoadjuvant ICIs in NSCLC.Despite the encouraging results from the clinical trials, there are several challenges still to solve.For example, PD-L1 expression level, which is the main biomarker of ICIs, has to be further investigated, since studies reported conflicting results on its role in predicting the curative effect of the drugs mentioned above. In fact, while in 2014 Velcheti and colleagues  stated tTiming and the model of neoadjuvant immunotherapy are still a matter of debate too. Clinical trials are simultaneously evaluating the efficacy of single drug neoadjuvant immunotherapy, a combination of different neoadjuvant ICIs and ICIs with standard chemotherapy, as well as adjuvant chemotherapy and maintenance immunotherapy.In conclusion, the data collected show that ICIs induce promising degrees of MPR  indicating that induction immunotherapy is now emerging as the new effective standard of care for patients with resectable NSCLC. The definition of the better schedule and patient selection remain fundamental to optimize the therapeutic effectiveness and, in addition, to limit useless therapy.


Multifocal micronodular pneumocyte hyperplasia (MMPH) is a rare pulmonary manifestation of the tuberous sclerosis complex (TSC) with distinctive histological characteristics. Most case reports of MMPH associated with TSC usually have a history and typical clinical features  of TSC. We present a peculiar asymptomatic MMPH case that lacked the history and typical clinical features of TSC.A 56-year-old man was referred to our hospital with bilateral ground-glass opacities (GGOs) on chest computed tomography (CT) lasting 8 months, with no complaint of any discomfort. Because of the lack of clinical manifestations, the diagnosis of MMPH and TSC was confirmed by lung biopsy histopathology and gene sequencing of nonsense mutations in the TSC1 gene. Considering the relevant literature review and that the prognosis of most patients with MMPH is generally stable, no special treatment was given. We followed up with the patient for three years after discharge, and the clinical manifestations and imaging features of the patient were stable.To our best knowledge, this is the first case of MMPH lacking typical clinical manifestations of TSC confirmed by histopathology combined with gene sequencing. MMPH should be considered as one of the differential diagnoses of multiple GGOs in the lung even when the findings of TSC are not recognized. Multifocal micronodular pneumocyte hyperplasia (MMPH) is a rare pulmonary manifestation of the tuberous sclerosis complex (TSC) and an autosomal dominant disorder characterized by seizures, facial angiofibroma and mental retardation . Some prA 53-year-old man without respiratory symptoms had undergone chest computed tomography (CT) during a routine physical examination 8 months prior, revealing multiple ground glass opacities (GGOs) in the bilateral lungs. Considering the endemicity of tuberculosis in China he was empirically treated with isoniazid, rifampicin, pyrazinamide and ethambutol (HRZE) at a local hospital. However, no radiologic improvement occurred, and bronchoscopy showed no special abnormalities after 6 months of anti-tuberculosis treatment. Additionally, bronchoscopy showed no abnormalities except for left upper lobe bronchial mucosa congestion at the local hospital. After one month of anti-infective treatment with roxithromycin at another hospital, the radiological changes were still not improved. Thus, the patient was referred to our department for further diagnosis and treatment. In addition to a history of hypertension, he had neither a history of other diseases nor a family history of diseases. The patient worked in a woolen mill with a history of dust exposure. No special abnormality was found on physical examination.Positron emission computed tomography (PET-CT) was performed, which showed multiple subependymal calcified nodules in bilateral ventricles Fig. a, b; difTo further explore the nature of the nodules in the bilateral lungs, a lung biopsy was performed with the consent of the family. Two wedge-shaped resection specimens of the right upper lung and right lower lung were sent for examination during the operation. In the section of the specimen, more than 10 scattered gray-white and gray-yellow nodules were observed with the naked eyes, the diameter of which was 0.2–0.7 cm. Microscopically, the alveolar epithelium in the diseased region proliferated actively. Additionally, some of them grew like spikes attached to the wall, many tissue cells gathered in the alveolar cavity, and widening of the alveolar space was observed in some nodules, with fibrous tissue hyperplasia and scattered lymphocyte infiltration Fig. . ImmunohBecause MMPH is extremely rare and its cell morphology is difficult to differentiate from lung AAH, AIS and even MIA, further genetic tests were performed to identify MMPH. Molecular pathology showed no mutations in ALK, ROS1, KRAS, NRAS, BRAF, PIK3CA, HER2, RET, or EGFR. The pathological results were consistent with the histological characteristics of MMPH. According to the relevant literature , MMPH isNext, we followed up with the patient for up to three years. During this period, the patient showed no respiratory symptoms. Additionally, no significant change was found in the diffuse nodules of the bilateral lungs within 1 year Fig. . CranialMMPH, first described by Popper et al. in 1991 , is a maMost patients with MMPH are asymptomatic, and the physiology or prognosis of MMPH is unclear, with the available literature suggesting an overall stable disease course , 10. HRCTSC is an autosomal-dominant neurocutaneous disease with high phenotypic variability and is characterized by classic Vogt’s triad of facial sebaceous adenoma, epilepsy, and mental retardation , 13. TheIn conclusion, we describe an asymptomatic case of MMPH secondary to TSC diagnosed by pathological biopsy combined with genetic sequencing. Thus, MMPH must be considered in the differential diagnosis of multiple ground-glass nodules in both lungs, even in cases lacking clinical manifestations of TSC, and the diagnosis can be further assisted by imaging examination, histopathology and genetic sequencing.


Staphylococcus aureus (S. aureus) on antibiotic consumption. Enhancing the use of narrow-spectrum beta-lactam antibiotics such as flucloxacillin/cefazolin/cefalexin is one of the main goals in optimising antibiotic therapy of S. aureus infections.Antimicrobial stewardship (AMS) strategies worldwide focus on optimising the use of antibiotics. Selective susceptibility reporting is recommended as an effective AMS tool although there is a lack of representative studies investigating the impact of selective susceptibility reporting on antibiotic use. The aim of this study was to investigate the impact of selective susceptibility reporting of S. aureus (MSSA) were restricted to flucloxacillin/cefazolin/cefalexin, trimethoprim-sulfamethoxazole, clindamycin, gentamicin and rifampin/fosfomycin, instead of reporting all tested antibiotics. The impact of implementing selective reporting was analysed by monitoring total monthly antibiotic consumption in our hospital and in a reference hospital (recommended daily dose/100 occupied bed days: RDD/100 BD), as well as on an individual patient level by analysing days of therapy adjusted for bed days (DOT/ 100 BD) for patients with S. aureus bacteremia (SAB) and respectively skin and soft tissue infections (SSTI).This interventional study with control group was conducted at a tertiary care hospital in Germany. During the one-year interventional period susceptibility reports for all methicillin-sensitive P < 0.001). The use of intravenous flucloxacillin/cefazolin for SAB rose significantly from 52 to 75 DOT/100 BD (plus 42%), just as the use of oral cefalexin for SSTI . Considering the overall consumption, there was no decrease in antibiotics omitted from the antibiogram. This was probably due to their wide use for other infections.MSSA-antibiograms were acquired for 2836 patients. The total use of narrow-spectrum beta-lactams more than doubled after implementing selective reporting (from 1.2 to 2.8 RDD/100 BD, S. aureus infections. On a patient level, this assumption was verified by a significant improved treatment of S. aureus infections in the subgroups of SAB and SSTI. As useful AMS tool, we recommend implementing selective reporting rules into the national/international standards for susceptibility reporting.As narrow-spectrum beta-lactams are not widely used for other infections, their increase in the overall consumption of the entire hospital was a strong indicator that selective reporting guided clinicians to an optimised antibiotic therapy of The online version contains supplementary material available at 10.1186/s13756-021-01021-7. Significant efforts have been made to implement antimicrobial stewardship (AMS) worldwide to improve antibiotic prescribing to prevent multidrug resistance and improve patient care. There are a number of policies, strategies and tools outlined in different guidelines –3 and syOne of the recommended tools is selective reporting of antibiotics in accordance with treatment guidelines to optimize antibiotic prescribing –3, 10–12S. aureus with its large number of pathogenicity factors causes severe infections that should be treated by optimal antibiotic therapy. Narrow-spectrum beta-lactam antibiotics such as flucloxacillin or cefazolin/cephalexin have better activity against S. aureus than broad-spectrum beta-lactams such as piperacillin-tazobactam, ceftriaxone or even cefuroxime [Clostridioides difficile infections and the risk of selecting multi-resistant bacteria e.g. extended spectrum beta-lactamase-strains (ESBL) or methicillin-resistant S. aureus (MRSA). Therefore, enhancing the use of narrow-spectrum beta-lactams is one of the main goals of AMS in S. aureus infections to optimize antibiotic therapy of the individual patient and to prevent the spread of multi-resistant bacteria.furoxime –15. In aS.aureus. Changes in other AMS tools were minimised during that period in order to focus on the impact of antibiotic reporting.The aim of this study was to investigate the effect of switching from non-selective reporting of all tested antibiotics to selective reporting of recommended antibiotics in case of culturing This interventional study was conducted at the Helios Clinics of Schwerin, a tertiary care hospital in Germany with more than 1200 beds. Helios Clinic Duisburg, a tertiary care hospital of comparable size and structure (more than 1000 beds), served as a reference without intervention. The study was approved by the Ethics Committee of the Faculty of Medicine at the University of Rostock (A 2017-0149).S. aureus (MSSA) were modified in the following way: Only recommended therapeutically appropriate antibiotics for S. aureus infections were reported , clindamycin, gentamicin, rifampin/fosfomycin for combination therapy); whereas all others, especially broad-spectrum antibiotics  were excluded. The laboratory operation system  was programmed to automatically omit these antimicrobials without operator intervention, in order to minimise effort and errors of the laboratory staff. The test results were still available on request. Before the intervention, all tested antibiotics were reported on the antibiogram, predetermined by industrial panels (Table S. aureus infections was given in every susceptibility testing report to guide the clinician’s selection of the most appropriate antibiotic depending on the severity of the disease: "First choice for severe S. aureus infections/bacteremia: high dose intravenous flucloxacillin/cefazolin (ceftriaxone/cefotaxime/vancomycin are less effective in the treatment of MSSA); mild infection/oral follow-up: cefalexin, trimethoprim-sulfamethoxazole or clindamycin depending on the indication, side effects and allergies".From November 01, 2017–October 31, 2018, reports on susceptibility testing (antibiogram) of all tested methicillin-sensitive The prescribing clinicians were not informed of the ongoing study.S. aureus (MRSA).We did not change the reporting of susceptibility testing in methicillin-resistant S. aureus was detected was recorded monthly in order to have a baseline of infections for the intervention. For overall consumption of the entire hospital, the monthly use (RDD/100 BD) of each antibiotic was analysed by linear regression using an interrupted time-series approach with group comparisons (Table Clostridioides difficile infections as recommended in the IDSA guideline [To measure the effect of the intervention we monitored the recommended daily dose/100 occupied bed days (RDD/100 BD) as a standardised method for measurement of antibiotic usage. To calculate RDD/100 BD, the total monthly use of every antibiotic in the entire hospital was devided by the occupied bed days and the assumed normal daily dose . SSTI were chosen, because there had not been any prior AMS interventions in the relevant departments.We evaluated records of all patients with skin and soft tissue infections (SSTI) caused by S. aureus bacteremia (SAB) selected by the statistics program of the microbiology laboratory .Furthermore we analysed records of all patients with S. aureus positive sample in the laboratory. This specific period was chosen; because this is the period it usually takes from the detection of S. aureus to a complete antibiogram being available in the electronic patient record in our hospital. For SAB, maximum follow up was 14 days per patient , as this is the recommended minimal duration of therapy in order to limit future antibiotic usage for secondary complications. Poisson regression was used to investigate the impact of implementing selective reporting on the individual patient level. The patient bed days (BD) were used as an exposure variable. The days of therapy / 100 bed days (DOT/100 BD) were estimated for each antibiotic in the period prior to and after the implementation of selective reporting. Incidence rate ratio (IRR) and corresponding p-values were calculated to compare DOT/100 BD in regard to the two periods.Days of therapy (DOT) were evaluated by three independent reviewers. They were counted commencing on day 2 after the receipt of the first Additionally, in case of SAB the number of patients with a "reasonable" therapy adaption (defined as replacing any other antibiotic by intravenous flucloxacillin or cefazolin within day 2 to 4) was counted prior to and after intervention.Differences in patient characteristics  were tested by exact Fisher-test / Mann–Whitney-U-test. All statistical tests were two-sided and the significance level was set at 0.05. For statistical analysis, Stata/IC 16.1 for Unix was used .S. aureus infections.We refrained from any other AMS interventions during the interventional period, except for restricting the use of oral cefuroxime due to its insufficient absorption rate of approximately 50%. This was implemented in both clinics at the same time. In particular, there was no additional consultative support by infectious disease specialists in S. aureus detection in the Helios Clinics of Schwerin was not significantly different during the year prior to and after implementing selective reporting . That were approximately 0.5 patients with S. aureus infections/colonisations per 100 occupied bed days (BD), if the total occupied bed days of 549 511 during the observed two-year period is taken into account. The pre- and post-intervention antibiotic use of all available antibiotics in the Helios Clinics of Schwerin and the reference clinic is shown in Table The monthly number of patients with S. aureus infections , as well as use of oral cefalexin for less severe infections or follow-up therapy . Furthermore, an increase in the use of trimethoprim-sulfamethoxazole (p < 0.017) was detected after selective reporting was introduced. Overall, there was a statistically significant increase in the usage of selectively reported antibiotics in the interventional clinic .Before the start of selective reporting, no difference in the total consumption of the narrow-spectrum beta-lactams  could be found between the two hospitals Table . After tRegarding the course of time there was a slight monthly increase of intravenous flucloxacillin/cefazolin usage in both clinics, but after implementing selective reporting in the interventional clinic the consumption rose immediately and consolidated on a higher level Fig. . For oraRegarding the antibiotics omitted from the report, no significant change in the consumption was observed after intervention Table . The onlC. difficile infections was significantly lower in our clinic even before intervention, without a relevant change after intervention.Considering the total overall antibiotic use, there was no relevant difference between boths clinics prior to and after intervention. The incidence of nosocomial S. aureus, we found 222 patients in the year before and 190 patients in the year after intervention. Excluding all patients with MRSA infection, colonization without treatment, mixed infection with other pathogens or death/discharge before susceptibility testing (day 2), the data of 85 versus 88 patients was analysed , without a concurrent reduction in the total use of cefuroxime —despite the withdrawal of oral cefuroxime. In sum, the use of all selectively reported antibiotics nearly doubled from 20 to 38 DOT/100 BDs, reaching statistical significance. In contrast, the use of third-generation cephalosporins and carbapenemes ceased completely.Comparing days of therapy (DOT) of different antibiotics before and after implementing selective reporting Table , we founSearching the Hybase program we found 96 patients with SAB (MRSA excluded) in the year prior to versus 98 patients after implementing selective reporting. All pediatric patients and patients dead, discharged or transferred to another hospital before day 2 were excluded, as were all patients with missing medical records or allergy to penicillin .We found a significant higher use of intravenous flucloxacillin/cefazolin (recommended standard therapy for SAB) after implementing selective reporting, rising from 52.4 to 74.6 DOT/100 BD (+ 42%). The sum of therapy days of all selectively reported antibiotics (except antibiotics for combination therapy only) rose in a similar way. In exchange, there was a sharp decline of the no longer reported ampicillin-sulbactam/amoxicillin-clavunate from 69 to 4 DOT/100 BD and a trend towards lower prescription of most of the other not reported antibiotics . The total proportion of patients on flucloxacillin/cefazolin rose from 58 to 89%, including all patients who had initially received flucloxacillin.As a measure of antibiotic consumption, we additionally calculated all DOT per 1000 admissions for SAB and SSTI—without any additional information.S. aureus infections by solely reporting the most effective antibiotics on the antibiogram? A consulting infectious disease specialist (ID)/clinical microbiologist has a huge impact on the optimized therapy of S. aureus infections, but this is costly and often not possible [S. aureus infections. To date, selective reporting is poorly implemented in Europe (only in about one third of European countries), predominantly for urine cultures; only in Ireland, Turkey, the UK and Sweden is it endorsed as a standard of care by the health care authorities [Is it possible to guide clinicians to prescribe the optimal antibiotic therapy for possible , 17, 18.possible , 19, 20,possible –25 or fopossible . To our horities –29.S. aureus with high dose intravenous flucloxacillin or cefazolin is associated with lower mortality rates compared to the treatment with broad-spectrum beta-lactams or vancomycin [S. aureus, as well as to omit vancomycin, linezolid or broad-spectrum beta-lactams [There is broad consensus that narrow-spectrum beta-lactams such as intravenous flucloxacillin or first generation cephalosporins  have better activity against MSSA than broad-spectrum beta-lactams. The treatment of bacteremia caused by ncomycin , 30–35. -lactams , 20, 36.We monitored the monthly consumption of different antibiotics using RDD/100 BD, a standardised method for measuring antibiotic use, which is not influenced by fluctuating patient numbers. We compared consumption in the year prior to and after implementing selective susceptibility reporting in our clinic to the reference clinic. We used RDD instead of the frequently used defined daily dose (DDD) , 37 as tAdditionally, we analysed antibiotic use on an individual patient level using “Days of therapy” (DOT), favoured by IDSA guideline 2016 [S. aureus infections .Regarding the overall consumption of the hospital there was a significant increase of antibiotics recommended for Especially the use of narrow-spectrum beta-lactams  rose significantly after selective reporting.S. aureus infections—were analysed as an entity because they were assessed to be equally effective and replaced each other depending on side effects [Intravenous flucloxacillin and cefazolin—used in SAB and the initial therapy of severe  effects , 31, 38.p = 0.001)  Table. This inS.aureus infections in both hospitals, the overall consumption data indicates that the selective antibiogram significantly increased their use for those infections.As the use of narrow-spectrum beta-lactams was restricted to targeted treatment of p = 0.020) in SSTI (Table p = 0.002) in SAB , whereas in SAB there was a significant increase in the use of intravenous flucloxacillin/cefazolin from 52 to 75 DOT/100 BD . Selective reporting obviously strongly supported clinicians to optimize the treatment of S. aureus infections after receiving the report. In 77% of cases with SAB, the therapy was converted to a flucloxacillin/cefazolin regime on day 2–4. This is in contrast to 42% conversion rate before the introduction of selective reporting. If you include cases where treatment had initially been started on a flucloxacillin/cefazolin regime, 72 of 81 (89%) of the patients with SAB received appropriate treatment (50 of 86 before intervention). This number could hardly be further increased, since amongst the SAB group there were patients requiring broader therapy spectrum due to further infections . We did not exclude these patients because they were difficult to determine (proven versus suspected infections).This assumption was confirmed by our individual patient level data. Days of therapy (DOT) of all selectively reported antibiotics rose significantly from 20 to 38 DOT/100 BD . Neither was there an impact on the number of C. difficile infections. We didn´t expect this anyways due to the wide use of these broader spectrum antibiotics for other infections, e.g. sepsis, pneumonia or meningitis. There was even a slight (partly significant) rise in the usage of broad-spectrum antibiotics such as carbapenems and vancomycin in our clinic. In effect this led to an aligning with the significantly higher baseline level of piperacillin-tazobactam, carbapenems, fluorochinolons and vancomycin usage reported by the reference clinic for the pre-interventional period .Considering the overall consumption of the hospital  did not reach statistical significance however. This was probably due to low case numbers (86 vs. 81) and polymicrobial infections (see above). In the SSTI group  and led to a higher consumption per BD . Additionally average age and number of comorbidities increased. More patients with sepsis needed more wide-spectrum antibiotics .S. aureus infections such as SAB. This is certainly true for our hospital. Therefore, it´s likely that the advantage of implementing selective reporting might have been shown more clearly in clinics without prior AMS activities.Furthermore, a rise of AMS counselling and interventions within the last decade has already had a significant impact on the treatment of S. aureus on antibiotic use. There is a strong indication that selective antibiotic reporting improves the therapy of S. aureus infections by enhancing the use of narrow-spectrum antibiotics.This interventional study is, to our knowledge, the first prospective study to prove the impact of selective reporting for Thus, selective reporting of recommended antibiotics is a useful AMS tool, which can be easily implemented with few personnel and technical efforts. We recommend implementing selective reporting rules in the national and international standards for susceptibility reporting.Additional file 1. Flow chart - exclusion criteria of skin and soft tissue infections SSTI.Additional file 2. Flow chart - exclusion criteria S. aureus bacteremia (SAB).


Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) are common pathogens encountered in infected cardiovascular-implantable electronic device (CIED). Continuous, in-situ targeted, ultra-high concentration antibiotic (CITA) treatment is a novel antibiotic treatment approach for localized infections. CITA provides sufficient local antibiotic concentrations to heavily infected cavities while avoiding systemic toxicity.In-vitro confirmation of the efficacy of the CITA treatment approach in simulated compartmentalized infections.™ , 4MIC, and CITA at 103MIC, for growth inhibition of high bacterial loads (106 colony-forming-units/ml) of ATCC strains of P. aeruginosa, E. coli, and S. aureus.A rapid automated bacterial culture analyzing system) Uro4 HB&LE. coli and P. aeruginosa. 4MIC level extended the delay of exponential bacterial growth. Increasing concentrations of vancomycin similarly temporarily delayed S. aureus growth. All tested antibiotics at CITA of 103MIC totally inhibited the exponential growth of the tested bacteria through 72 hours of exposure. (P<0.001).The addition of gentamicin and amikacin at 1MIC concentrations only temporarily inhibited the exponential growth of 3MIC effectively inhibited exponential bacterial growth of high loads of P. aeruginosa, E. coli, and S. aureus. This model offers preliminary laboratory support for the benefit of the in-situ antibiotic treatment, providing ultra-high concentrations directly at the compartmentalized infection site, not achievable by the conventional intravenous and oral routes.In this in-vitro model, CITA at 10 The conventional intravenous and oral routes of antibiotic delivery are limited as a single treatment modality of infected cardiovascular implantable electronic devices (CIEDs) and other prosthetic biofilm-related infections such as prosthetic joint infection , 2. This2–103 are required to eradicate high bacterial loads and biofilm [The systemic toxicity of the various antibiotic agents limit local levels at the infection site to below those effective in treating heavily infected cavities and the minimum biofilm eradication concentration (MBEC) for local biofilm growth . Ultra-h biofilm –10. It i biofilm .2–103-fold higher than the respected standard serum MIC  [The continuous, in-situ targeted, ultra-high concentration antibiotic (CITA) treatment method delivers antibiotics directly to the infection site at concentrations in the range of 10stitute) . The CITS. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) are the most prevalent soft tissue pathogens with the first two are known to form biofilms in soft tissues infection associated with the presence of foreign bodies [The rationale of local antibiotic delivery is to enhance local antibiotic soft tissue concentration and eradicate the remaining biofilm and prevent recolonization. Staphylococcus aureus  in an in-vitro conceptualized model. The conventional antibiotic susceptibility testing (AST) assays (e.g. The disk defusion test) are designed for the determination of the susceptibility of bacteria to varying antibiotic concentrations [2–103 MIC. Alternative laboratory tests and tooling are required to evaluate the efficacy of the CITA treatment method. A fast, automated bacterial culture Uro4 HB&L™ analyzing system (HB&L) was introduced by Alifax  for bacteriuria, bacteremia, and biological fluid screening [In this study, we evaluated the efficacy of the 10trations . Most cucreening . The dev3MIC, in suppressing bacterial growth of pathogens commonly encountered in localized infected wounds at high bacterial loads, 106 colony-forming units (CFU/ml). Bacterial growth rates without antibiotics (0MIC), bacterial growth rates at antibiotic concentrations representing the standard dosage for intravenous or oral routes (1MIC), and bacterial growth rates at 4MIC served as controls.The main objective of this study was to determine in-vitro the efficacy of selected antibiotics, at concentrations of 106 CFU/ml) of P. aeruginosa, E. coli, and S. aureus in order to simulate localized infected wounds. The inocula were then exposed to various concentrations of appropriate antibiotics in order to determine the effect of concentration on the rate and time to exponential bacterial growth (EBG).This was an in-vitro model using standard HB&L vials with growth medium at 37°C that were inoculated with high concentrations . The light scattering technology detects the change in bacteria concentration within a few hours of initial bacterial growth. The signals are processed by the software, which monitors the turbidity of the solution generated by the growth of bacteria during the time frame and converts the light signals to bacterial CFU/ml. A magnetic stirrer bar homogenizes the vial content before each reading, and the growth of microorganisms is monitored in real-time by displaying growth curves on the computer screen. The time of the EBG is derived from the growth curve, or the list of optical density data reads.3MIC) for each antibiotic at the respected standard MIC concentrations for each bacterial strain [Antibiotic solutions of gentamicin , amikacin , and vancomycin  were diluted in 0.9% w/v sodium chloride solution  to the desired 1MIC, 4MIC and ultra-high concentrations , E. coli (ATCC® 25922™ and 35218™) and S. aureus (ATCC® 25913™ and 25923™) were investigated. Three to five colonies of overnight grown cultures on blood agar at 35°C were suspended in sterile normal saline. Standardized organism suspensions were adjusted to 0.5 McFarland (~108CFU/ml) and diluted (1:10) in sterile normal saline solution to obtain an inoculum of bacterial concentration of 107 CFU/ml. A 0.1ml suspension of the inoculum was then added to 2 ml standard HB&L-enriched medium vials . A 0.6ml solution of each antibiotic, at final concentrations of 1MIC gentamicin at (4mg/L) for P. aeruginosa and E. coli, amikacin at (16mg/L) for P. aeruginosa and E. coli and vancomycin at MIC (2mg/L) for S. aureus, was added to the experimental vial. 4MIC and 103MIC were prepared, respectively. A 0.6ml of normal saline was added to the 0MIC controls. The vials were then placed in the incubation chamber of the HB&L device, at 37°C, with stirring. Samples were continuously monitored by the HB&L for 72 hours to determine EBG. All experimental test samples, together with controls, were prepared and monitored in parallel by the HB&L.The study design is schematically presented in E. coli and P. aeruginosa at a concentration of 106 CFU/ml were evaluated in standard vials containing 0, 1, 4, and 103MIC of either gentamicin or amikacin, respectively. EBG of ATCC strains of S. aureus at 106 CFU/ml was evaluated in standard vials containing 0, 1, 4, and 103MIC of vancomycin. The timing of EBG was established from the list of optical density data readings. EBG was determined if two consecutive absorbance readings increased by >10% over 50minutes. Vials with cultures expressing EBG were counted as an event. Since this study aimed to determine in-vitro the efficacy of selected antibiotics at 1MIC and 103MIC in suppressing EBG, this was evaluated by comparing EBG prevalence (rates) at 72 hours and time-to-event analysis between cultures. Differences in EBG rates in samples exposed to antibiotic concentrations of 1MIC and 103MIC at 72 hours were analyzed with Fisher exact probability test. Time-to-event curves were constructed for each of the bacterial strains and their corresponding antibiotic treatments. The log-rank test was used to test the differences between the time-to-event curves in cultures exposed to 1MIC and 103MIC.EBG of ATCC strains of 3MIC, were analyzed with either the Mann-Whitney test or the Kruskal-Wallis test. Data was analyzed with the aid of a dedicated software program . Proportions were approximated to the nearest decimals and P values to the nearest thousandth.To further evaluate the sensitivity of bacterial growth inhibition to antibiotic treatment, the differences in the timing of EBG were compared between the various antibiotic concentrations for each of the strains. Only cultures exhibiting EBG within 72 hours were included in this analysis. Variations in timing between the different antibiotic concentrations: 0MIC, 1MIC, 4MIC, and 10P. aeruginosa incubated with gentamicin (n = 26); P. aeruginosa incubated with amikacin (n = 18); E. coli incubated with gentamicin (n = 28); E. coli incubated with amikacin (n = 20); and S. aureus incubated with vancomycin (n = 33). In six (4.8%) cultures, there were technical errors leading to premature termination without achieving either one of the endpoints set for this experiment . The premature termination was caused by device technical failure or inappropriate interpretation of the growth pattern graph as EBG. Of these, one S. aureus culture incubated with 1MIC vancomycin and three S. aureus cultures incubated with 4MIC of vancomycin demonstrated non-EBG growth pattern as defined for this study. Two other S. aureus cultures incubated with 103MIC of vancomycin showed no growth at 48 hours when the experiment was prematurely terminated. Since these six cultures did not achieve either one of the two endpoints set for this experiment, these were excluded from the contingency analysis, but were included in the time-to-event analysis.One hundred and twenty-five cultures were set up: 3MIC.3MIC. Since only a few data points were available for the evaluation of P. aeruginosa exposed to amikacin, differences in timing of EBG observed between cultures exposed to 0MIC and 1MIC did not reach statistical significance (p = 0.057).P. aeruginosa was cultured with gentamicin at 1 MIC, this delayed EBG onset compared to 0 MIC. However, all cultures eventually developed EBG. Increasing the gentamicin to 4MIC both decreased the rate of EBG and further delayed its onset. Gentamicin at 103MIC, totally inhibited EBG in this experiment. Similar results were observed in the rest of the experiments. E. coli was exposed to different MICs of gentamicin. In this specific experiment, onset of EBG was delayed in inocula exposed to 1MIC and 4MIC compared to 0MIC. In the inocula exposed to 103MIC, no EBG was observed.E. coli and P. aeruginosa, with a similar temporary effect demonstrated for treatment of S. aureus by vancomycin. CITA at 103MIC of gentamicin and amikacin totally inhibited the growth of high bacterial loads of E. coli and P. aeruginosa throughout the entire 72 hours of the incubation period. CITA at 103MIC of vancomycin had the same effect on S. aureus.In this in-vitro model, we show that 1MIC of gentamicin and amikacin only temporarily inhibited high bacterial loads of 2–103 MIC, as a simulation of our clinical experience treating infected CIEDs with expected biofilm bacterial growth over the hardware . The minimum biofilm eradication concentration (MBEC) is within the 102-103MIC range. Our objective was to evaluate the maximum antibiotic dose, which would be both effective and safe in case of rapid drug absorption from the wound into the systemic circulation. It is for this reason that in the clinical scenario, we chose the recommended daily intravenous doses. These provide 102-103MIC of antibiotic concentration within the wound. Reducing the concentration may expose the pathogens to sub-inhibitory levels of antibiotics allowing the emergence of tolerance/resistant strains.We applied CITA in the range of 10Increasing local antibiotic concentrations was found to be directly related, not only to decreased EBG occurrence but also to the increased latency period till EBG. Our findings reemphasize the need for high antibiotic concentration at the infection site for bacterial growth inhibition of high bacterial loads. The mechanism of EBG latency and inhibition with increased antibiotic concentrations should be further explored.2-103MIC antibiotic levels at the infected cavity and 1MIC range serum levels, avoiding systemic toxicity [The maximal systemic antibiotic administration dose by either an intravenous or oral route is conventionally limited to systemic levels in the order of 1MIC depending on the antibiotic and clinical considerations . When high concentrations of antibiotics at the infection site are required, both the intravenous and the oral routes may be restricted since systemic toxic levels will be exceeded. Unlike the intravenous and oral antibiotic administration routes, the CITA method allows for the reversal of the concentration gradient, providing localized, 10toxicity .Localized infections associated with implantable devices, osteomyelitis, and heavily infected wounds are at times life-threatening conditions, imposing a substantial risk for the patients and are associated with significant medical and financial burden on the global health systems , 19, 20.2–103MIC was safe and effective [2-103MIC at the intraarticular cavity. A similar treatment pattern was observed in our patients treated for all the above indications of compartmentalized infections by CITA with vancomycin and gentamicin irrespective to tissue vascularity. Our current in-vitro findings provide early laboratory proof-of-concept justifying this clinical approach of introducing ultra-high concentrations of antibiotics to the infection site, enabling the inhibition of bacterial growth of even high levels of bacteria and localized biofilm.Various methods of local antibiotic administration have been reported. Most literature published to date deals with prevention , 26. Somffective . The senCertain limitations should be considered when extrapolating the results of this study to clinical treatment. Differences exist between a compartmentalized infection in clinical situations and the environment within a vial containing a growth medium, as used in this experiment. Differences may include: pH, oxygen partial pressures, presence of proteins, enzymes, and the presence of foreign materials. All may potentially reduce the efficiency of antibiotics in preventing EBG. However, whatever these differences may be, the antimicrobial efficacy will still be related to the concentration of antibiotics achieved within the infected cavity. It is expected that substantially higher concentrations within the infected cavity achieved via CITA will perform more effectively than lower doses achieved by the conventional intravenous or the oral routes.invitro experiment, the antibiotics were added to the vial once. Continuous administration of antibiotics at 103MIC could further delay EBG compared to a one-dose regimen, through the local buildup of antibiotic concentrations, with slow defusion release to the surrounding tissues. For example, continuous administration of vancomycin at the site of infection leads to drug accumulation. This was observed in clinical cases, where serum levels of vancomycin could still be measured 5–7 days after CITA was terminated . Higher volume compartments require increased doses in order to attain local 103MIC of the applied in-situ antibiotics on wound healing. We already referenced our clinical experience in the treatment of over 80 infected CIEDs with no evidence of wound healing impairment. Another scenario includes patients with combat injuries of the extremity terated by CITA with immediate and early wound closure [Ultra-high concentrations of antibiotics may potentially inhibit or alter wound healing. Our clinical experience demonstrates no adverse effect of 10 closure . Clinica2–103 MIC CITA concentrations, which requires alternative laboratory tests and tooling. The H&BL system allows for detecting bacterial growth in a translucent culture medium within a relatively short period of time. The time latency for EBG can serve as an effective, early indicator for the sensitivity of the infective bacteria to a specific antibiotic. The same principle may apply for early detection of bacterial resistance to empirical treatment provided by the traditional intravenous and oral routes. Further studies to determine the efficacy of additional antibiotic agents, wider range of antibiotic concentrations, combination antibiotics effect, and the adequate exposure time required to reach both bacteriostatic and bacteriocidal effects is recommended. In addition, further evaluation of the value of the CITA approach in treating additional bacteria, polymicrobial infections and biofilm growth, are advocated.Though not the main objective of this experiment, the results of this study indicate that the H&BL system allows for the rapid determination of ineffective empirical antibiotic treatment irrespective of the identification of the drug-resistant pathogen causing the infection. Early adjustment of an appropriate antibiotic treatment thus can be achieved before pathogen identification and sensitivities are determined. Determination of drug sensitivity and the optimal antibiotic therapy for various microorganisms by the conventional, standard plate culture analysis are relatively slow and are not standardized for the 102-103MIC levels in an in-vitro model simulating localized infection with high bacterial loads. These findings provide initial laboratory support and a proof-of-concept for the application of CITA as a novel approach for antibiotic treatment of compartmentalized bacterial infections.This research demonstrated the growth inhibition of different bacteria species by various antibiotics by means of in-situ administration of 10S1 File(DOCX)Click here for additional data file.