This study aims to address the issues posed by frost damage to concrete structures in cold regions, focusing on reinforcement and repair methods to increase the service life of existing structures instead of costly reconstruction solutions. Due to the limitations of conventional concrete in terms of durability and strength, this research focused on ultra-high-performance concrete (UHPC) by replacing part of the cement with recycled brick powder (RBP) to strengthen ordinary C50 concrete, obtaining UHPC-NC specimens. Mechanical tests investigated the bonding performance of UHPC-NC specimens under various conditions, including interface agents, surface roughness treatments, and freeze–thaw after 0, 50, 100, and 150 cycles with a 30% replacement rate of RBP. Additionally, a multi-factor calculation formula for interface bonding strength was established according to the test data, and the bonding mechanism and model were analyzed through an SEM test. The results indicate that the interface bonding of UHPC-NC specimens decreased during salt freezing compared to hydro-freezing, causing more severe damage. However, the relative index of splitting tensile strength for cement paste specimens showed increases of 14.01% and 14.97%, respectively, compared to specimens without an interface agent. Using an interface agent improved bonding strength and cohesiveness. The UHPC-NC bonding model without an interfacial agent can be characterized using a three-zone model. After applying an interfacial agent, the model can be characterized by a three-zone, three-layer bonding model. Overall, the RBP-UHPC-reinforced C50 for damaged concrete showed excellent interfacial bonding and frost resistance performance. In cold regions, frost damage seriously impacts the safety and durability of concrete structures . CompareHowever, conventional concrete still has limitations, including low durability and strength. These limitations and disadvantages restrict its use as a material for repair purposes ,8,9. Whi2 emissions [2 and Al2O3 content, micro-aggregate properties, volcanic ash-like characteristics, alignment with auxiliary cementitious materials, and ability to be utilized as supplementary cementitious materials [However, the high costs and environmental impacts of UHPC due to its raw materials pose challenges. Notably, the cement industry alone contributes to approximately 5–7% of global COmissions ,17,18. Tmissions ,20,21; tmissions . Relevanaterials ,24,25. Raterials ,27. Lin aterials  and Xue aterials  studied aterials  demonstrHowever, previous experimental studies have primarily focused on the interface bonding performance of UHPC with existing concrete in room-temperature and freeze–thaw environments , and reiHe Yan et al.  evaluateMARC-ANDR E et al.  used UHPThis study addresses these gaps by preparing recycled brick powder UHPC (RBP-UHPC) through partial cement replacement and assessing its bonding behavior at the interface with frost-damaged concrete. The practical significance of using recycled brick powder in high performance concrete (RBP-UHPC) is its durability and cost effectiveness. The primary purpose is to obtain the best reinforcement method and the change law of bonding performance, which provides a basis for the reinforcement and repair of frost-damaged concrete. Additionally, a multi-factor calculation formula for interface bonding strength was established according to the test data. A micro-mechanism analysis of the RBP-UHPC using scanning electron microscopy (SEM) was carried out, with the aim of gathering data to develop the engineering applications of RBP-UHPC; specifically, there are broad implications in the repair of concrete structures damaged by water freezing, salt freezing, and salt-induced stresses.52.5) produced by Mengdian Cement Industry  was utilized as the primary binder, and the main components are shown in 52.5 OPC, was employed for the interface agent. The swelling agent in the interface agent was the UEA-type expander produced by Wuhan Angsi Manpen Building Materials Co., Ltd., . It exhibited a longitudinal limit expansion rate greater than 0.02% after 15 days and a vertical limit drying rate less than 0.02% after 180 days.When producing the RBP-UHPC specimens, ordinary Portland cement , cement net slurry (A2), and cement expansion slurry (A3).These treatments were performed on the specimens placed in a triple test mold measuring 100 mm × 100 mm × 100 mm, with RBP-UHPC poured sideways. After vibrating and forming for 48 h, specimens were subjected to steam curing in a steam-curing box after removing the molds. After completing these steps, the specimens were transferred to a standard curing room for 28 days. Finally, the UHPC-NC specimens required for the test were obtained, and the YAW-2000B pressure testing machine and prismatic steel block produced in Jinan, China were used for splitting and tensile tests. All the above preparation procedures are detailed in The design test procedure for assessing the interface bonding strength of RBP-UHPC-reinforced frozen concrete was carried out in accordance with the specifications “Standard for Test Methods of Long-term Performance and Durability of Ordinary Concrete” (GB/T50082-2009)  and “TecAccording to the “Technical Requirements for Ultra-high Performance Concrete” (T/CECS 10107-2020)  and the βn for splitting tensile strength and the index γn for the rate of decrease in splitting tensile strength were introduced. βn represents the ratio of splitting tensile strength in the UHPC-NC specimens to that in ordinary C50 concrete after n freeze–thaw cycles.The interface bonding strength γn represents the ratio of splitting tensile strength in the UHPC-NC specimens after n freeze–thaw cycles to the splitting tensile strength in the UHPC-NC specimens without any freeze–thaw cycles.n freeze–thaw cycles; The interface bonding of the UHPC-NC specimens is the weakest part. The splitting tensile test specimen failure is usually near this position. Its failure location and failure form are related to many factors. By observing the interface bonding failure state, UHPC-NC specimen interface failure can be divided into the following types according to freeze–thaw cycle numbers , as illuClass C failure affects ordinary concrete failure without an interface agent. It is characterized by steel fibers protruding from the UHPC bonding surface, substantial attachment of frost-damaged concrete, significant damage to the frost-damaged concrete bonding surface, and some fibers adhering to it. Class D failure indicates the destruction of the interface agents, as evidenced by the presence of interface agents attached to the frost-damaged concrete and UHPC failure surfaces, along with a limited amount of steel fibers protruding at the UHPC interface bond. Class E failure is the destruction of interface agents and ordinary concrete, which is manifested by the adhesion of interface agents on the surface of frost-damaged concrete and UHPC, and the steel fiber protruding and partial frost-damaged concrete aggregate attached to the surface of UHPC. Class F failure is the failure of ordinary concrete when there is an interface agents, which is manifested as the fracture position of the specimen is on the side of ordinary concrete; here, there is an interface agent and a large amount of concrete crushed stone adhered to the surface of UHPC, the ordinary concrete bonding surface is seriously damaged, and a large amount of aggregate is exposed. Class G failure is failure without the interface agents; the concrete is crushed, which is manifested as concrete gravel and steel fibers adhering to the UHPC side, and the ordinary concrete damage has been crushed if it is serious. Class H failure is failure when there is an interface agent; the concrete is crushed, which manifests as an interface agent on the UHPC side, the concrete gravel is attached to it, and the ordinary concrete side has been crushed.Generally, when the failure location is nearer to the regular concrete side, the bonding effect tends to be better due to higher interface bonding strength. Conversely, when failure occurs directly at the bonding surface, the bonding effect is weaker, resulting in the lowest splitting strength for the reinforced specimen.βn and γn. Analyzed the effects of interface agents and roughness on the splitting tensile strength of specimens under water freezing cycles. This study conducted splitting tensile strength tests on UHPC-NC test blocks after water freezing and calculated the corresponding The use of interface agents was observed to enhance the interface bonding strength of the reinforced specimens. This enhancement was primarily attributed to numerous pores and microcracks within the bonding interface, resulting from frost damage to the ordinary concrete. Without interface agents, the cement slurry in UHPC could only partially fill the voids within the ordinary concrete. However, after applying the interface agents, the interface agents can make the hydration reaction of cement more complete. The hydration reaction products will grow into these holes and microcracks, filling most of the holes in ordinary concrete and increasing the mechanical bite force between the interfaces. The influence of the interface agents on the specimen decreases with an increase in the freezing cycles. By the end of the hydro-freezing cycle, the splitting tensile strength decreases at a rate that is the same or even weaker than that of the specimen without the interface agents. This is because other materials, such as steel fibers in UHPC, will reduce their shrinkage so that there is a shrinkage difference between UHPC and NC, resulting in tensile and compressive stress. This is superimposed with the tensile compressive stress caused by temperature deformation during the hydro-freezing cycle, resulting in a decrease in bonding strength and the inability of the interface agents to play an important role . Therefoγn of UHPC-NC specimens; this impact is less than the effect on the relative index βn of split tensile strength. Initially, the hydro-freezing cycle of the splitting strength of the specimen under the four interface roughness treatments aligns with the βn curve. However, during the later stage of the hydro-freezing cycle, the splitting strength of UHPC-NC specimens treated by the splitting method at the interface decreased the fastest relative index βn because the NC in the specimen was frost-damaged concrete. When the hydro-freezing cycle test was repeated, the performance worsened, resulting in its strength declining faster and faster. In the initial stage of the hydro-freezing cycle, the specimen subjected to the roughness method showed the highest splitting tensile strength with the smallest reduction rate (γn), and its slope was the smallest. As the number of freezing cycles increased, the reinforced specimens treated with the four roughness methods displayed a pattern of initially rising and subsequently declining in the rate of reduction for their splitting tensile strength. After 150 hydro-freezing cycles, splitting- and pulling-method-treated specimens decrease at splitting tensile strength γn rate, while the other three roughness treatments are closer and closer, and its slope becomes the largest. Increasing freezing cycles degrade the C50 concrete state, decreasing the splitting strength of the UHPC-NC specimens. The splitting strength of the reinforced specimen becomes smaller and smaller; the impact of the interface roughness treatment method on the splitting strength becomes smaller and smaller; the specimen treated with the splitting method gradually loses its advantage; the splitting tensile strength decreases faster and faster. This leads to an acceleration in the splitting strength reduction rate index; the reinforcement specimens under the other three kinds of interface roughness treatment become increasingly similar.βn and γn values were measured. Salt freezing cycles ranged from 0 to 50; the relative index of the splitting tensile strength in the cement paste specimens was higher than that of specimens without an interface agent. These showed 14.01% and 14.97% higher values, respectively, as shown in The splitting tensile strength test was carried out after exposing the UHPC-NC test block to salt freezing; during testing, the corresponding In contrast, specimens without an interface agent exhibited the highest reduction in tensile strength at this stage. This result highlights the pivotal role of the cement expansion slurry and interface agent in maintaining tensile strength at this specific salt freezing cycle count. At 150 cycles of salt freezing, the two indices under three different interface agents are almost the same. Because the interface agent is greatly affected by salt freezing, the cement slurry interface agent is more affected by salt freezing, and the salt freezing cycle accelerates the failure rate of the reinforced specimen.βn index of the interface bond splitting tensile strength, especially in the early stages of the salt freezing cycle. However, the influence of interface roughness on the γn index of splitting tensile strength is limited, and this effect becomes even more negligible as the salt freezing cycles progress. The decline rate indices of the splitting tensile strength of the reinforced specimens under four kinds of roughness were found to be very close. In the early stages of the salt freezing cycle, the two indices of the specimen obtained by the splitting method were found to be significantly better than those of the other three methods. After 150 salt freezing cycles, the two indices of the four interface roughness treatment methods become smaller and closer, within 10%~30%; this is less than the corresponding index of the UHPC-NC specimen underwater freezing cycle. This phenomenon can be attributed to the fact that, as the count of freeze–thaw cycles escalates, the salt solution’s water retention also escalates, resulting in a heightened water saturation within the concrete. This elevated saturation intensifies the damage caused by salt freezing in the concrete. The freezing of the solution within the pores generates concealed stresses inside the concrete, which eventually lead to substantial internal damage; this observation corroborates previous experimental research carried out by Tahmureszadeh, K. et al. [According to the findings presented in In terms of the overall freeze–thaw cycle process, both indices of UHPC-NC specimens treated by splitting method are far higher than those of ordinary C50 concrete. The splitting tensile strength is also far higher than the splitting tensile strength of frozen C50 concrete specimens, and the reinforcement effect is good.According to the previous test data analysis, a method for evaluating the interface bonding strength between RBP-UHPC and frozen concrete is proposed, which provides theoretical guidance for practical engineering applications.Researchers have introduced approaches to determine bonding strengths at the interfaces between newly placed and preexisting concrete. Wang Mengwei  establisIn this paper, through studying the influencing factors such as roughness, interface agent, and freeze–thaw conditions, we concluded that the interface bond strength of UHPC-NC is affected equally by the roughness, the interface agent, and the freeze–thaw conditions. According to the content of previous research, the multi-factor calculation formula of interface bonding strength at room temperature was established:τ represents the interface bond strength; In the formula, Based on the ratio of splitting tensile strength observed in specimens using different interface agents, the ratios are as follows: when no interface agent is present, the ratio The test data were analyzed through a regression experiment, and the relationship between splitting tensile strength and roughness was obtained as follows:2 was found to be 0.85846, and the fitting was found to be good. Therefore,The determination coefficient RAccording to the ratio of splitting tensile strength of specimens under water freezing and salt freezing environments, the following were found to be true: when the working condition is water freezing, 2 is 0.998971, and the fitting line is very close to the experimental value. Through an examination of various factors affecting the interface bond strength of UHPC-NC specimens, we determined the combined effect of the interface agent, the roughness, and the freeze–thaw conditions on the interface bond splitting tensile strength; the results are depicted in Finally, combined with all the data, the calculation formula of bond splitting tensile strength of the UHPC-NC interface was performed, as follows:According to the strength calculation formula under the freeze–thaw cycle state, the multi-factor calculation formula of interface bonding strength in the freeze–thaw cycle state was introduced. In this formula, Regression analysis used to validate experimental data, it can be concluded that the relationship between the tensile strength of splitting and the number of freeze–thaw cycles is:2 was found to be 0.945, and the fitting was found to be good. Therefore,The determination coefficient RThrough assessing the influence of different factors on the interface bonding strength of the UHPC-NC specimens, the comprehensive impact of the interface agent, the roughness, and the freeze–thaw conditions on the interface bonding splitting tensile strength is depicted in The interface bonding mechanism between RBP-UHPC and frost-damaged C50 concrete was studied by an SEM microscopic test to analyze its microscopic characteristics. The UHPC-NC bonding interface was scanned by high-magnification light microscopy, the state of the two materials at the bonding interface was analyzed, and the UHPC-NC bonding model was established.A cube test block with a length of 10 mm was taken on the bonding interface of UHPC-NC and observed using scanning electron microscopy (SEM) and the micromorphology of the UHPC-NC specimen interface under the three different interface agent treatments. The goal was to gain insights into the arrangements and distributions of various components, including the recycled brick powder, the cementitious matrix, and any other relevant constituents.It can be seen from Regarding the model of the interface bonding between preexisting and newly placed concrete, the widely acknowledged framework is the three-zone bonding model, which was introduced by Emmnos et al. . The modLyubmiov  introducThis research shows that the mechanical performance of the transition layer is poor, and the damage is severe. The thickness is generally 10–50 μm. The microscopic morphologies and mechanical properties of the transition layer determine the success of the reinforced specimen. The transition layer is the weak layer of the whole bonding area, and stress damage usually occurs in the transition layer. When we study the interface failure characteristics of the reinforced specimen, it can be attributed to the study of the failure performance of the transition layer concrete.The bonding surface of the specimen is scanned by a high-magnification optical microscope, as shown in The interface agent layer L2 is composed of the interface agent itself, and its performance is also closely related to the UHPC and ordinary concrete present. The interface agent is generally only about 3 mm deep. During the pouring and hydration process, some sodium ions and calcium ions in the UHPC will enter the interface agent layer, and the steel fiber in the UHPC will also enter the transition layer. The UHPC-NC interface agent layer can be observed through an optical microscope. The presence of fibers in the interface agent can be seen, as shown in The interface agent–UHPC bonding transition layer L3 is a gradual transition layer from the newly poured UHPC to the interface agent. The number and shape of crystals in this area are similar to those of the UHPC. However, due to the simultaneous vibration and hydration with the interface agent and the presence of fibers in the UHPC, the layer is closely bonded to the interface agent, which has little effect on the performance of the interface. It can be predicted that a three-zone–three-layer bonding model can characterize the bonding characteristics of the UHPC-NC specimens coated with interface agents.(1)Based on the findings of this study, the 30% recycled brick powder replacement rate into UHPC shows significant improvement in durability and frost resistance. The strength and relative dynamic elasticity modulus remained unchanged even after 150 freeze–thaw cycles. Therefore, the recycled brick powder UHPC can form a good adhesion with ordinary concrete, making it a valuable option for reinforcing existing frost-damaged concrete in cold areas.(2)During the initial phase of the freeze–thaw cycle, the specimens treated with the interface agent show stronger bonding at the interface as compared to specimens without the agent. However, as the number of freeze–thaw cycles increased, the beneficial impact of the interface agent on the specimens diminished gradually.(3)The bonding strength at the interface of the UHPC-NC specimens is weaker during salt freezing cycles compared to water freezing cycles. Additionally, the rate of decline in bonding strength is faster in the salt freezing environment, indicating more severe damage to the UHPC-NC specimens.(4)This study used a multi-factor formula and linear regression equation to accurately predict interface bonding strength in the UHPC-NC specimens based on factors like the interface agents used, the roughness, and the freeze–thaw conditions, aligning well with experimental results showing its accuracy.(5)Microscopic analysis of the bonding interface revealed that the bonding interface was not coated with the interface agent and showed poor bonding, due to the absence of the interface agent, large cracks, and insufficient cement hydration in the UHPC-NC. A three-zone–three-layer bonding model provides valuable insights into the intricate bonding mechanism between UHPC and NC.


Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease. Despite the promising anti-fibrotic effect, the toleration of pirfenidone (PFD) by the patients in full dose is low. Combination therapy is a method for enhancing the therapeutic efficiency of PFD and decreasing its dose. Therefore, the present study evaluated the effect of a combination of losartan (LOS) and PFD on oxidative stress parameters and the epithelial-mesenchymal transition (EMT) process induced by bleomycin (BLM) in human lung adenocarcinoma A549 cells. The non-toxic concentrations of BLM, LOS, and PFD were assessed by the MTT assay. Malondialdehyde (MDA) and anti-oxidant enzyme activity including catalase (CAT) and superoxide dismutase (SOD) were assessed after co-treatment. Migration and western blot assays were used to evaluate EMT in BLM-exposed A549 after single or combined treatments. The combination treatment exhibited a remarkable decrease in cellular migration compared with both single and BLM-exposed groups. Furthermore, the combination treatment significantly improved cellular anti-oxidant markers compared with the BLM-treated group. Moreover, combined therapy markedly increased epithelial markers while decreasing mesenchymal markers. in vitro study revealed that the combination of PFD with LOS might be more protective in pulmonary fibrosis (PF) than single therapy because of its greater efficacy in regulating the EMT process and oxidative stress. The current results might offer a promising therapeutic strategy for the future clinical therapy of lung fibrosis. This  Idiopathic pulmonary fibrosis (IPF) is an irreversible, chronic, and progressive age-based disease with unknown causes worldwide. The overall prevalence of IPF is estimated to be 3 to 9 cases per 100,000 person-years in Europe and North America . There iin vivo and in vitro studies, PFD with anti-proliferative, anti-oxidant, and anti-fibrotic features postpones IPF via attenuating EMT, collagen deposition, and fibroblast proliferation -pyridone or pirfenidone (PFD) has been proven by the US Food and Drug Administration (FDA) to treat IPF patients . More imferation , 9. Howeferation . Hence, Combination treatment is one of these choices to overcome the progression of PF regarding its higher therapeutic effects besides lower side effects and drug resistance. Compounds with the same target protein or the same pathway can benefit from combination therapy . . LOS can hinder the TGF-β/Smad signaling pathway significantly in several different tissues (Losartan (LOS) is an angiotensin II receptor antagonist and an effective agent for stabilizing lung function in IPF patients, with a relatively low toxicity profile . TGF-β c. LOS can tissues , 16. In in vitro model of lung fibrosis. BLM has been widely used in chemotherapy for many years  was used to imitate an ny years . Howeverny years . The BLMny years . TherefoMaterialsA549  was provided by Pasture Institute Cell Bank, Tehran, Iran. Dimethyl sulfoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), Malondialdehyde (MDA), Phosphate buffered saline , and penicillin-streptomycin (P/S) were purchased from Sigma-Aldrich Co. Ltd., St. Louis, MO, USA. Dulbecco’s Modified Eagle’s Medium F12 (DMEM/F12) and fetal bovine serum (FBS) were supplied from Gibco-BRL Co. Ltd., Waltham, MA, USA. Polyvinylidene difluoride membrane (PVDF) was acquired from Bio-Rad Laboratories, CA, USA. EMT antibodies against α-SMA, β- catenin, Vimentin, Zo-1, Snail1, Slug, and E-cadherin were purchased from Cell Signaling Technology Inc., USA. Antibody against GAPDH  was purchased from Santa Cruz Biotechnology, USA. BLM was purchased (MW=1512.6 g/mol) from Selleck Chemicals, Houston, TX, USA, and PFD (MW=185.22 g/mol) from Intermune Company, USA. LOS (MW=461 g/mol) is manufactured by Sigma -Aldrich Co. Ltd., St. Louis, MO, USA.Cell culture 2. A549 cells were cultured and passaged in DMEM F12 supplemented with 10% inactivated FBS and 1% solution of P/S at 37 °C in a humid atmosphere containing 5% COCell viability assay4 cells/100 μl for 24 hr. Next, the cells were treated with various concentrations of BLM (0.05 to 0.5 µg/ml), PFD (0.025 to 0.5 mM), and LOS (0.05 to 1 mM) alone for 48 hr to determine the non-toxic concentrations of compounds. After adding 10 μl of MTT (5 mg/ml) to the wells, it was incubated further three hours at 37 °C and 5% CO2. After that, formazan crystals were dissolved in DMSO. The absorbance of each group was recorded (n=3) at 570 nm utilizing a microplate reader . This absorbance is proportional directly to the number of living cells. Finally, results were presented as a percentage of control values. We selected the non-toxic concentration of BLM for further evaluation (0.1 µg/ml). Besides, we chose the non-toxic and tolerated concentration of LOS and PFD on A549 cells for the next steps. Generally, the cells were then divided randomly into five main classes: the control group ; the BLM-treated group; the BLM-LOS-treated group; the BLM-PFD treated group, and BLM with the combination of LOS and PFD (LOS+PFD)-treated group.Cell viability was examined by the MTT assay. Briefly, cells were seeded in 96-well plates at 10Scratch assay4 cells/well to measure cell migration. In this study, using in vitro scratch assay, the protective effects of LOS (0.2 and 0.5 mM) and PFD (0.2 mM) on the A549 cell-treated BLM for 48 hr were evaluated and compared. For this purpose, by using a sterile 10-100 µl pipette tip, the cell monolayer culture (~ 100% confluence) was scratched. Then, the cells were rinsed with PBS twice and substituted with fresh medium, either supplemented with test reagents or medium alone (control). After 0 and 48 hr, an inverted microscope was utilized to capture images from five independent, random microscopic fields (20). Image J analysis software was used to measure gap areas and calculate the relative cell migration of the treated to untreated control cells.A549 cells were seeded onto a 24-well plate at a density of 6× 10Measurement of MDA content, superoxide dismutase (SOD), and catalase (CAT) activities4 in a 24-well plate, and after treatment with BLM and LOS and/or PFD for 48 hr, they were collected to measure oxidative stress markers. MDA concentration was measured by washing the cells with ice-cold PBS and homogenizing them in 400 µl of 0.5% Triton X-100. The cell homogenates were utilized in the TBARS assay. MDA level was measured colorimetrically as the degraded product of thiobarbituric acid at 532 nm  determination, the homogenized cells were incubated with H protein .2•¯. A unit of Cu, Zn-SOD activity is determined as the quantity of enzyme needed for causing 50% inhibition of pyrogallol autoxidation. At 412 nm, absorption was read for SOD in U/ml , the ability of the enzyme to hinder the pyrogallol autoxidation was measured. This technique is based on the dismutation of this radical by SOD and the competition between the pyrogallol autoxidation by O in U/ml , 24.Western blottingA549 cells were treated and then homogenized using RIPA (Radioimmunoprecipitation assay) lysis buffer containing phosphatase and protease inhibitor cocktails. The Bradford method was used to measure total protein . The celStatistical analysisOne-way analysis of variance (ANOVA) was used to assess the statistical analyses after Tukey’s multiple comparisons, then Dunnett’s test for selected pairs if appropriate with Prism 8.0 . Data were presented as the mean ± standard deviation (SD). Effect of BLM, PFD, and LOS on A549 viabilitySeveral concentrations of BLM  were used in A549 cells and cell viability was evaluated by MTT assay to determine the optimal concentration of BLM for treating A549 cells to prevent excessive cell death. BLM was non-toxic at a concentration below 0.2 µg/ml, so BLM at 0.1 and 0.2 µg/ml was chosen for the next steps. Furthermore, MTT was used to examine the effect of PFD and LOS on the viability of A549 cells. We measured the cytotoxicity of five serial dilutions of PFD  and LOS  to determine the non-toxic concentrations. It was inferred from the current results that the maximum non-toxic concentration of PFD was 0.2 mM. To introduce LOS as an intervention in BLM-induced EMT, we also selected two non-toxic concentrations (0.2 and 0.5 mM) of LOS alone, as shown in Effect of BLM, PFD, LOS, and PFD+LOS on A549 cells migrationAmong 0.1 and 0.2 μg/ml BLM concentrations, the concentration of 0.1 μg/ml was better through scratch assay (data not shown). As shown in Effect of BLM, PFD, LOS, and PFD+LOS on A549 oxidative stress parametersAfter exposure of A549 cells to BLM (0.1 μg/ml), the level of MDA was significantly higher than in the control group. However, treatments significantly reversed the effect of BLM on MDA levels in A549 cells . BLM alsEffect of BLM, PFD, LOS, and PFD+LOS on EMT- involved proteins in A549 cellsAccording to vs control). LOS, PFD, and LOS+PFD reduced the expression and activity of above mentioned transcription factors (vs BLM group). Together, these results suggest that co-treatment of LOS with PFD had the most pronounced effect on BLM-induced EMT in A549 cells.In et al.’s study, combination therapy was performed as a new therapeutic strategy for IPF treatment . This study was approved by the Ethics Committee of Kerman University of Medical Sciences (IR.KMU.REC.1398.422).This research was supported by Consent for publication submission is given by all authors.No potential conflicts of interest were reported by the authors.


Relevant studies published from 2013 up to March 2023 were selected by searching Scopus, PubMed, and Web of Science. Selected imaging studies were analyzed using a modified version of the critical Appraisal Skills Programme (CASP). Ten studies encompassing 335 patients were selected. On a patient-based analysis, MET sensitivity ranged between 75.6% and 100%, resulting higher than that measured for FDG (0–100%). MET outperformed FDG for the detection of focal lesions, diffuse bone marrow involvement and mixed patterns. PET-derived parameters resulted higher for MET than for FDG, with a strong correlation with clinical variables , although FDG maintained a prognostic impact on outcome prediction. When compared to other tracers or imaging modalities, MET showed stronger correlation and inter-observer agreement than FDG. Although biased by the small cohorts and requiring confirmation through multicenter studies, preliminary findings suggest that MET–PET should be preferred to FDG for PET imaging of MM, or alternatively used as a complementary imaging modality. Some issues, such as tracer availability and the role of MET with respect to other emerging tracers , should be the topic of further investigations.The aim of this systematic review is to provide a comprehensive overview of the existing literature, comparing  Multiple myeloma (MM) represents a complex and life-threatening condition, with a wide spectrum of manifestation mainly due to the activation of two distinct pathological pathways. The first is represented by the production and accumulation of monoclonal antibodies that can precipitate in the urine and cause renal failure. The second is the activation of skeletal osteoclasts through the nuclear factor kappa-B ligand (RANKL), leading to the formation of lytic lesions and, as a further step, the appearance of pain, fractures and hypercalcemia ,2. PatieMM accounts for 0.9% of all cancer cases and is slightly more prevalent in men than in women. In recent years, we have been witnessing a dramatic drop in MM mortality rate mainly thanks to meaningful progresses in clinical management and the implementation of molecularly targeted therapies . HoweverThere are two main staging systems for MM: the international staging system (ISS) and the Durie–Salmon system (DSS). DSS takes into account immunoglobulin levels, hemoglobin and calcium concentration and the number of bone lesions to predict MM prognosis, while ISS is based on the serum beta-2 microglobulin (Sβ2M) and albumin, resulting in being easier to be computed than DSS . 18F-fluorodeoxyglucose (FDG), for the assessment of therapy response and the evaluation of minimal residual disease (MRD). Recently, new visual descriptive criteria for PET interpretation  have been gaining consensus among clinicians for use in both clinical workflow and multicenter trials  40-thiothymidine (ng = 43) . Patientn = 3, progressive disease n = 2, response evaluation n = 39) MM patients who underwent FDG and mpWB MRI for staging or response assessment, within a 10-day interval, were included. Of the enrolled subjects, MET–PET was also available for comparison in 24 cases. The following criteria were identified to categorize mpWB MRI as positive: fat fraction < 20%, diffusion-restriction, and hypointensity on the T2-weighted images. Concerning MET and FDG images, aside from qualitative analysis, the authors extracted the following parameters: SUVmax, SUVmean, SUVpeak. In addition, PET-images were scored according to DS criteria. In 37 cases, bone marrow biopsy was available as a reference. An excellent grade of agreement was registered among MRI-readers for the classification of medullary lesions as vital/non-vital. Notably, 94.1% of lesions categorized as vital on the basis of MRI also received a DS4 or DS5 on FDG and MET images. However, it has to be underlined that by comparing mpWB MRI and PET findings, the interobserver agreement was fair (k = 0.53) and strong (k = 0.79) for FDG and MET, respectively. An interesting study compared the diagnostic performance of multi-parametric (mp) whole body (WB)-MRI for medullary lesions’ detection with respect to MET and FDG–PET . An over68Ga-Pentixafor, a radiopharmaceutical targeting chemokine (C-X-C motif) receptor 4 (CXCR4), for the imaging of SSM in a retrospective analysis carried out by Zhou and colleagues [68Ga-Pentixafor were enrolled. Images of each PET imaging modality were analyzed both qualitatively and quantitatively: a ROI was placed in the center of each L2-L4 vertebra, and maximum and mean tumor-to-background ratios (TBRmax and TBRmean) were calculated by comparing vertebral SUVmax and SUVmean with the SUVmean measured in the right atrium. SUVmean and TBRmean measured in L2-L4 on MET and 68Ga-Pentixafor correlated with bone marrow (BM) plasma cell (PC) infiltration, while this relationship was not found for FDG. FDG and MET were both compared with lleagues . Ten patFrom the analysis of the selected papers, some considerations about the role of FDG and MET for the imaging of MM can be made. MET outperformed FDG in terms of sensitivity and diagnostic accuracy for the PET imaging of MM at staging and for the detection of a viable tumor after therapy. In particular, MET was more capable to accurately identify MM extramedullary localizations and define the grade of bone marrow involvement, with a strong correlation with the histological findings. Notably, overall tumor burden determined on MET images showed a strong correlation with clinical variables and impacted on patients’ prognosis, thus reflecting the severity of MM involvement ,22. http://www.pharmacopeia.cn/v29240/usp29nf24s0_m13060.html, accessed on 27 March 2023), MET did not find a widespread application in clinical practice due to several issues. First of all, the short half-life of 11C limits the use of these tracers to the PET centers equipped with an in situ cyclotron. Second, 11C-radionuclide has a suboptimal positron energy for PET imaging, thus negatively impacting on image quality and spatial resolution [18F-fluorine, namely 18F-fluciclovine or 18F-FACBC [18F-ethyl-tyrosine (18F-FET), another amino-acid tracer used in neuro-oncology, in 32 MM patients; although it was not compared to FDG, 18F-FET showed good diagnostic performance, detecting a greater number of lesions with respect to CT in subjects with a newly diagnosed disease [However, in spite of these promising results and although it can be prepared “in house” and employed for clinical use, insofar, as it has been established by current pharmacopeia (8F-FACBC , recentl8F-FACBC . On the It has to be underlined that, although MET showed superior diagnostic performance with respect to FDG for MM staging and post-treatment evaluation, FDG maintained a prognostic impact to predict survival in relapsing patients and correlated with high-risk cytogenetic ,28,29. IAn interesting role for MET would be found in the identification of “high-risk” SMM rather than FDG; indeed, both Zhou et al. and Lapa et al. ,19 descrNone of the selected manuscripts employed hybrid PET/MRI for MET and FDG imaging . The com68Ga-pentixafor/177Lu-pentixather” holds promise to move the field forward [Finally, MM cannot be left behind in the so-called “theranostic revolution”. Theranostics combines diagnosis and therapy in a unique approach, since it involves the sequential administration of a couple of identical or very similar radiopharmaceuticals, both targeting a specific tumor-associated biomarker; the first one labeled with a nuclide suitable for imaging with PET or gamma-camera, the second one conjugated with a nuclide emitting beta or alpha particles to exert anti-tumor effects ,40,41. I forward ,43. HowePreliminary results emerging from the analysis of the existing literature, although biased by the small cohorts of the included studies and requiring further confirmation through multicenter cooperations, suggest that MET should be preferred to FDG for the assessment of MM patients at staging and after therapy, although FDG might be considered in well-selected, high-risk subjects for prognostic stratification. Some issues, concerning tracer availability and its role with respect to other emerging imaging modalities, should be addressed by further investigations.


Antibiotic resistance among hospitalized patients is a growing concern due to irrational antibiotic consumption. Exploring the dynamics of antibiotic consumption including healthcare-seeking behavior is indispensable to minimizing the antibiotic resistance burden. This study explored the pattern of antibiotic consumption and healthcare-seeking behavior of patients before being admitted to tertiary hospitals in Bangladesh.Between August and September 2022, we conducted this cross-sectional study in four tertiary care ( 2 public and 2 private) hospitals. A total of 943 admitted patients were randomly enrolled and face-to-face interviews were conducted to obtain data on antibiotic usage history, healthcare-seeking patterns, and duration of medical consultation. Descriptive statistics were used for the analysis and interpretation.Prior to hospitalization, 32.8%  of patients had antibiotic consumption history within the previous two months without lab confirmation. About one-quarter (25.6%) of the participants sought antibiotic treatment from unregistered physicians. Patients admitted to private hospitals were found to be higher to seek treatment from unregistered sources compared to public hospitals . Less than two-thirds (63.8%) of patients completed their entire course of antibiotics before admission to the hospitals. The average duration between symptom onset and medical consultation was 5.6 (SD 1.3) days before hospitalization. In terms of place for healthcare seeking, the majority of participants (52.0%) visited government hospitals, followed by private hospitals (22.6%), and pharmacies (20.1%). Self-medication was found in 5.3% of participants before being admitted to the hospital. However, 72.2% of patients were prescribed antibiotics after admission and only 5.3% had a laboratory diagnosis before receiving antibiotics.Hospitalized patients were found to consume antibiotics more frequently before and after admission. Furthermore, healthcare seeking from unregistered sources, including delayed and incomplete treatment could hindrance to combating against antibiotic resistance among admitted patients.All Authors: No reported disclosures


EuroLung Risk scores were established to predict postoperative morbidity and mortality in patients undergoing anatomic lung resections. We aimed to perform an external validation of the EuroLung scores, which were calculated from data of the European Society of Thoracic Surgeons database, in our video-assisted thoracoscopic surgery cohort.All available EuroLung scores were calculated for 718 patients scheduled for anatomic video-assisted thoracoscopic surgery resections between 2009 and 2019. Morbidity and mortality according to the definitions of the EuroLung scores were analyzed in a prospectively maintained database.η = 0.155–0.174). The EuroLung1 app score showed the biggest area under the receiver operative characteristic (ROC) curve with 0.660. Binary logistic regression analysis showed that predicted postoperative forced expiratory volume in 1 s was associated with increased complications in both EuroLung1 and parsimonious EuroLung1 scores. Thirty-day mortality was 0.7% (predicted 1.10–1.40%) and was associated with predicted postoperative forced expiratory volume in 1 s for both EuroLung2 and parsimonious EuroLung2 scores. The EuroLung2 (2016) showed the biggest area under the ROC curve with 0.673. Only a very weak eta correlation between predicted and observed mortality was found for both aggregate EuroLung2, EuroLung2 (2016), EuroLung2 (2019), and parsimonious EuroLung2 (2016) (η = 0.025/0.015/0.011/0.009).Overall observed complication rate was 10.45%. Scores showed weak individual correlation (EuroLung scores help to estimate postoperative morbidity. However, even with the highest aggregate EuroLung scores possible only 50% suffer from postoperative morbidity. Although calibration of the scores was acceptable, discrimination between predicted and observed events was poor. Therefore, individual correlation between predicted and observed events is weak. Therefore, EuroLung scores may be best used to compare institutional quality of care to the European Society of Thoracic Surgeons database but should not be used to preclude patients from surgical treatment.The online version contains supplementary material available at 10.1007/s00464-022-09786-7. In the coming years the rate of surgically resectable early stage lung cancers will be on the rise, as lung cancer screening routines are established , 2. As aTreatment planning in resectable lung cancer usually relies on an algorithm evaluating the fitness for surgery and is mainly focused on cardiac and pulmonary function testing. However, there are other medical conditions increasing the risk of postoperative complications and surgical mortality that are currently not routinely considered. Thoracic surgeons are trying to establish risk scores which may provide some decision guidance when interdisciplinary tumor boards have to weigh a primary surgical therapy approach against other treatment modalities, such as stereotactic body radiotherapy or radiofrequency ablation. The ESTS EuroLung scores, based on the ESTS database, represent such a tool for the risk calculation of 30-day postoperative morbidity and 30-day postoperative mortality based on clinical values –6. The En = 4) were excluded. To preemptively avoid possible confounders patients over the age of 80 (n = 20) were excluded, because this patient cohort represents a very preselected group with above-average performance status at our department . No other exclusion criteria were used. 718 patients remained for further analysis. Permission for retrospective data analysis of our VATS cohort was granted by the local ethics committee .All patients scheduled for an anatomic VATS resection for primary lung cancer at our department from 02/2009 to 07/2019 were retrospectively analyzed. Due to our ethical directive patients under the age of 18 , chronic kidney disease (CKD), cerebrovascular disease (CVD), body mass index (BMI), type of resection , predicted postoperative forced expiratory volume in one second (ppoFEV1), postoperative morbidity, death.Postoperative morbidity was defined according to the EuroLung score and included, but was not limited to: respiratory failure, prolonged mechanical ventilation (> 24 h), acute heart failure, reintubation, pneumonia, atelectasis requiring a bronchoscopy, pulmonary edema, prolonged air leak, embolism, acute respiratory distress syndrome (ARDS), stroke, transient ischemic attack (TIA), acute kidney failure, arrhythmia requiring treatment, myocardial infarction. In accordance with the definition of the EuroLung scores postoperative morbidity and mortality was only included if it occurred during the first 30 days after surgery.Pneumonia definition in our database matches the definition of Fernandez et al. .EuroLung1 and EuroLung2 score from 2016 and 2019, seen in Table Statistical analysis was performed using IBM SPSS Statistics 26  and included the methods recommended by Altman et al. for external validation of prognostic tests .Pearson’s chi-squared test or Fisher’s exact test were used for identifying relationships between categorical variables. One-way analysis of variance was used for comparing means between various numerical variables. In all EuroLung scores binary logistic regression was used to test their computational variables for significance between our patient groups with and without EuroLung morbidity/mortality. This is to examine whether the variables of our cohort differ from the study by Brunelli et al. . Hosmer–p-value < 0.05.Results were expressed as means. Statistical significance was assumed for a A total of 718 patients were analyzed. Overall patient characteristics and respective morbidity and mortality characteristics are shown in Supplementary Table 1, 2, 3, and 4.Every patient in the cohort was scheduled for a primary anatomic VATS resection for primary lung cancer (100%). Our observed 30-day morbidity was 10.45% and observed 30-day mortality was 0.70%.In our cohort 75 out of 718 patients 10.45%) suffered from postoperative morbidity, as defined by Brunelli et al., in the first 30 days after surgery and this rate was lower than the calculated EuroLung scores Table  . 5% sufferp =  < 0.001; EuroLung1 (2019): p =  < 0.001; parsimonious EuroLung1 (2016): p =  < 0.001; parsimonious EuroLung1 (2019): p =  < 0.001; EuroLung1 App: p =  < 0.001). Patients with morbidity also showed a significantly higher EuroLung1 aggregate score , vs 5.54 ; p = 0.001).Using the various EuroLung scores, the calculated morbidity ranged from 11.11 to 20.85%. The parsimonious EuroLung1 (2019) showed the most accurate prediction with 11.11%  in comparison to the cohorts observed morbidity rate of 10.45%. Patients with a postoperative morbidity showed significantly higher EuroLung scores in all available morbidity scores than patients without (EuroLung1 (2016): η = 0.155; EuroLung1 (2019): η = 0.168; parsimonious EuroLung1 (2016): η = 0.156; parsimonious EuroLung1 (2019): η = 0.174; EuroLung1 App: η = 0.173; EuroLung1 aggregate score: η = 0.122). In accordance with these results the AUROC was 0.660 for the EuroLung1 App, 0.646 for the EuroLung1 (2019), 0.645 for the EuroLung1 (2016), 0.642 for both parsimonious EuroLung1 scores (2016 & 2019), 0.599 for the EuroLung1 aggregate score and did not proof high discrimination. The parsimonious Eurolung1 (2019), which showed the most accurate prediction and the highest η-value, had a statistically different AUROC than the EuroLung1 aggregate score (p = 0.010) and the EuroLung1 App (p = 0.032). The rest of the EuroLung scores showed no statistically different AUROC. The EuroLung1 App showed a significantly better discrimination than the EuroLung1 aggregate score (p = < 0.001) and both parsimonious EuroLung1 scores (p = 0.032/0.032), but not for the EuroLung1 (2016) (p = 0.220) and EuroLung1 (2019) (p = 0.217). Respective ROC curves are shown in Fig. All EuroLung scores only showed a weak individual correlation (EuroLung1 (2016): p = 0.958; parsimonious EuroLung1 (2016 & 2019): p = 0.996; EuroLung1 aggregate score: p = 0.919].The Hosmer–Lemeshow test for goodness of fit was not significant for all morbidity scores and therefore valid [EuroLung1 (2016 & 2019): a = −0.007/−0.007). Moreover, they also showed the tightest estimation spread with their respective calibration slopes being the closest to 1 (b = 0.935/0.911).Calibration-in-the-large showed a graphical trend toward systematically too high predictions, while at the same time showing too extreme risk estimations in the calibration slope, as visualized in Fig. p = 0.041, parsimonious EuroLung1 (2016 & 2019): p = 0.042). Male gender showed to be a significant risk factor for the aggregate EuroLung1 score (p = 0.025).For further investigation of the impact of risk factors for morbidity a binary logistic regression analysis was performed for each risk score. For the EuroLung1 and parsimonious EuroLung1 (2016 & 2019) lower ppoFEV1% was associated with a higher risk for postoperative complications (EuroLung1 (2016 & 2019): The relationship between the EuroLung1 aggregate score and our observed morbidity rate is shown in Fig. p = 0.547).A subgroup analysis did not show a difference in observed morbidity for patients with neoadjuvant therapy  and was lower than predicted in any EuroLung score. The closest result was estimated by the parsimonious EuroLung 2 with 1.10% , followed by EuroLung2 (2019) with 1.11% , EuroLung2 App with 1.29% , and the EuroLung2 (2016) with 1.40%  in comparison to the cohorts observed mortality rate of 0.7%. The relationship between 30-day mortality and demographic data, risk scores, and perioperative morbidities are shown in Table p = 0.695; EuroLung2 (2019): p = 0.769; parsimonious EuroLung2: p = 0.811; EuroLung2 App: p = 0.983). Also, EuroLung2 aggregate scores (2016 & 2019) did not differ between groups .Patients with observed mortality did not show significantly higher EuroLung scores (EuroLung2 (2016): η = 0.025; EuroLung2 (2016): η = 0.011; EuroLung2 (2019): η = 0.015; parsimonious EuroLung2: η = 0.009, EuroLung2 App: η = 0.000). In accordance with these results the AUROC was 0.673 for the EuroLung2 (2016), 0.656 for the EuroLung2 (2019), 0.645 for the parsimonious EuroLung2, 0.641 for the EuroLung2 App, 0.610 for the aggregate EuroLung2 (2016) and 0.596 for the aggregate EuroLung2 (2019) and did not proof high discrimination. The AUROC of all available EuroLung scores showed no statistically significant difference, when compared between themselves. Respective ROC curves are shown in Fig. All EuroLung scores showed only a very weak individual correlation (both EuroLung2 aggregate score (2016 & 2019): p = 0.937; parsimonious EuroLung2: p = 0.961; EuroLung2 aggregate score (2016): p = 0.926; EuroLung2 aggregate score (2019): p = 0.313).The Hosmer–Lemeshow test for goodness of fit was not significant for all mortality scores and therefore valid (EuroLung2 (2016 & 2019): p = 0.040, p = 0.033). For the parsimonious EuroLung2 only ppoFEV1% showed significance (p = 0.030) and for the EuroLung2 aggregate score (2016) CAD showed significant impact (p = 0.025). For the EuroLung2 aggregate score (2019) no significant variable was found.For the computational variables, binary logistic regression for the EuroLung2 (2016 & 2019) scores showed lower ppoFEV1% and CAD being significant risk factors for mortality . We found a statistical significant difference in postoperative mortality in the group of patients after solid organ transplantation, compared to the group of non-transplant patients (p < 0.001).Two patients died of ARDS, two patients suffered a lethal sepsis and one patient suffered from both complications and died subsequently. Noteworthy, all patients had low EuroLung2 aggregate scores see Fig. . Interesp = 1.000).A subgroup analysis did not show a difference in observed mortality for patients with neoadjuvant therapy , showing insufficient precision. Although the EuroLung1 (2019) showed a rather good calibration with an intercept of −0.007 and a calibration slope of 0.935 the discrimination was weak with a c-statistics of 0.646.Our results show that the parsimonious EuroLung1  displays the best correlation with our cohort´s observed morbidity rate of 10.45%. Despite this, the correlation with individual patient morbidity was only weak  was lower than the one predicted with ESTS EuroLung2 scores. Further analysis showed that lower ppoFEV1% correlated with higher 30-day mortality. Also, we found a high rate of mortality in patients with a history of solid organ transplantation (23.1%). A higher 90-day mortality after surgical treatment of lung cancer in patients after solid organ transplantation was also described recently by Drevet et al. . Solid oTo investigate possible confounders for this discrepancy between expected and observed morbidity and mortality we compared the patient characteristics of the ESTS database with our own VATS database. Our patients showed a lower ppoFEV1% (72.7 vs. 62.9) and a higher amount of diabetes (2.7% vs. 12.5%). In contrast to the EuroLung database our cohort consists of only VATS patients (vs. 13.1% and 26% in the ESTS database at the time of publication of the EuroLung scores 2016 and 2019), which might decrease postoperative complication rate, as a VATS approach has shown to reduce postoperative morbidity such as pneumonia, intensive care admission, bleeding or the need of reoperation. Even in the case of conversion to open surgery primary VATS cases do not show higher complication rates , 14–16. Regarding postoperative mortality, the lowest predicted number of events was 50% higher than the actual observed mortality (1.1% vs. 0.7%), again showing only weak individual correlation. The reason for the discrepancy is unclear. On the one hand, benefits of minimally invasive surgery might be underestimated in the EuroLung scores due to the low number of VATS procedures in the ESTS database. On the other hand, as shown by Decaluwe et al., almost 25% of 30-day mortality after a scheduled anatomic VATS resection is linked to major intraoperative complications, which cannot be predicted . HoweverPerhaps future EuroLung scores will perform better on VATS cohorts, as the number of VATS data in the ESTS database is growing. As Moons et al. recommend, a prognostic model not performing well in new populations should rather include the new patient data than establish a new model . Also, wAccording to our results, the EuroLung scores can be used to benchmark quality of care in Europe, but should not be used to preclude patients from surgical treatment of lung cancer due to its weak individual correlation. The various risk scores can be used for a more detailed patient consenting, to set expectation within reason, but also to screen for patients who might benefit most from preoperative rehabilitation efforts. The inclusion of other clinical factors such as frailty scores, or sarcopenia screening might improve the accuracy of the risk scores.The fact that our database consists only of primary VATS patients might influence study outcome, as the prognostic EuroLung scores have been established on a mixed cohort with a rather high thoracotomy rate.The retrospective character is no limitation of this study as the study design was set as an external model validation study. Although treatment methods and patient selection throughout the years might have changed, it should not impact the validity of our result, because the ESTS database, on which the EuroLung scores are based on, includes patients between June 2007 and December 2018.Interpretation of our validation of EuroLung2 scores in our study has to be undertaken with caution, as the study population had a rather low number of events. Therefore, also no adequate calibration analysis was possible.Decision for or against surgery for lung cancer remains a highly individual decision for each patient and should not be based upon currently available risk scores. A calculated risk score should not inhibit patients from receiving surgery for lung cancer. Risk score calculation should rather be used for improved patient consenting and comparison of postoperative outcome with other departments. Currently, many large retrospective databases, such as the ESTS database, lack promising new risk factors making it difficult if not impossible to establish more precise risk prediction models with these databases. Future efforts should aim at including these variables, such as sarcopenia or history of solid organ transplantation, for further adaptions of the risk score.Supplementary file1 (DOCX 17 kb)Supplementary file2 (DOCX 17 kb)Supplementary file3 (DOCX 17 kb)Supplementary file4 (DOCX 14 kb)Below is the link to the electronic supplementary material.


This study aimed to characterise antibiotic prescribing and dispensing patterns in rural health facilities in China and determine the community prevalence of antibiotic resistance. We investigated patterns and drivers of antibiotic use for common respiratory and urinary tract infections (RTI/UTI) in community settings, examined relationships between presenting symptoms, clinical diagnosis and microbiological results in rural outpatient clinics, and assessed potential for using patient records to monitor antibiotic use. This interdisciplinary mixed methods study included: (i) Observations and exit interviews in eight village clinics and township health centres and 15 retail pharmacies; (ii) Urine, throat swab and sputum samples from patients to identify potential pathogens and test susceptibility; (iii) 103 semi-structured interviews with doctors, patients, pharmacy workers and antibiotic-purchasing customers; (iv) Assessment of completeness and accuracy of electronic patient records through comparison with observational data. 87.9% of 1123 recruited clinic patients were prescribed antibiotics (of which 35.5% contained antibiotic combinations and >40% were for intravenous administration), most of whom had RTIs. Antibiotic prescribing for RTIs was not associated with presence of bacterial pathogens but was correlated with longer duration of infection (OR = 3.33) and presence of sore throat (OR = 1.64). Fever strongly predicted prescription of intravenous antibiotics (OR = 2.87). Resistance rates in bacterial pathogens isolated were low compared with national data. 25.8% of patients reported antibiotics use prior to their clinic visit, but only 56.2% of clinic patients and 53% of pharmacy customers could confirm their prescription or purchase included antibiotics. Diagnostic uncertainty, financial incentives, understanding of antibiotics as anti-inflammatory and limited doctor-patient communication were identified as key drivers of antibiotic use. Completion and accuracy of electronic patient records were highly variable. Prevalence of antibiotic resistance in this rural population is relatively low despite high levels of antibiotic prescribing and self-medication. More systematic use of e-records and in-service training could improve antibiotic surveillance and stewardship in rural facilities. Combining qualitative and observational anthropological methods and concepts with microbiological and epidemiological investigation of antibiotic resistance at both research design and analytic synthesis stages substantially increases the validity of research findings and their utility in informing future intervention development. Antimicrobial resistance (AMR), and especially resistance of bacterial pathogens to antibiotics , is recognised as a major global health concern –3, with This paper presents findings from an interdisciplinary study to characterise antibiotic prescribing and dispensing patterns in rural health facilities of Anhui Province and measure the community prevalence of antibiotic resistance. Our aims were to identify key drivers of and patient pathways to antibiotic use (ABU) for common infections, assess relationships between presenting symptoms, clinical diagnosis and microbiological diagnosis in rural healthcare settings (which do not have laboratory facilities for culturing samples), and evaluate the potential for using patient records to monitor ABU. Such evidence is essential to support the development of effective interventions to promote antibiotic stewardship and reduce the burden of antibiotic resistance, as well as to identify accurate methods for monitoring changes in ABU and ABR over time. We chose to focus on outpatients presenting with urinary tract infections (UTI) and respiratory tract infections (RTI) as they comprise a high proportion of outpatient consultations for which antibiotics are used in general practice/primary (first-level) care, a setting which accounts for roughly three-quarters of antibiotics prescribed worldwide . This paAt the base of the medical system in rural China are village clinics (outpatient only) and township health centres (which have inpatient beds);  in urbanVillage clinics are staffed by village doctors who trained and are certified as Village Practitioners but do not have degree level medical qualifications, unlike the licensed doctors who work at township health centres and in higher tier health facilities . Around This interdisciplinary study comprised three main components: microbiological sampling and laboratory testing, clinical study and record review, and qualitative study and quantitative survey of antibiotic prescribing and purchasing practices. We used a convergent mixed-methods approach,  with datData collection took place consecutively in four counties of Anhui Province, at one village clinic and one township health centre in each county. Health facilities were selected randomly from a list provided by the provincial health board of all potential facilities fulfilling set criteria  in each county. Microbiological sampling and semi-structured observations were conducted in all eight health facilities, while exit surveys, interviews and observations in retail pharmacies were conducted in the first three study areas . We also completed a complementary systematic scoping review to identify key influences on antibiotic use in China, the results of which are published separately .The full research protocol was reviewed and approved by the Biomedical Ethics Committee of Anhui Medical University (reference number: 20170271). Participation of patients and doctors was voluntary and written informed consent was sought from all participants.https://doi.org/10.5523/bris.1waogd70b72v2s329r0tajapp.Data referred to in this paper are available at the University of Bristol data repository, data.bris, at Eligible patients were recruited using consecutive sampling in each participating health facility. Inclusion criteria were ≥18 years, presenting at the recruitment site for their current illness for the first time during the study period, and diagnosis of exacerbation of COPD, upper RTI with cough, sore throat, or UTI , ‘tummy dropping’ pain, turbid urination, incontinence, hematuria, or a diagnosis of urethritis). Specimen collection of sputum from RTI patients with productive cough, throat swabs from patients with sore throat and urine from patients with a UTI were performed by the attending doctor and placed into a sterilised tube for refrigeration and transportation to the participating laboratory within four hours according to a standard operating procedure. Vitek 2 compcat  was used for drug susceptibility testing and results were interpreted according to CLSI reference guidelines . Full deFollowing recruitment, participants were accompanied through their outpatient journey by a researcher who completed a recruitment proforma, clinic observation worksheet and exit survey. Presenting symptoms, tests ordered, diagnosis given and treatment prescribed were recorded through direct observation. At retail pharmacies or medicine shops, interactions between customers and pharmacy workers including symptoms presented, medicines and information requested/provided and medicines purchased were recorded over a two-week period in each shop.All consenting patients recruited at health care facilities completed a researcher-administered questionnaire after their consultation to elicit details of symptoms, prior treatment, views on treatment and lay diagnoses. At retail pharmacies customers purchasing antibiotics were asked to complete a similar short structured exit survey on leaving the shop.A representative subsample of patients recruited at healthcare facilities were contacted for a follow-up interview at home, between one and two weeks after their clinic visit. A subsample of customers purchasing antibiotics over the counter were also interviewed. Semi-structured interviews were conducted with attending doctors at all participating health facilities at the end of the recruitment period and with workers at retail pharmacies.After study recruitment had been completed in each of the first four participating medical facilities, researchers revisited each facility to identify the electronic records (e-records) for the observed consultations. Researchers then compared data from clinic observations and recruitment proformas with the information in the e-records, using a bespoke template to document degree of agreement. Full methodological details of this study component are provided elsewhere .Questionnaire responses were double-entered into a database using EPI DATA 3.1, then extracted and analysed using SPSS and Microsoft Excel 2013. Quantitative data analysis comprised descriptive estimations and multivariate logistic regression modelling to assess factors associated with antibiotic prescription from health care professionals and test relationships between observational data on clinical diagnoses and microbiological results. Initial coding of a subsample of interview transcripts in Mandarin and translated English versions was carried out by three interviewers and three UK-based researchers; codes were compared in Skype meetings to derive a dual-language coding framework for each qualitative dataset. Remaining transcripts were coded in Mandarin by interviewers using NVivo and analysed thematically.We first provide a descriptive summary of our research activities by study setting and then present findings on patterns of antibiotic use drawing on clinic recruitment data and patient exit surveys, and on identification of bacterial pathogens and rates of antibiotic resistance from the microbiological study component. This is followed by statistical analysis of relationships between antibiotic prescribing, patient symptoms and microbiological results. Research findings from direct observations, exit interviews and in-depth interviews are then presented to shed light on reasons for the patterns of antibiotic use. The final section presents results from a comparison between directly observed clinical consultations and patient record data that has important implications for future monitoring and surveillance of antibiotic use.84.3% to 93.2% of all recruited patients received a prescription containing antibiotics in the four project sites (87.8% of RTIs and 89.1% of UTIs); of these, between 45.5% and 41.1% of prescriptions were for intravenous (IV) antibiotics see . PatientK.pneumoniae (n = 71), H.influenzae (52), H.parainfluenzae (28), P.aeruginosa (20), H.parahaemolyticus (14), S.aureus (11), M.catarrhalis (10) E.aerogenes (8), S.pneumoniae (8), E.cloacae (6) and 41 other bacteria. 385 of RTI patients provided throat swab samples, from which bacterial isolates were obtained of H.parainfluenzae (n = 24), K.pneumoniae (14), S.aureus (7), E.cloacae (6), β-Haemolytic Streptococcus (5), H.influenzae (5), H.parahaemolyticus (3), M.catarrhalis (2), P.aeruginosa (2), H. haemolyticus (2) and 13 other bacteria. 55 patients with UTI provided mid-stream urine samples which yielded 23 strains of E.coli and 5 other bacteria.683 of the 1068 RTI patients seen in clinics provided sputum samples, from which bacterial isolates were obtained of E.coli from urine specimens was higher than CARRS data for almost all antibiotics tested, with particularly large differences in resistance seen for aztreonam (86.9% vs 58.3%), ceftriaxone (91.3% vs 76%) andciprofloxacin 73.9% vs 45.3%) and and 100% for amikacin, imipenem and meropenem  shows that rates of antibiotic sensitivity were higher for many bacterial species collected from sputum, swab or urine specimens when compared to the published data, as shown in eropenem . Among KRelationships between antibiotic prescribing (using clinic recruitment and patient exit survey data) and bacterial isolation and antibiotic resistance  were then explored statistically. Because numbers of patients presenting to outpatient clinics with UTI symptoms were unexpectedly low , the statistical analysis in this section focuses only on RTI patients . MultivaClinical consultations were generally short (<5 minutes) and contained minimal exchange of information; history-taking was rare. Patients typically described their symptoms briefly and throat or pulse was sometimes examined before the doctor wrote a prescription or  an order for a blood test or X-ray. Consultations took place in the presence of other patients waiting their turn, returning with test results, or waiting to receive guidance on medication use. Most over-the-counter purchases of antibiotics at retail pharmacies also involved limited consultation, with nearly half (47%) of medicines purchased without prescription being selected by customer request and only 53% decided by the pharmacy worker after symptoms were described.observed to receive antibiotic prescriptions reported that their prescription included antibiotics, while a similar proportion of antibiotic-purchasing pharmacy customers (46.98%) said the medicine they had just bought contained antibiotics  of clinic patients who had been ibiotics . A higheAmong the 30 doctors interviewed from all participating clinical facilities, over half indicated that diagnostic uncertainty drives antibiotic prescribing. The perceived need to provide effective treatment and ensure patient recovery frequently resulted in combined prescribing  of an antibiotic and a Traditional Chinese Medicine preparation regarded as anti-viral:kang sheng su), anti-inflammatories (xiao yan), when in fact it is a viral infection, then you won’t have good results. So all you can do is use the two together in combination” (2-1-20180131).“As a doctor, I think that if you aren’t sure then all you can do is choose to use both types together. Because if you can’t confirm what it is and if you only use, say, antibiotics  it is, then generally you would use broad-spectrum antibiotics (kang sheng su)… sometimes combining two or three is definitely a bit better. Why do I say a bit better? It’s because you aren’t certain which type of antibiotic (kang sheng su) is better for the infection” (2-1-20180201).“…if you aren’t certain what type of microbial infection (Such prescribing practices were described as protecting patient safety (including preventing risk of secondary infection), satisfying perceived patient demand for rapidly effective treatment, and safeguarding doctors against the potential reputational risk of failing to treat successfully.”Now, the patients are anxious to achieve quick success and get instant benefits, if you do not use antibiotics, if the effect is not good, the patient will bring you trouble” [1–2–20170626].Some doctors linked this concern, along with the difficulty of refusing to prescribe antibiotics, to the low social status of both rural doctors and the poor populations they serve:“The social status of the base-level clinicians is low. […] If the medical treatment works to [the patient’s] recovery, it is your job done; but if it doesn’t work, you are to be blamed […]. Many families here are the ‘left-behind’ households, with young parents working as migrants away in the city [while grandparents look after the children]. Clinicians end up bearing the brunt if any social problems arise. (2-1-20180115).Some doctors also highlighted the need to earn income for themselves and their health facility as driving their prescribing practices. Following the successful implementation of the Essential Medicines Policy (2009), health facilities cannot mark up the costs of oral medicines, but provision of clinical services including tests and parenteral drug administration are economically important: “But now the hospital[s], in order to raise income, they give patient[s] infusion treatment easily” [2–1–20180115]; “If you give the patient infusion [IV antibiotics], the effect is faster, it will attract more patients, the income will be better.”[1–2–20170625] The influence of social position and economic considerations on antibiotic prescribing among village doctors is further explored in a separate publication .xiaoyan yao, literally ‘anti-inflammation medicine’) or the specific drug name (e.g. amoxicillin) rather than the biomedical term for ‘antibiotic’ in patient consultations, explaining this as necessary for communication:Another driver of frequent antibiotic use is the characterisation of antibiotics as ‘anti-inflammation medicine’. As described elsewhere in greater depth,  biomedickang sheng su). They do not know ‘antibiotics’ (kang sheng su), they only know ‘anti-inflammation medicine’ (xiaoyan yao)” (2-1-20180115).“The common people won’t understand if I say ‘antibiotics’ , patients logically regard antibiotics as appropriate treatment and frequently referred to encounters with healthcare personnel as a source of this knowledge:Interviews with patients, however, overwhelmingly confirmed that common symptoms of infection including sore throat, coughing, fever, redness and swelling are recognised as indicating the presence of inflammation. Since antibiotics are ‘anti-inflammation medicine’  and between physicians, as well as variability in when records are completed . E-records are used much less frequently in village clinics than in township health centres, due to low computer literacy levels and poor connectivity. E-records were created only for individuals with health insurance. E-records were found for 781 (75.7%) of the 1030 patients who had been observed in these clinics; the remaining 25% either did not have e-records or were not retrievable from patient identifiers . E-record accuracy was better in relation to antibiotics (82.8% of e-records accurately recorded what was prescribed in clinic) than for diagnosis (45.0% accuracy) and symptoms (1.1% accuracy) . E-recorGuidelines for Rational Use of Drugs in Primary Care for Acute Upper Respiratory Tract Infection (2020) state that bacterial infection only accounts for 20–30% of diagnosed patients [Assessment Indicators and Requirements for clinical application management of Antibacterial Drugs, [This study confirmed that rates of antibiotic prescribing and consumption remain high in rural eastern China. The Chinese Medical Association’s patients . The Chipatients –35 but al Drugs,  no more The results from different methodological approaches converge to enhance their validity; thus our exit survey finding that roughly a quarter of those attending low level health facilities for common RTIs  or UTIs had received antibiotics for previous illness episodes in the past year, and that two-thirds of these were from a hospital pharmacy, concords with our observational data on antibiotic prescribing practices in these facilities. The findings that a further third of clinic patients had obtained antibiotics outside hospital pharmacies, and that nearly 90% of retail pharmacy customers had not previously sought treatment elsewhere, further suggest that over-the-counter purchasing of antibiotics as a first resort for everyday health problems is not uncommon, and this is consistent with previous studies that provide evidence for purchasing of antibiotics without prescription in various settings and population groups in China despite ongoing government efforts to restrict this . From thE.coli and 54% for H.influenzae, for example. Interventions to optimise prescribing practices among rural doctors need to address patient understandings of antibiotics and, given the availability of over-the-counter antibiotics, be accompanied by measures to involve retail pharmacies in antibiotic stewardship. However, analysis of our clinic observations contradicts assertions made by some doctors in our interviews that their antibiotic prescribing is a response to patient demand. While we found that direct patient requests for oral and especially IV antibiotics were strongly linked with antibiotic prescribing, such requests were only observed in a small minority of clinical consultations , and doctors were often seen to prescribe antibiotics spontaneously. This suggests that, as reported elsewhere, antibiotic prescribing is more closely associated with doctors’ assumptions about what patients want than with patients’ actual expectations [Analysis of clinic patient presenting symptoms in relation to prescriptions suggests that empirical prescribing of antibiotics is particularly associated with presence of sore throat and, in the case of intravenous antibiotics, fever. Our findings further indicate that these symptoms are viewed by both patients and doctors as indicating the presence of ‘inflammation’, for which antibiotics (as ‘anti-inflammation medicine’) are considered appropriate. The lack of an association between presence of bacterial pathogens and prescription of antibiotics reinforces the difficulty of determining clinical need for antibiotics in the absence of microbiological diagnostic tests. Interviews with doctors confirmed that while they know these conditions may not all be bacterial, they are concerned about the possibility of secondary infections and, in these settings where patients have low incomes and can rarely afford time off work and domestic duties, they regard patient recovery as most reliably ensured by prescribing broad spectrum antibiotics, in combination with an anti-viral traditional Chinese medicine to cover all possible causes. Previous studies have identified uncertainty about treatment and lower levels of professional training as associated with higher levels of antibiotic prescribing . Suboptictations .per se that works faster than oral medication. Less than half (47%) of those prescribed intravenous antibiotics knew what medicines they were receiving, while doctors explained that providing this ‘service’ helps to generate income for their health care facility. In the short term, doctors could be advised to reduce the use of antibiotics in infusions  while in the longer term, patient-level measures to increase awareness of the risks of IV antibiotic administration and system-level interventions to modify economic incentives for prescribing infusions could be considered.The implications of our findings regarding the use of intravenous antibiotics in outpatients for antibiotic stewardship warrants particular attention. Our interview and exit survey data suggest that patients value IV infusion as an effective medical intervention Importantly, our microbiological data demonstrate that despite high rates of antibiotic prescribing and dispensing, resistance rates are low in comparison with published surveillance data. The participating laboratory in our study is a member unit of CARRS national surveillance system using similar tests and standards so our drug sensitivity results are comparable to CARRS. The lower resistance rates found in our study are therefore probably due to the presence of hospital-acquired resistant infections and referred patients who have already acquired resistance being overrepresented in the data from urban tertiary hospitals which participate in the CARRS national surveillance system.Our study was limited to eight health facilities in rural areas of one Province and therefore may have limited generalisability for rural China as a whole, although study sites were randomly sampled within the parameters of our selection criteria. Our selection of study sites was also geographically limited by the need to transport samples to our reference laboratory in Hefei  by public transportation within four hours; however, this microbiological study has nonetheless demonstrated that it is feasible to assess antibiotic resistance rates in bacterial pathogens from patients in rural China. A further limitation of our study was very limited recruitment of UTI patients, who unexpectedly did not often present at these frontline health facilities. Possible reasons for this may include self-referral to more distant higher-level facilities, perhaps because of stigma arising from a perceived association between UTI symptoms and those of sexually transmitted infections , resulting in patients avoiding seeking treatment at facilities where they are known, or low prevalence of UTI in this population, but this issue requires further investigation.Our record review study has important implications for use of e-records for ABR surveillance and monitoring effectiveness of antibiotic stewardship interventions. E-records facilitate reimbursement from state medical insurance and accordingly, are created using a patient’s medical insurance number; where the patient has no insurance ID or has exceeded the individual annual reimbursement allowance for state-funded care, doctors may create e-record(s) using the ID of one or more family members to help cover costs. The use of e-records primarily for the purpose of insurance reimbursement rather than for patient care accounts for the high degree of accuracy and completeness of antibiotic prescriptions (82.8%) and doses (68.4%) as compared with that for symptoms (1.1%) and diagnoses (45%). These findings are explored in greater detail in a separate paper . CombiniIn the first study from China on antibiotic resistance in rural communities, we demonstrated the feasibility of obtaining and culturing microbiological samples to ascertain community prevalence of resistance in rural settings. Our findings show that while antibiotic prescribing rates were very high for common respiratory tract infections, prevalence of antibiotic resistance was consistently lower than that reported through national surveillance. This implies that population prevalence in China is probably lower than previously assumed, consistent with antibiotic resistance prevalence rates being elevated in the large urban tertiary facilities that participate in the national surveillance system.We have also identified diagnostic uncertainty, limited doctor-patient communication, perception of antibiotics as anti-inflammatory, and (for intravenous antibiotics) financial incentives as key drivers of antibiotic use. While self-medication with antibiotics was not unusual and a quarter of recruited patients had consumed antibiotics prior to their first visit to the recruiting clinic, only a small minority of patients directly requested antibiotics in clinical consultations; and only half of clinic attenders and pharmacy customers were able to verify whether their prescription or purchase included antibiotics. This evidence suggests that unwarranted antibiotic prescribing in clinical settings does not result principally from specific patient demand. Measures to optimise antibiotic prescribing and consumption in rural settings need to address health system drivers, including insurance reimbursement mechanisms, remuneration for clinical services that indirectly incentivises intravenous administration of antibiotics, and socioeconomic position of rural doctors; improved diagnostic support, tailored clinical guidelines and in-service training for rural doctors to manage clinical uncertainty and patient consultations; better regulation of retail pharmacies and supply chains; and public engagement to address the perception of antibiotics as inflammation-reducing and the valorisation of intravenous administration of medicines.Comparison of electronic patient records with directly observed doctor-patient consultations shows that e-records are often incomplete and inaccurate, particularly in village health facilities where some doctors have limited expertise in the use of computerised systems. E-records’ variable accuracy and completeness have important implications for monitoring and surveillance of antibiotic prescribing. Analyses of the effectiveness of policies aiming to change antibiotic prescribing practices that are based on such data should be interpreted with caution.Our study demonstrates the value of a conceptually unified cross-disciplinary approach, in which different methodological approaches provide insights beyond those which can be derived from solely epidemiological, microbiological, or social science study designs. The use of qualitative interviews provided insights into the reasons for frequent antibiotic prescribing and retail purchasing, while the use of direct observation drawing on ethnographic approaches made visible structured differences between recorded and actual diagnoses and prescriptions, and between local and national patterns and prevalence of antibiotic resistance. Combining qualitative, observational and survey methodologies with microbiological and epidemiological approaches to the study of antibiotic resistance from research design stage substantially increases study robustness and the validity of research findings for use in the development of future antibiotic stewardship interventions.