Sugarcane bagasse (SCB) is a significant agriculturalresidue generatedby sugar mills based on sugarcane crop. Valorizing carbohydrate-richSCB provides an opportunity to improve the profitability of sugarmills with simultaneous production of value-added chemicals, suchas 2,3-butanediol (BDO). BDO is a prospective platform chemical withmultitude of applications and huge derivative potential. This workpresents the techno-economic and profitability analysis for fermentativeproduction of BDO utilizing 96 MT of SCB per day. The study considersplant operation in five scenarios representing the biorefinery annexedto a sugar mill, centralized and decentralized units, and conversionof only xylose or total carbohydrates of SCB. Based on the analysis,the net unit production cost of BDO in the different scenarios rangedfrom 1.13 to 2.28 US$/kg, while the minimum selling price varied from1.86 to 3.99 US$/kg. Use of the hemicellulose fraction alone was shownto result in an economically viable plant; however, this was dependenton the condition that the plant would be annexed to a sugar mill whichcould supply utilities and the feedstock free of cost. A standalonefacility where the feedstock and utilities were procured was predictedto be economically feasible with a net present value of about 72 millionUS$, when both hemicellulose and cellulose fractions of SCB were utilizedfor BDO production. Sensitivity analysis was also conducted to highlightsome key parameters affecting plant economics. Sustainable production of 2,3-butanediolusing differentcarbon fractions of sugarcane bagasse and its economic feasibilityare studied. Cellulose and hemicellulose are inexpensivesources of fermentable sugars. Cellulose is a homopolymer of glucose,while hemicellulose is a heteropolymer and contains a mixture of C5(xylose and arabinose) and C6  sugars.Xylose is the major monosaccharide in hemicellulose, making it thesecond most abundant sugar in lignocellulosic biomass after glucose.The depolymerization of these two polysaccharides generates sugarplatform for sustainable biorefineries.3The gradual depletion of fossil resourcesand the associated environmentalpollution problems are posing a severe threat to the world. Industrialbiotechnology making use of microbial cell factories has emerged asa potential alternative to the petrochemical route for sustainablemanufacturing of building block chemicals. Though the production ofthe platform chemicals from first-generation biomass has been verysuccessful, it gives rise to the food versus feed debate. The biologicalroute provides the opportunity for using both non-edible as well aswaste biomass, which leads to reduced waste generation with simultaneousproduction of platform chemicals and achieving the goal of a carbon-neutralsociety.8 Being a rich source of fermentable sugars, SCB can be valorizedto high-value products with a circular biorefining approach. Likeother lignocellulosic feedstocks, the majority of the literature hasfocused on using cellulosic sugars from SCB for fermentative productionof fuels and chemicals, with limited research on the hemicellulosefraction rich in xylose.1Sugarcaneis one of the most cultivated crops across the worldto meet sugar and liquor demands. It is grown in more than 100 countrieswith an annual production of 1907 million MTs. Brazil and India arethe leading producers of sugarcane in the world. The two main productscoming from sugarcane mills are sugar (sucrose) and ethanol, whilethe major waste stream is sugarcane bagasse (SCB) which is a dry fibrousresidue obtained after the extraction of juice from sugarcane. Crushingone MT of sugarcane generates approximately 0.3 metric MTs of SCB,leading to an annual global production potential of ∼570 millionMTs. SCB is thus one of the largest agricultural residues in the world.The current practices by sugar mills, particularly in India, involveburning SCB to generate heat and electricity for the plant. SCB islignocellulosic biomass with the following composition, cellulose:40–50%; hemicellulose: 25–35%; and lignin: 20–30%.n-butanol are C4 alcohols. However,low solvent titer (∼20 g/L) together with complex acetone–butanol–ethanol(ABE) separation is the major challenge involved in ABE fermentation,making bio-n-butanol highly expensive than the petrochemicalroute. On the contrary, the fermentative route has been reported toachieve a much high BDO titer (>100 g/L), making it an ideal candidatefor manufacturing from renewable sources.10 Despitethis, even today, the dominant route for BDO production is the petrochemicalone. The current market price of BDO is ∼$2.8–3.5/kg,and a cheap production via the microbial route can be cost competitiveto fossil-based production.13 One of the possible solutionsto circumvent the high production cost is to manufacture BDO fromlow-cost feedstock, such as SCB, brewer’s spent grain (BSG),sugar beet pulp, bread waste, etc. For example, Amraoui and associatesachieved an accumulation of 118.5 g/L BDO using the cellulosic fractionof BSG with yield and productivity of 0.43 g/g and 1.65 g/L h, respectively.10 In another report, Amraoui and associates useddetoxified xylose-rich hydrolysate from SCB for BDO production andreported a BDO titer of 63.5 g/L with yield of 0.36 g/g.14 The results were comparable to pure xylose,where 71.1 g/L BDO was achieved with a conversion yield of 0.40 g/g.Furthermore, the BDO obtained from pure xylose and detoxified SCBhydrolysate was extracted and recovered by the aqueous two-phase systemmethod using isopropanol as the extractant and (NH4)2SO4 as a salting-out agent. The recovery of BDOin both cases was more than 85%. In a recent work, Narisetty and associatesgenerated BDO from bread waste and amassed 138.8 g/L titer with yieldand productivity of 0.48 g/g and 1.45 g/L h, respectively.2 All these reports indicate that using non-edibleand waste biomass can result in developing an industrial process forBDO manufacturing.One suchhigh-value chemical is 2,3-butanediol (BDO), which isa straight chain C4 diol with hydroxyl groups attached to the secondand third carbon atom. BDO finds a multitude of applications in food,pharmaceutical, and chemical industries and shows potential as a biofueldue to its high heat of combustion. BDO is a gateway molecule to avariety of chemical products with vast commercial potential. The totalmarket of BDO and its derivatives is estimated to be ∼$43 billion.Both BDO and 25 Munagala et al. (2021) described the overall sustainability of SCBvalorization to produce lactic acid based on the life cycle and TEAof biorefinery annexed to a sugar mill.23 Shaji et al. (2021) also focused on utilizing SCB and performeda detailed sustainability assessment of a biorefinery producing succinicacid from SCB.21 Studies by Mesa et al.(2016), Gubicza et al. (2016), and Ntimbani et al. (2021) have describedTEA of ethanol production using SCB.22 There are some studies describing the economic feasibilityof BDO production from various carbon sources, such as glycerol, sucrose,sugarcane molasses, BSG, food waste, and lignocellulosic biomass.27 TEA by Koutinas et al. (2016) focused on the estimation of the minimumselling price (MSP) of BDO using glycerol, sucrose, and sugarcanemolasses. This study reported that the complex nutrient supplements,raw material market price, and fermentation efficiency were majorcontributors to MSP.13 Haider et al. (2018)conducted a techno-economic evaluation of BDO production from fermentationbroth based on four different distillation designs for separationand purification.29 Harvianto et al. (2018) alsocompared the economic feasibility of BDO separation from fermentationbroth based on conventional distillation with that of a hybrid extraction-distillation(HED) scheme. It was shown that adding an extraction column beforedistillation improved the process economics.30 Mailaram et al. (2022) described TEA of BDO production using C5and C6 sugars derived from BSG.26 Theyused the pinch technology as a process integration tool to minimizethe external utility consumption and calculated the unit productioncost and MSP for different BDO titers and plant capacities correspondingto the centralized and decentralized biorefinery. However, to thebest of our knowledge, there is no study on TEA to produce BDO usingSCB as feedstock.The prospect of microbial production of BDOindustrially, as suggestedby these experimental studies, necessitates validation by detailedtechno-economic performance analysis of the proposed processes. Techno-economicanalysis (TEA) is an effective and standard methodology to evaluatethe economic feasibility of a product or process. TEA has been previouslyreported for utilizing SCB as a feedstock in the production of liquidbiofuels, lactic acid, succinic acid, bioethanol, furfural, biosurfactant,xylitol, activated carbons, or simply for energy generation.31 but before furtherinvestment in pilot-scale studies could be made, it will be criticalto investigate if this process can lead to cost-effective productionof BDO and profitability of the proposed biorefinery system.The current study has been carried out toevaluate the economicfeasibility of fermentative BDO production from SCB. The technicalfeasibility of this process has been established experimentally, asdescribed in our recent papers,In this study, a 96 MT/day SCB processingplant was modeled toevaluate the techno-economic performance of BDO production using SuperProDesigner process simulator . Experimental datafrom our previous studies were used to describe mass and energy balancesin the different stages of product formation, such as pre-treatment,fermentation, and subsequent downstream processing (DSP). Design specificationswith appropriate thermodynamic property methods were used to modelauxiliary processes, such as grinding, washing, screw-pressing, steam,and power generation. This work proposes to build the plant in Indiaas an annexure to an existing sugar mill that produces SCB waste.The construction phase of the plant will be one year with a start-upperiod of three months. The operating lifetime of the plant was assumedto be 20 years, with 312 operating days every year and the remainingdays for maintenance. Specific details of the production process areexplained below.31 A simplified scheme of the proposed plant is depicted in 25 The process starts with size reduction of SCB, followedby dilute acid pre-treatment using sulfuric acid (5% w/w) and subsequentsteam explosion. The pre-treatment step converts hemicellulose toxylose, leaving the cellulose and lignin fractions mostly intact.The conversion percentages vary among the different studies.33 In this analysis, we have assumed 100% conversion of hemicelluloseto xylose with trace amounts of other sugars and acetic acid. We alsoconsidered 10% conversion of cellulose to glucose and 1% conversionof xylose obtained from hemicellulose to furfural. In all these reactions,we have kept the final xylose concentration and mass flow rate consistentwith the pre-treatment data obtained from Nova Pangaea TechnologyLimited, who have developed and optimized the pre-treatment processfor treating 4 dry MT/h of SCB.25 The outputfrom pre-treatment at high pressure is sent to a flash vessel andthen to a washer unit where wastewater containing minor impuritiesand organic acids are separated. The other stream is sent to a screwpress unit, which separates xylose in the liquid form from the solidresidue containing remaining cellulose and lignin. The solid cakeis then forwarded to a boiler for steam generation, while the xylose-richstream is sent to a detoxification unit to remove any remaining impurities.Detoxified xylose-rich hydrolysate derived from SCB is then transferredto a fermenter for BDO production. An adequate quantity of mutantstrain of Enterobacter ludwigii (10%v/v) was first sent to a seed fermentation unit with the supply ofessential nutrients before transferring to the fermenter.The BDO production process fromSCB-derived xylose was reported in our previous publications.The BDO concentration was 63.5 g/L in the brothoutput from thefermenter, and it was fed to a centrifugation unit for solid–liquidseparation. The reaction stoichiometry for xylose and glucose conversionto BDO is as follows:Xylose to BDOGlucose to BDO2 during BDO fermentationis inevitable, and besides, a substantial amount of carbon is lostin the form of byproducts. In our previous work, ∼30% carbonwas lost in the form of CO2 largely generated during biosynthesisof BDO and acetoin, and about 22–24% was lost due to the byproductformation including ethanol, acetic, succinic, and lactic acid.34 For TEA, we have not considered the formation of byproducts in ouranalysis; however, the BDO yield is considered in the process model,which forms the basis of our work and does take care of the carbonloss in form of CO2 and byproducts.The carbon loss in the form of CO30 Based on the study by Harvianto et al. (2018),oleyl alcohol, which has high selectivity for BDO, was used as thesolvent in this work. BDO was extracted to the solvent-rich organicphase, which was separated from the water-rich phase by decantation.30 The organic phase leaving the extractor wassent to a distillation column, where BDO was recovered as the distillateand the solvent as the bottom product. The solvent was recycled backto the extraction column (10% loss is assumed). Here, it should benoted that the impact of large amount of unreacted sugars in the performanceof the hybrid extraction distillation system is not entirely known,and how this would affect the efficiency of the extraction and thepurity of the product would need to be evaluated based on a comprehensiveexperimental study. The extractant used in our study, i.e., oleylalcohol, demonstrated a high BDO distribution coefficient and lowwater solubility. The high distribution coefficient ensured the maximumrecovery of BDO from fermentation broth during extraction. Our extractionprocess model showed that more than 99% BDO could be recovered. Theoleyl alcohol has low water solubility and vice versa. Therefore,the organic and aqueous phases contained only a small amount of waterand oleyl alcohol, respectively. On the other hand, the unreactedsugars, being highly polar with high water solubility, remained mostlyin the aqueous phase. Therefore, we selected the oleyl alcohol asan extractant for the extraction of BDO from the fermentation broth,from which pure BDO was recovered by distillation.The HED separationmethod, as described by Harvianto et al. (2018),was used for downstream separation and purification of BDO from thefermentation broth. Liquid stream from the centrifuge was first sentto an extractor unit, where it was mixed with a solvent.1.The first scenario  assumes that SCB is obtained free of cost. It also assumes thatall the utilities required in the process are generated in the samecombined facility. Part of the steam requirement is fulfilled fromthe co-generating steam by burning the solid residue obtained fromthe screw-press unit during pre-treatment, with additional requirementsmet by the combined heat and power units in the attached sugar mill.Cooling water requirement is also met through the facilities in thesugar mill. However, it is assumed that natural gas needs to be purchasedseparately.2.23 Other factors remain same as scenario 1.Unlike thebase case, the second scenarioassumes that SCB is procured at the cost of US$ 50/MT.3.The third scenario considers that theBDO production plant is no longer annexed to a sugar mill but is astandalone facility. Therefore, all costs associated with utilitiesthat cannot be generated in-house would need to be included. In thisscenario, part of the required steam is generated using the solidresidue from the screw-press unit during pre-treatment in a boiler/steamgenerator, and the remaining is met by using additional SCB. Here,SCB is still assumed to be free.4.The fourth scenario is a combinationof the second and third scenarios, where the costs of both SCB andutilities are included. This scenario refers to a standalone facilityfor BDO production using SCB, which is purchased at US$ 50/MT.5.10 This scenario thus allows full utilization ofthe carbohydrate fractions of SCB toward product formation, with onlythe lignin fraction being sent to the boiler.The fifth scenario buildsup on thefourth scenario. This represents a standalone production plant wherethe cellulose fraction, which was previously being used for steam/powergeneration, is now used for BDO production. This, however, requiresan additional step of enzymatic hydrolysis to convert the cellulosefraction to glucose before it can be used for BDO production.In this work, fermentative BDO productionfrom SCB-derived xylose was studied considering five different processscenarios. These scenarios will eventually help us determine the cut-offfor the plant becoming profitable and the range of economic feasibility.The hypothetical plant is situatedin Maharashtra, India. Hence, the corresponding material costs andhourly wage rates are applied. However, the currency used for theeconomic assessment is US$ for easy comparison with the literature.The costs are adjusted to the year 2021 based on Chemical EngineeringPlant Cost Index. The main economic inputs and assumptions adoptedfor economic analysis are presented in Total capital investment (TCI) for the plant is calculatedby addingdirect fixed capital (DFC) cost, working capital cost, and startupcost. DFC is based on direct costs, indirect costs, and contractor’sfee and project contingency, which are taken as 5 and 10% of the sumof direct and indirect costs. Direct cost is made up of equipmentpurchase cost and costs related to piping, instrumentation, insulation,electrical facilities, buildings, and yard improvement which are 40,15, 6, 12, 15, and 10% of total equipment purchase cost, respectively.The indirect cost includes engineering and construction costs, whichare both 10% of direct costs. Working capital is estimated based onexpenses to cover 15 days of raw materials and 30 days of labor, utilities,and waste treatment. The startup cost is calculated as 5% of DFC.The total operating cost of the plant is calculated by adding costsassociated with raw materials, labor, waste treatment/disposal, utilities,facility maintenance, depreciation, and miscellaneous costs, includinginsurance and local taxes. Additionally, we need to include the overheadcost incurred by the operation of non-process-oriented facilitiesand organizations, such as accounting, payroll, fire protection, security,cafeteria, etc., which are all clubbed together as factory expenses.The unit cost of individual raw materials, chemicals, and utilitiesused in the process is described in 13The net present value (NPV),a measure of establishing a project’s potential profitability,is calculated assuming a discount rate of 7% and 20 years projecteconomic life. Positive NPV suggests that the plant will see financialgains over its lifetime and establish its economic feasibility tojustify the present-day investment. NPV, payback time, and returnof investment (ROI) were calculated to assess the profitability ofBDO production from SCB in the proposed biorefinery. The BDO sellingprice was assumed to be US$ 3/kg.Any change in economic parameters,such as cost of feedstock, utility cost, BDO selling price, discountrate, etc., could potentially affect the final economic performanceof the proposed biorefinery. A sensitivity analysis was thus conductedfor a % change in these parameters, and the corresponding NPV in eachscenario was calculated.Estimates of TCI for the 96MT/day SCB processing plant under the five scenarios are presentedin As can be seen from Among thefour stages, the biggest contributor to DFC is fermentation,covering about 80% of costs in scenarios 1 and 2, close to 70% inscenarios 3 and 4, and about 78% in scenario 5. The high DFC costsin fermentation are mainly originating from the high equipment costsof fermenters. Looking closely at working capital, we can see thatthese costs are lowest in scenario 1  and increase successivelyas we move from scenario 2 to 5. Start-up costs, which are directlydependent on DFC, are again lowest for scenarios 1 and 2 ,and the percentage increase for scenarios 3, 4, and 5 is similar tothat observed for DFC.TCI for the BDO production plant underscenario 1 is US$ 22,400,821.Since the contribution of working capital to the total TCI is quiteless, the large variations in working capital are not transferredto TCI. Therefore, we observe less than 1% increase in TCI for scenario2 when compared to scenario 1. However, with the additional costsassociated with utilities, scenarios 3 and 4 show a jump of about14% in TCI compared to scenario 1. For scenario 5, costs associatedwith additional processing result in a significant increase in TCI, which is 37% higher than that in scenario 1.The individual percentage contributions of the four processingstages to TCI in the five scenarios are shown in For the five scenarios, the breakdownof annual operating cost (AOC) for the fermentative production ofBDO is illustrated in For scenario 1, depreciation, maintenance, and factoryexpensesare the major cost contributors, with 31, 21, and 19% contributionsto the total AOC. Raw materials only account for about 13% of AOCin scenario 1, mainly because the feedstock SCB is assumed to be obtainedfree in this case. As opposed to this, raw materials become the majorcontributor (42%) to AOC in scenario 2, largely due to the procurementcost of SCB. This drives the total AOC of scenario 2 to increase 1.5times compared to scenario 1 to a value of US$ 4,640,000. Depreciation,maintenance, and factory expenses become the second, third, and fourthhighest contributors.When considering a standalone facilitythat is no longer annexedto the sugar mill, as described in scenario 3, the cost of utilitiesas expected becomes the major contributor to AOC, accounting for almost38% of the total, again followed by depreciation, maintenance, andfactory expenses. This decentralization, where utilities can no longerbe accessed from the sugar mill, leads to about 1.7 times increasein AOC compared to the annexed facility in scenario 1.For scenario4, which considers procurement of SCB in additionto being a decentralized standalone facility, the high costs of rawmaterials (30%) and utilities (29%) dwarf all the other contributions.This factor also leads to an overall increase of AOC by about 2.3times compared to scenario 1.With US$ 9,050,000, the AOC ofscenario 5 is the highest comparedto all previous scenarios. Similar to scenario 4, the cost of rawmaterials (30%) and utilities (30%) are the major contributors, followedby depreciation (14%). The cost of enzymes for converting celluloseto glucose leads to an increase in the cost of raw materials, andoverall increase in system volume drives up the expenses on utilities.13The main revenue of the plantcomes from the sale of BDO, and in scenarios 3, 4, and 5, additionalsavings are accrued with the generation of power which is recycledback to the plant. The detailed economic performance of the differentscenarios is illustrated in detail in As can be seen from Compared toproduction in an annexed facility (as in scenarios1 and 2), moving the plant to a standalone decentralized locationresults in substantial drop in NPV to negative values for scenarios3 and 4. It is largely due to the increased capital investment andoperating costs. Looking at scenario 3 specifically, the net unitproduction cost of BDO is lower than that for scenario 2 due to thepower savings. However, these savings are still not sufficient tooffset the increased investment and operating costs which result inoverall negative NPV . Results for scenario4 are even less favorable as the higher costs involved in SCB procurementand utilities lead to a highly negative NPV ,deeming the plant economically unviable.Scenario 5, on theother hand, shows favorable economic performance, provingthat it is possible to turn a standalone facility profitable by utilizingall carbohydrate fractions of SCB for BDO production. With a moreuseable substrate, the higher overall yield of BDO leads to increasedrevenue (2.7 times) compared to the first four scenarios, which helpsin outweighing the higher capital investment and operating costs associatedwith scenario 5, making it economically feasible even if SCB and utilitiesare not available for free.13Previously, Koutinas et al. (2016)presented the techno-economicevaluation of BDO production via fermentation of three different carbonsources: glycerol, sucrose, and sugarcane molasses. They calculatedthe MSP of BDO (unit price for zero NPV) for each feedstock and reportedthat MSP varied from 2.1 to 2.9 US$/kg for glycerol, 1.97 to 5.26US$/kg for sucrose, and 2.6 to 4.8 US$/kg for sugarcane molasses.When using the same economic parameters  as usedby Koutinas et al. (2016), the MSP of BDO from the current plant variesfrom US$ 1.86/kg (for scenario 5) to US$ 3.99/kg (for scenario 4).MSPs for other scenarios of the current study are US$ 2.37/kg forscenario 1, US$ 2.96/kg for scenario 2, and US$ 3.37/kg for scenario3. Thus, scenario 5 in this proposed BDO production plant using SCBshows better economic viability than BDO production using either glycerol,sucrose, or sugarcane molasses.26 A similar decrease in unit production cost couldbe achieved for the plant proposed in this work. For example, if theplant capacity for the SCB to BDO plant is hypothetically increasedto 2000 MT/day, the unit production cost of BDO in scenario 1, whichwas 1.13 US$/kg (for 96 MT/day capacity), would fall to US$ 0.48/kg.These numbers are very promising; however, it should be noted thatthe decision for the real plant capacity would depend on several factorssuch as local availability of the feedstock, regulations, space availablefor expansion, etc. Therefore, these decisions would require furtherdeliberation.Some studies have reported TEA results based on unit productioncost. For example, Mailaram et al. (2021) described a techno-economicassessment for BDO production from BSG with a plant capacity of 100MT/day. They reported unit production costs to be in the range ofUS$ 1.736/kg to US$ 1.842/kg. Current analysis based on fermentativeBDO production using SCB with a plant capacity of 96 MT/day [whichis comparable to the one used by Mailaram et al. (2021)] predictsthe unit production cost to be in the range of US$ 1.13 US$/kg (scenario1) to US$ 2.28/kg (scenario 4). Mailaram et al. (2021) also showedthat economies of scale would reduce the production cost to US$ 1.07/kgif the plant capacity was increased 20 times (2000 MT/day).39 Maina et al. (2019) worked on optimization ofBDO production in fed-batch cultures using very high polarity sugarfrom sugarcane mills and also presented techno-economic evaluations.Based on the analysis, they estimated BDO MSP to be varying betweenUS$ 3.12/kg and 2.67/kg for annual production capacities of 10,000and 50,000 MT, respectively.40 Comparedto Maina et al. (2019), microbial BDO production from SCB in the currentwork with annual production capacity of 29,952 MT  predicts a much lower MSP of BDO US$ 1.86/kg (scenario5).Benalcázar et al. also performed economicassessments forthe production of bio-based BDO from the syngas platform involvinggasification of lignocellulosic biomass  followed by syngas fermentation. They reportedthe MSP of BDO varied between US$/kg 2.75 and US$/kg 2.9 using thishybrid process (biomass gasification followed by syngas fermentation).The results from our study show that with complete utilization ofcarbohydrate fractions, BDO produced using microbial fermentationof SCB could be more profitable than using the hybrid syngas platform,41 This reported MSP is onlyslightly below the lowest MSP (US$ 1.86/kg for scenario 5) obtainedin our analysis. Also, it should be noted that economic analysis ofZang et al. (2020) is based on plant life assumption of 30 years,whereas the same in our proposed plant is assumed to be 20 years.Zang et al. (2020) also conducted a detailed TEA for conversionof switchgrass to BDO and co-products such as furfural and technicallignin, enabled by high-solid loading deep eutectic solvent (DES)pre-treatment. Based on their analysis, MSP of BDO was estimated tobe US$ 1.7/kg–1.74/kg, depending on the solid loadings duringthe process.The effectiveness and economic viability of SCB as a potentialfeedstock for fermentative BDO production are thus highlighted whencompared to both bio-based approaches as well as conventional fossil-derivedBDO from previous studies.The above analysis is largelydependent on the specific economic parameters selected for the study.Therefore, sensitivity analysis is performed to understand the effectof variation in important parameters, such as cost of SCB, sellingprice of BDO, utility cost, plant operational life, and discount rate,on the economic performance of the different scenarios.Theresults of this sensitivity analysis are presented in 42 It can be seen that changes in the SCB cost only affect the NPVof scenarios 2, 4, and 5 because SCB was considered to be free inscenarios 1 and 3. As expected, NPV was found to decrease with increasingSCB cost. For scenario 2, as the cost of SCB increases to US$ 75/MT,the estimated NPV of the plant goes down, reaching a low value ofUS$ 463,470. It is clear that even a small further increase in SCBprice would lead to a negative NPV and make the project economicallyunsustainable. For scenario 4, the decrease in SCB cost to US$ 25/MTimproves the performance, but it is still not sufficient to make theplant economically viable. Scenario 5 maintains large positive NPV even when the SCB cost is increased to US$ 75/MT.For the first case, as can be seen in It can be seen from In the secondcase 5b, thecThe third factor,i.e., the selling price of BDO, has a very stronglinear impact on the NPV of the plant. It can be seen in 43 For scenario 5, whenthe BDO selling price is reduced to US$ 1/kg, NPV falls to a negative46.8 million US$, implying that the plant would no longer be economicallyviable. Therefore, for the current plant capacity with the reportedyield and productivity, fermentative BDO from SCB would not qualifyas a platform chemical even if both cellulose and hemicellulose fractionsare utilized.For a fermentationproduct to classify as a platform chemical,the MSP should reach 1 US$/kg.The fourth factor, i.e., plant operational life,was changed ±5years from the original assumption of 20 years 5d. It caThe fifth factor, i.e., discount rate, is changed ±2 fromthe originally assumed value of 7%. As can be seen in BDO is a versatile chemical with huge derivativepotentials andhas promising fuel properties for blending with motor fuels. Furthermore,high BDO titers make fermentative production feasible from biomass-derivedsugars. This work demonstrates the techno-economic feasibility ofBDO production from SCB. The process model is developed for processing96 MT of SCB per day under five different scenarios. Overall lookingat the profitability and sensitivity analysis, a standalone facilityfor BDO production from SCB could show financial gains and becomeeconomically feasible if both cellulose and hemicellulose fractionsare utilized for BDO production. If BDO is to be produced only usingxylose derived from SCB, it may still be possible to run an economicallyviable plant if either one of the conditions is met: the plant isannexed to a sugar mill which can provide the utilities, or the feedstockis free, or the cost of utilities can be subsidized, or BDO can besold at a higher price, or plant operational life can be increasedto 25 years, or if the discount rate can be lowered to 5%.Amongthe five plant scenarios considered in this work, scenario1 predicted the lowest unit production cost for BDO, 1.13 US$/kg,while scenario 5 showed the best performance in terms of economicviability with ROI of about 30% and NPV of almost 72 million US$.It was also seen that compared to other scenarios, scenario 5 wasleast susceptible to any changes in the selected economic parameters.The future work will be directed toward the techno-economic feasibilitystudies and life cycle analysis for expanding this plant to convertBDO derived via a fermentative route to higher value chemicals suchas 1,3-butadiene and methyl ethyl ketone.


The PIK3CA-related overgrowth spectrum (PROS) encompasses a group of rare disorders characterized by the overgrowth of various body parts, driven by mutations in the PIK3CA gene. This study presents a case of a Moroccan female patient with PROS, demonstrating a phenotype associated with genetic mosaicism in the PIK3CA gene. A multidisciplinary approach, involving clinical examination, radiological assessment, and genetic and bioinformatic analyses, was employed for diagnosis and management. Next-generation sequencing and Sanger sequencing identified a rare variant, c.353G>A, in exon 3 of the PIK3CA gene, not detected in leukocyte DNA but confirmed in tissue biopsy samples. The comprehensive analysis of this case furthers our understanding of PROS and highlights the importance of a multidisciplinary approach to the diagnosis and management of this rare disorder. The PIK3CA-related overgrowth spectrum (PROS) is a group of rare disorders characterized by the overgrowth of various body parts, including bones, skin, and blood vessels. The disorders are caused by mutations in the PIK3CA gene, which encodes the p110α catalytic subunit of the phosphoinositide 3-kinase (PI3K) enzyme. This gene is essential for regulating cell growth, proliferation, and survival, and mutations in PIK3CA can lead to abnormal activation of the PI3K/AKT/mTOR signaling pathway .Several studies have reported the clinical and genetic characteristics of PROS in different populations ,4. A molThe treatment of PROS involves the use of low-dose sirolimus  and alpeIn this paper, we report a Moroccan female case of PROS presenting a phenotype associated with genetic mosaicism in the PIK3CA gene. This mosaicism was identified by DNA analysis of tissue biopsy samples but was not present in lymphocyte DNA.The patient was a seven-year-old female born with a birth weight of 4.400 kg, length of 54 cm, and head circumference of 41 cm. She was diagnosed with congenital hip dislocation in the neonatal period. The patient showed developmental delay, with sitting achieved at seven months and no standing or walking. She also had a speech delay. Physical examination revealed global developmental delay, left-sided hemihypertrophy, skin and joint hyperlaxity, and multiple cutaneous anomalies, such as angiomas on the back and limbs, hamartomas on the neck and left upper limb, livedo-like lesions on the left lower limb, and painful tumefaction of the left forearm. She also had an umbilical hernia  and left lower limb circumference of 36 cm . She had a limited range of motion of her left elbow and knee and a painful tumefaction of the left forearm that had been present for several months.Laboratory examinations, including complete blood count and renal function tests, were normal. Radiological examination showed normal renal and abdominal ultrasound, but an ultrasound of the soft tissue (tumefaction of the left forearm) showed a vascular formation likely a thrombosis, and brain magnetic resonance imaging (MRI) revealed left cerebral hemisphere hypertrophy with midline shift to the right, dilatation of the ventricular system, and cortical fissures.A genetic analysis was performed using next-generation sequencing and confirmed by Sanger sequencing. The analysis revealed a variant in exon 3, c.353G>A, leading to a p.Gly118Asp change Figure . This vaPROS is a group of rare and heterogeneous disorders characterized by somatic mosaic mutations in the PIK3CA gene -14. The The clinical presentation of PROS is highly variable, ranging from isolated overgrowth to multisystem involvement ,17,18. CThe diagnosis of PROS is primarily based on clinical findings and confirmed by molecular testing ,19. In oManagement of PROS is multidisciplinary and primarily focused on symptom relief and improving the quality of life ,8,18. FoIn conclusion, this case study of a Moroccan female with a mosaic PIK3CA mutation illuminates the complex clinical manifestations associated with PROS and the critical role played by molecular testing, such as next-generation sequencing, in achieving a definitive diagnosis. By employing a multidisciplinary approach, involving specialists in genetics, radiology, and pediatrics, the comprehensive evaluation and management of patients with PROS can be ensured. Additionally, this case emphasizes the potential of targeted therapies, such as PI3K/AKT/mTOR inhibitors, in providing more effective and individualized treatment for patients with PROS. Further research is warranted to explore the molecular mechanisms underlying PROS pathogenesis and to advance the development of novel therapeutic strategies tailored to the specific molecular alterations present in affected individuals.


Chronic respiratory insufficiency can result from respiratory infections like pneumonia, which can permanently harm the lungs and respiratory system. A 21-year-old female patient arrived at our emergency medicine department (ED) complaining of acute lower-limb pain that worsened when she walked. She also reported feeling weak and having an acute, undiagnosed fever that was resolved by taking medicine two days after the day of admission. She was found to have a body temperature of 99.4°F, decreased air entry on the left side of the chest, and diminished bilateral plantar responsiveness. With the exception of a low calcium level and an increased liver function test, her biochemical indicators were normal.The left lung's basal region had fibrosis, and the right lung's hyperplasia served as a compensatory mechanism, according to the chest radiograph and CT scan of the thorax. The patient underwent treatment with intravenous pantoprazole, ondansetron, ceftriaxone, multivitamin supplementation, gabapentin, and tablets of amitriptyline. On Day 7, her lower limb pain had significantly recovered. After an eight-day hospital stay, she was discharged with instructions to follow up with the pulmonary medicine outpatient department (OPD) and the neurology OPD. A well-known occurrence known as compensatory hyperinflation of the lung happens when one lung is severely injured or rendered inoperable, leading the other lung to enlarge to make up for the loss of respiratory function. This case demonstrates the ability of the respiratory system to compensate for significant damage to one of the lungs. Pulmonary hyperinflation is defined as an abnormal rise in functional residual capacity (FRC) of the lung, which is the volume of the lung at the end of tidal expiration. To a radiologist, however, hyperinflation implies an increase in total lung capacity. Several conditions that cause significant unilateral destruction of the lung can also result in significant compensatory hyperinflation on the other side. When a lung is severely hyperinflated, it frequently herniates anteriorly across the midline and may almost completely fill the opposite hemithorax. This phenomenon is referred to as pseudo-horseshoe lung . HorseshA 21-year-old female presented to the emergency medicine department (ED) with complaints of pain in bilateral lower limbs for one month, acute in onset, progressive in nature, aggravated on walking, non-relieving, and associated with generalized body weakness. Furthermore, she added an undocumented fever, acute in onset, on and off in nature, without diurnal variation, chills, or rigors, and relieved on taking acetaminophen 500 mg twice a day, two days prior to the day of admission. There was no history of trauma. Upon further review of history, it was found that the patient had suffered from severe pneumonia at the age of three years. The patient had no respiratory discomfort. The patient had no history of diabetes, hypertension, tuberculosis, asthma, thyroid disorder, or significant surgical intervention if any. There was no history of allergy or contact with tuberculosis patient.On physical examination, she looked unwell, with a physical temperature of 99.4°F, heart rate of 98 beats per minute, blood pressure of 119/79 mmHg, and oxygen saturation of 98% on room air. On respiratory examination, air entry was reduced on the left side of the chest at the basal region. On cardiovascular examination, normal S1 and S2 sounds with no added sounds or murmur were present. The central nervous system examination revealed a grade 1 sensorium with a Glasgow Coma Score (GCS) of 15. Both pupils were reactive to light. She was moving her limbs on command but could not walk due to pain in her bilateral lower limbs. Her deep tendon reflexes were intact but bilateral plantar response was reduced. Detailed motor and sensory system examinations were carried out and found to be intact. There was no neck stiffness. We closely kept monitoring related parameters until our patient was discharged.On Day 1 of admission, her biochemical markers were normal except for the liver function test, where serum glutamic oxaloacetic transaminase (SGOT) was 64.9 IU/L (0-40 IU/L) and serum glutamic pyruvic transaminase (SGPT) was 54.5 IU/L (0-45 IU/L). The serum electrolyte showed a reduced calcium level of 3.56 mg/dl (4.5-5.5 mg/dl). The serum vitamin B12 level was 2134 pg/ml .The chest radiograph indicated the presence of fibrosis in the basal area of the left lung, as depicted in Figure Treatment consisted of intravenous (IV) pantoprazole 40 mg BD, IV ondansetron 4 mg BD, IV tramadol 1 ampule in 100 ml normal saline (NS) stat, multivitamin supplementation, and IV ceftriaxone 1 gm BD, on Day 1 of admission. On Day 2, we revoked tramadol, as she was found allergic to it. Her color Doppler test was normal and without any evidence of deep venous thrombosis (DVT).On Day 3, the patient underwent a neurology consultation, during which compressive radiculopathy was suspected. As a result, gabapentin tablets of 100 mg strength were added to the patient's regimen to be taken once daily at bedtime, along with amitriptyline tablets of 10 mg strength, also to be taken once daily at bedtime. An X-ray of the lumbosacral (LS) spine (in anteroposterior (AP) and lateral views) was subsequently conducted for further assessment and was found normal , asthma, and cystic fibrosis. In this case, there was no evidence of any congenital defects or any chronic pulmonary diseases, and the patient had no history of asthma or cystic fibrosis. Furthermore, there was no evidence of any acute exacerbation of COPD .The patient was diagnosed with left-sided pleural effusion with fibrosis, which could be the result of a previous infection or inflammation. Fibrosis can develop as a result of prolonged inflammation in the pleural space, which can lead to decreased lung compliance and restrict the lung's ability to expand, thereby resulting in compensatory hyperinflation of the contralateral lung.Compensatory hyperinflation is a physiological phenomenon where a functional lung expands to compensate for the loss of respiratory function in a damaged or non-functional lung. In this case, the patient had left-sided pleural effusion and fibrosis due to pneumonia when they were three years old. The fibrosis had affected the left lung, rendering it non-functional, but the right lung underwent compensatory hyperinflation to maintain adequate respiratory function. Despite the severity of the fibrosis, the patient's right lung exhibited an impressive compensatory capacity, resulting in complete functional recovery.This case highlights the adaptability and compensatory ability of the lungs, even after a history of pneumonia and lung fibrosis. Understanding this compensatory ability can be useful in managing and treating lung diseases, as it may inform therapeutic strategies aimed at promoting the growth and development of lung tissue to enhance respiratory function. Although the patient presented with complaints related to a neurological disorder, a significant respiratory disorder was diagnosed. While there is limited information on the relationship between these two conditions, this case report could provide valuable insights and contribute to further understanding.


Immune-checkpoint inhibitors (ICIs) have emerged as a core pillar of cancer therapy as single agents or in combination regimens both in adults and children. Unfortunately, ICIs provide a long-lasting therapeutic effect in only one third of the patients. Thus, the search for predictive biomarkers of responsiveness to ICIs remains an urgent clinical need. The efficacy of ICIs treatments is strongly affected not only by the specific characteristics of cancer cells and the levels of immune checkpoint ligands, but also by other components of the tumor microenvironment, among which the extracellular matrix (ECM) is emerging as key player. With the aim to comprehensively describe the relation between ECM and ICIs’ efficacy in cancer patients, the present review systematically evaluated the current literature regarding ECM remodeling in association with immunotherapeutic approaches.This review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines and was registered at the International Prospective Register of Systematic Reviews . PubMed, Web of Science, and Scopus databases were comprehensively searched from inception to January 2023. Titles, abstracts and full text screening was performed to exclude non eligible articles. The risk of bias was assessed using the QUADAS-2 tool.After employing relevant MeSH and key terms, we identified a total of 5070 studies. Among them, 2540 duplicates, 1521 reviews or commentaries were found and excluded. Following title and abstract screening, the full text was analyzed, and 47 studies meeting the eligibility criteria were retained. The studies included in this systematic review comprehensively recapitulate the latest observations associating changes of the ECM composition following remodeling with the traits of the tumor immune cell infiltration. The present study provides for the first time a broad view of the tight association between ECM molecules and ICIs efficacy in different tumor types, highlighting the importance of ECM-derived proteolytic products as promising liquid biopsy-based biomarkers to predict the efficacy of ICIs.ECM remodeling has an important impact on the immune traits of different tumor types. Increasing evidence pinpoint at ECM-derived molecules as putative biomarkers to identify the patients that would most likely benefit from ICIs treatments.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022351180, identifier CRD42022351180. Nearly half of all patients with metastatic cancer are eligible to receive ICIs, with an increasing use of these agents seen in several (neo)adjuvant and maintenance settings –3. ICIs Immune checkpoint (IC) pathways are physiologic mechanisms aimed at attenuating T cell responses to prevent autoimmunity and maintain immune homeostasis. Tumors can viciously take advantage of the immune-inhibitory pathways to limit the extent of T cell activation and maintain immune tolerance. Indeed, ICs and their ligands are frequently upregulated in the tumor microenvironment (TME) of various cancer types, thus hindering the anti-tumor immune responses . Hence, It is now well established that the efficacy of ICIs in cancer treatment is strongly affected not only by the specific characteristics of cancer cells and the expression levels of the immune checkpoint ligands, but also by other components of the TME. Indeed, the response to ICIs highly relies on the innate immune TME constituents, e.g. macrophages and natural killer cells (NK), on the tumor hypoxic levels, as well as on the efficiency of tumor-associated vasculature . A key cThe ECM is a complex network of molecules which, thanks to its mechanical as well as biochemical properties, strongly impacts on all the cellular TME components, thus affecting tissue homeostasis , 9. The Contrary to what previously thought, the ECM is not a mere static TME component, rather it undergoes continuous dynamic remodeling as well , 21. ECMIn the light of these findings, some ECM molecules, as well as fragments deriving from their proteolytic remodeling, are emerging as putative biomarkers to delineate the immune traits of the tumors, as well as the efficacy of immunotherapies. The aim of this systematic review is to identify and summarize all the published human research studies in this context. In particular, we aim to address the following questions: Can ECM remodeling regulate the tumor immune response? Is the ECM composition impacting on the efficacy of immune checkpoint inhibitors? Can ECM molecules/fragments represent a valuable biomarker to predict the outcome of cancer patients treated with immune checkpoint inhibitors?22.1https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=351180).The systematic review was designed based on the Preferred Reporting Ideas for Systematic Review and Meta-analyses [PRISMA ] systema2.2Original research articles written in English and published before 20 January 2023 were eligible for inclusion. We included studies reporting any relation between ECM molecules and the immune traits of the tumors, as well as the response to immune checkpoint inhibition. We included studies regarding patients diagnosed with cancer, regardless of the cancer type, disease staging and PD-L1 expression status. Our inclusion criteria did not involve any age restrictions, since we wished to comprise both young and older patients. Articles not published in English, whose full text was not available, letters to the editor, case reports, and poster presentations were excluded.To ensure a comprehensive retrieval of all the studies relative to ECM and ICIs efficacy, we chose to exploit three relevant and reliable databases: PubMed (MEDLINE), Scopus and Web of Science. The combination of mesh terms searched in the databases were “extracellular matrix molecules” or “extracellular matrix remodeling” and “immune checkpoint inhibitors” or “immunotherapy”. Searches have also been performed using the names of the specific immune checkpoint inhibitors  and some specific ECM molecules .2.3Duplicate articles were removed from the results of the literature search. Two independent authors screened the titles and abstracts of the remaining articles to ensure that the eligibility criteria were met. Any discrepancies between the authors were identified and discussed (with inputs from a third author if required). The remaining included articles assessed by full-text screening by two independent authors, using the same eligibility criteria.2.4Study quality and risk of bias were assessed by using the QUADAS-2 tool. The risk of bias in the studies was categorized based on the “yes” scores in the QUADAS-2 checklist. In particular, papers with all “yes” or maximum one “unclear or no” responses were classified as low risk. Instead, if two or more responses on the checklist were “unclear” or “no”, papers were attributed as unclear or high risk, respectively. If two or more responses were “unclear” and at least one response as “no” the paper was attributed as high risk. Finally, we considered the last question of the QUADAS-2 checklist  as an important key factor for the evaluation of the study quality, therefore papers in which the response was “no” were classified as high risk papers.33.1in vitro/in vivo only studies. During the screening process, nine articles not identified by the database searches but relevant for the present review were added manually to the list. As a result, 47 articles were included and analyzed in this systematic review . Two independent authors screened a total of 1,009 articles for their relevance in the topic by assessing the title, abstract or full-text. Among the 1,009 articles, 971 studies were excluded since unrelated to ECMs, relative to immunotherapy employed to treat other diseases, and c review Figure 1The qualitative analysis of the selected papers indicated that three points were mainly exposed to considerable bias: the design of case-control studies, the definition of a threshold and the inclusion of all the patients in the analysis. Anyway, the overall risk of bias of the included studies assessed by QUADAS-2 was low for 62% of the papers, unclear for 23% and high for 17% Figure 2The literature search spanned from inception to 2023, however most of the papers included in the present systematic review dated from the last five years. All tumor types in adult as well as in pediatric patients were included in the search, however none of these papers dealt with pediatric cancer. Among the tumor types, the majority of the papers were related to melanoma , breast cancer (BC) , colorectal cancer (CRC)  and hepatocellular carcinoma (HCC) . Half of the identified studies were carried out exploiting the patients’ cohorts available to the research teams, whereas the other half was exclusively based on bioinformatic analyses.3.2The ECM undergoes radical remodeling during tumor growth and progression, thereby replacing normal ECM with tumor-associated ECM , 75, 76.3.2.13.2.1.1Among the ECM components that have been associated with the traits of the tumor immune microenvironment, collagens are the most represented 2.The association between collagen deposition and the infiltration of immune cells has been well described in different cancer types. In triple negative breast cancer, high Th1 infiltration has been related to low collagen I content, whereas high Th2 and regulatory T cells (Treg) infiltration has been observed in collagen-rich lesions . Similar+ T cells, CD8+ T cells, B cells, neutrophils, macrophages, and dendritic cells), but also with the expression level of the checkpoint molecules CTL4, PD-1, PD-L1 and PD-L2  and procollagen C-endopeptidase enhancer (PCOLCE), were shown to correlate with the survival of patients with advanced melanoma treated with neoadjuvant immunotherapy combining high-dose interferon α-2b with the anti-CTLA-4 antibody ipilimumab . Similar+ T cell exhaustion through the binding with the leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) acting as an immune checkpoint molecule  exerts multiple functions by interacting with other ECM components and cell types impacting on tissue development, wound healing and cancer. Increased VCAN expression has been shown in solid tumors including ovarian, pancreatic, breast, lung, esophageal, bladder and colorectal cancer and to associate with patient’s prognosis , 87. Maner types . In 2022er types . In accoer types . In detain NSCLC , myelomain NSCLC , CRC , a highly expressed glycoprotein in malignant solid tumors, including breast cancer and oral squamous cell carcinoma (OSCC) , 89, 90.mor nest . In accorognosis . Furtherrognosis .3.2.1.4Collagen triple helix repeat containing-1 (CTHRC1) is a secreted ECM protein transiently expressed during the repair process of injured arteries  and skin3.2.1.5+ T memory cell rest, Tregs, CD8+ T cells, CD4+ T cells, and CD activation. Even if the molecular mechanisms affecting the immune response are still unknown, these data suggest that increased ABI3BP expression may impact on tumor progression also by modulating the tumor immune microenvironment. In accordance with this hypothesis, the expression of ABI3BP in lung cancer correlates with patient’s prognosis, with low expressing patients having a poorer outcome (ABI family member 3 binding protein (ABI3BP) is an ECM protein expressed in multiple organs, including the heart, kidney, lung, pancreas, and placenta, with low or variable expression in the spleen, liver, brain, bone, and skeletal muscle . ABI3BP  outcome .3.2.1.6Elastin microfibril interfacer 2 (EMILIN-2) belongs to the EDEN protein family –107 and Contrasting results have been found in other tumor types, as in low grade glioma  and clea3.2.1.7+ T cells and associate with poor prognosis (Biglycan (BGN) is an ECM proteoglycan with an essential role in mediating morphology, growth, differentiation and migration of epithelial cells and it is a well-known player in tumor development and progression –115. Sevrognosis .3.2.1.8In vitro and in vivo studies highlighted the role of OPN in determining the immune phenotype of the TME, since SPP1 expression directly correlated with CD8+ T cell activation and M2 macrophage polarization , encoded by the SPP1 gene, is a non-collagenous bone matrix protein involved in the development of different organs . Many strization –123. Howrization . The samutations .3.2.1.9+ T cells and immune cell infiltration in melanoma patients  is an ECM linker protein involved in the binding of proteoglycans to hyaluronic acid . HAPLN3 patients . When appatients .3.2.2The ECM properties, due to post-translational modifications such as the bio-physical structure and the stiffness, not only affects the recruitment/activation of immune, but also per se profoundly shape the tumor immune microenvironment . The majIn cancer, the alteration of this complex and multistep process leads to abnormal mechanical and physical properties of the ECM. The higher stiffness and density of tumor-associated ECM constitute a mechanical barrier which protects the tumor from immune cell infiltration and immune-mediated destruction. Overall the TME is less permissive to leukocyte invasion, favoring the establishment of a more tolerant immune environment and also impairing the efficacy of ICIs .This aspect is well represented in the study from Byers et al, in which the authors measured the stromal fibrillar morphology within the ECM in basal cell carcinomas (BCC) . The aut+ T cells, follicular helper T cells and Tregs; on the other hand, it was positively correlated with macrophages, activated mast cells, resting NK cells, CD4 memory activated T cells and CD4 memory resting T cells. Overall, PLOD2 was shown to be significantly associated with the tumor immune infiltration and with a poor patients’ outcome.In the same view, Xu et al. showed that PLOD2 , a member of PLOD family which mediates the formation of stabilized collagen cross-links generating a stiffer ECM, is overexpressed in a variety of tumors including gastric, bladder, lung, breast, and head and neck squamous cell cancer . Notably+ T, CD8+ T, NK cell infiltration. As well, Lecchi et al. developed a gene expression signature to identify high‐grade breast cancer patients with poor prognosis , Biglycan, EGF Containing Fibulin Extracellular Matrix Protein 2 (EFEMP2) and the basal membrane component Nidogen 2 (NID2). In breast cancer, the ECM3+/IFN− signature was associated with low tumor‐infiltrating lymphocytes, high levels of CD33+ cells, absence of PD‐1 expression or low expression of PD‐L1.Another enzyme driving collagen rearrangements and recently associated with immune infiltrating cells is PCOLCE, which localizes in the TME of several cancer types. Bioinformatic analyses highlighted that PCOLCE is a prognostic predictor for PAAD, thymoma and CES . Even ifrognosis . PCOLCE + and CD4+ T cells and macrophages, as well as with the expression of immune checkpoint molecules such as PD-L1 and CTLA-4. A functional enrichment analysis demonstrated that this effect was mainly based on ECM organization and regulation of cell−cell adhesion  1-4 . Due to adhesion . Howeveradhesion . In accoImportantly, not only the density and stiffness of the collagen matrix, but also the fiber alignment represents a barrier for immune cell infiltration. This aspect has been described in breast cancer by Sun et al. Their study reported for the first time the implication of discoidin domain receptor 1 (DDR1), a tyrosine kinase collagen receptor, in shaping the immune infiltrate of breast cancer . DDR1 in3.2.3ECM remodeling occurs on one side through the altered expression of the molecules, on the other side through their degradation mediated by the activation of target-specific proteases such as MMPs, disintegrins and ADAMs . Cancer 3.2.3.1+ T cells, as well as with the presence of MDSC cells, which, on the contrary, suppress the immune response , C3M (collagen III) and C4M (collagen IV) which were shown to be increased in cancer patients compared to healthy individuals , 148, anThe degradation of collagen IV by granzyme B generates a fragment distinct from C4M named C4G . In metaCirculating fragments are generated not only by the degradation of collagen but also other ECM molecules. For example, extracellular vimentin is citrullinated and cleaved by MMPs giving rise to a fragment known as VICM (citrullinated and MMP-degraded vimentin) . VICM isTaken together, these studies highlight a prominent role of the ECM in affecting the immune response. From the evaluation of the 47 papers taken into account, we can infer that collagens are the most studied ECM components in this context, impacting on the infiltration and activation of immune cells by constituting a physical barrier to effector cells’ infiltration and by influencing immune cells phenotype. Moreover, collagen remodeling represents a crossing-edge process among different tumor types and provides promising valuable biomarkers for ICIs efficacy. Nonetheless, from this study we can also conclude that other ECM components as glycoproteins and proteoglycans exert a prominent role in shaping the tumor immune response despite their effect is tumor-type specific.4As a key component of the TME, the ECM is becoming a crucial source of novel diagnostic and prognostic biomarkers . Due to The literature search strategy was intended to retrieve studies dealing with both adult and pediatric patients. However, none of the papers were related to pediatric cancers, likely because, in terms of absolute numbers, pediatric cancers are relatively rare and the use of ICIs is still under evaluation for these patients . Also, tOverall, the main processes and changes driving ECM remodeling in cancer have been well documented. However, it has become clear that each cancer type displays an unique ensemble of ECM molecules, ECM-remodeling enzymes and ECM-associated growth factors, collectively referred to as matrisome . This waOn the other side, the activity of some ECM components is strongly tumor-type dependent. This can be at least partially explained by the fact that ECM molecules display multimodular structures able to simultaneously modulate various biological functions and cell types, such as CAFS, immune cells and vascular cells. Thus, the overall association between the abundance of specific ECM proteins and the tumor immune traits are the result of a tight and complex molecular crosstalk between these cell types, through mechanisms that in part still remain elusive and need further investigation. In recent years, the crosstalk between immune and endothelial cells has been investigated to assess the impact of tumor associated vascularization on ICIs efficacy. These studies highlighted the synergic beneficial effect due to the simultaneous blockage of IC and the normalization of the vascular bed, leading to the design of novel therapeutic approaches based on the combination of ICIs and angiogenic drugs. On these grounds, it would be interesting to evaluate if the levels of ECM molecules exerting a role in both immunomodulation and angiogenesis may function as valuable biomarkers to stratify and identify the patients who benefit from the combination of anti-angiogenic therapy and ICIs.The identification of tumor-specific matrisomes suggests that tumor ECM might not only represent a valuable reservoir of predictive biomarkers but also a new therapeutic target to improve ICIs treatments. The ECM components, indeed, may be exploited as new druggable targets to act on the bio-physical properties of the matrix and, in turn to synergize with ICIs therapy. The tumor-associated ECM may be therapeutically modulated in several ways, including the targeting of single ECM molecules or ECM-remodeling enzymes. For example, the administration of recombinant hyaluronidase to reduce hyaluronan accumulation has been used in phase I and II clinical trials in combination with pembrolizumab and atezolizumab for the treatment of stomach, lung and pancreatic cancer , 162. ThThe potential weakness of the present systematic review resided in the fact that many of the studies are based on bioinformatic analyses. This represents a major limit since the altered mRNA levels not always coincide with the same alterations in the protein content. And this is particularly true when dealing with ECM molecules, which are extensively regulated not only at the transcriptional, but also post-translational level and undergo continuous remodeling. Nonetheless, we chose to comprise these studies since they were based on solid and strong results and provided deeper insights in the association between ECM and immune response, building the grounds for the development of new putative markers. Studies base on proteomic databases would certainly serve better this purpose, however these databases are limited compared to the RNAseq-based datasets. On these bases, we consider that more efforts should be put to attain a comprehensive proteomic profiling of the TME.The use of ICIs represents an important therapeutic option for cancer treatment, with subgroups of patients gaining major and long-term benefits. Nonetheless, a large number of patients showing scarce response to ICIs and some experiencing unwanted side effects. For these reasons, the identification of the patients that would better benefit from immunotherapies is key to avoid over-treatments and unnecessary side effects. In addition, this approach would allow the National Health Systems to optimize more efficiently the resources. Indeed, many investigations aimed at identifying reliable predictive biomarkers for ICIs efficacy are ongoing . These ahttps://doi.org/10.5281/zenodo.8348665.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: AF: Data curation, Writing – original draft, Formal Analysis, Investigation, Methodology. GC: Data curation, Formal Analysis, Investigation, Writing – original draft. EP: Formal Analysis, Writing – review & editing. LC: Formal Analysis, Writing – review & editing. GS: Formal Analysis, Writing – review & editing. ED: Formal Analysis, Writing – review & editing. GR: Writing – review & editing, Funding acquisition, Project administration. MM: Funding acquisition, Supervision, Writing – original draft. EA: Conceptualization, Data curation, Writing – original draft.


Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage damage and synovial inflammation and carries an enormous public health and economic burden. It is crucial to uncover the potential mechanisms of OA pathogenesis to develop new targets for OA treatment. In recent years, the pathogenic role of the gut microbiota in OA has been well recognized. Gut microbiota dysbiosis can break host-gut microbe equilibrium, trigger host immune responses and activate the “gut-joint axis”, which aggravates OA. However, although the role of the gut microbiota in OA is well known, the mechanisms modulating the interactions between the gut microbiota and host immunity remain unclear. This review summarizes research on the gut microbiota and the involved immune cells in OA and interprets the potential mechanisms for the interactions between the gut microbiota and host immune responses from four aspects: gut barrier, innate immunity, adaptive immunity and gut microbiota modulation. Future research should focus on the specific pathogen or the specific changes in the gut microbiota composition to identify the related signaling pathways involved in the pathogenesis of OA. In addition, future studies should include more novel interventions on immune cell modifications and gene regulation of specific gut microbiota related to OA to validate the application of gut microbiota modulation in the onset of OA. It has been reported that over 22.7 million individuals suffer from arthritis-induced activity limitations and 300 million people worldwide are diagnosed with OA , 2. OA cThe gut microbiota comprises multiple microorganisms, including bacteria, yeast, viruses, phages, parasites, and archaea, and due to its important endocrine and immune functions in pathophysiological modulation, the intestinal microbiome has been considered to be a “vital organ” . The gutAs mentioned above, more studies have focused on the role of the gut microbiota in OA. Many possible mechanisms of the gut microbiota and OA, including low-grade inflammation, metabolic changes and immune modulation, have been proposed. The gut microbiota interacts with risk factors for OA, including obesity, estrogen, age, sex, diet, metabolic syndrome, genetics, inflammation, mechanical loading and exercise , 21. Liu2The gut barrier has selective permeability for nutrients, water and electrolytes, while preventing harmful substances from transiting through the gut mucus membrane into other tissues, related organs and the circulatory system. The chemical barrier, biological barrier, mechanical barrier and immune barrier form the gut barrier. Bacterial membranes, tight junctions and intestinal epithelial cells form the mechanical barrier, so deleterious entities, such as microbes, intestinal antigens, and intestinal proinflammatory cytokines, are prevented from entering the blood circulation . The cheGut microbiota dysbiosis can cause changes in bacterial metabolism products and abnormal nutrient absorption, thus affecting gut permeability to allow bacterial products and inflammatory factors into the systemic circulation. Currently, the most studied categories of metabolism in association with host-microbiota interactions are bile acids (BAs), SCFAs, and metabolites of tryptophan. Dietary fibers are fermented by the anaerobic microflora in the gut with the generation of SCFAs as the end products, which indicates a healthy microbial community . When guFor many microflora, tryptophan is an important biosynthetic precursor . TryptopCholesterol in the liver can synthesize BAs via two mechanisms. The main mechanism is catalysed via cholesterol-7 α-hydroxylase (CYP7A1), finally generating cholic acid and chenodeoxycholic acid . The alt3Innate immunity is a primitive mechanism using germline-encoded proteins to recognize pathogens, thereby inducing immune responses. When a pathogen is encountered, the innate immune cell either kills the pathogen or stimulates the adaptive immune response to deal with the pathogen. The activation of the innate immune system plays a crucial role in the pathogenesis and progression of OA by recognizing DAMPs through interactions with pattern-recognition receptors (PRRs) , 65. PRR3.1In the knee synovium of OA patients, macrophages are the most widespread cell types , and areCD14 exists in various immune cells, such as monocytes and macrophages, and interacts with complexes of LPS and LBP , 85. LPSAccording to the research by Kraus et al., the number of activated macrophages was significantly associated with pain symptoms in the knee as well as the radiological severity of knee OA. In addition, they reported that macrophages are the cause of OA-related pain at the affected joint site . Analogo3.2As the body’s first line of defense against microorganisms, neutrophils play a crucial role in innate immunity . Brotfai3.3DCs are antigen-presenting cells derived from monocytes and can recognize and react to pathogen-associated and danger-associated signals, therefore bridging the innate and adaptive immune systems. Mature DCs mainly activate T cells to prime the adaptive immune system. Unlike macrophages, DCs do not have typical surface markers and are divided into plasmacytoid DCs (pDCs) and myeloid DCs (cDCs), and myeloid DCs are composed of myeloid cDC1 and myeloid cDC2 on the basis of cell lineage and correlate with the differential expression of essential transcription factors, such as interferon regulatory factors 8 and 4 . Now, a 3.4NK cells are CD56+CD3- lymphocytes comprising approximately 15% of all circulating lymphocytes, and can kill microorganisms through death-inducing receptors or the release of soluble molecules, including perforin and granulysin, so NK cells are vital components of the innate immune system. Based on the density of CD56 on the cell surface, NK cells are distributed into two cell subsets: CD56(bright) and CD56(dim) . CD56di. In contHuss et al. found that NK cells contained almost 30% of the CD45+ lymphocytes in the synovium of OA patients and expressed the chemoattractant receptors CCR5 and CXCR3. Compared with blood NK cells, NK cells in the synovium of OA patients show a silent phenotype consistent with post-activated exhaustion, which is impaired by cytokine-stimulated IFN-γ production . Jaime e3.5Mast cells act as sentinels in the innate immune system and respond to endogenous danger signals as well as exogenous pathogens rapidly . VariousIt is widely known that mast cells and their mediators are distributed in the synovial fluids and synovial tissues of OA patients . In addi4As described above, most studies have focused on the effect of innate immunity on the gut microbiota and osteoarthritis, especially on macrophages. Recently, some studies have focused on the effect of adaptive immunity on the intestinal microbiome and OA. Lymphocytes account for 10% of nonadipocyte cells in human adipose tissue and include B cells, T cells, innate lymphoid cell group 2 cells, NK cells and NK T cells . De Lang4.1Among adaptive immune cells, the predominant immune cells are T cells, which are crucial in the progression of OA. To date, it has been widely accepted that significant alterations in Th17 cells, Th9 cells, T memory cells, cytotoxic T cells, regulatory T (Treg) cells and Th1 cells are found in the synovial fluid, synovial tissues and peripheral blood of people with OA . However−CD69+), while fewer Treg cells were detected in peripheral blood concurrently with resting central memory cells (CD62L+CD69−)  . So et aL+CD69−) . Jhun anL+CD69−) . In a raL+CD69−) . Shan etL+CD69−) . FurtherL+CD69−) . ButyratL+CD69−) . In a rhL+CD69−) , and it L+CD69−) . Overall4.2in vivo and in vitro studies are needed.B cells are known to generate immunoglobulins and to regulate immunity. As immunosuppressive cells, regulatory B cells (Bregs) can maintain immunological tolerance via IL-10, IL-35, and TGF-β1 . Bregs c5The main characteristics of OA are chronic and progressive joint cartilage destruction and osteophyte proliferation, which ultimately result in chronic disability and a crucial burden on health care systems globally. To date, the most recognized clinical therapy for OA is palliative treatment, which ameliorates the symptoms of OA . Present5.1The selective utilization of prebiotics by the host’s gut microbiome is good for health, and the major components of prebiotics are part of carbohydrate groups, mostly oligosaccharides; however, prebiotics are not limited to carbohydrates only . Accordi5.2Probiotics, composed of live microorganisms, play a crucial role in the modulation of the intestinal microflora by enhancing the generation of antimicrobial compounds as well as immunoglobins and reducing the release of bacterial endotoxins , 145. LaSo et al. revealed that coadministration of Lactobacillus casei and type II collagen/glucosamine could significantly relieve the pain of OA and reduce cartilage destruction in a rat OA model compared to type II collagen/glucosamine or Lactobacillus casei alone; furthermore, oral supplementation with Lactobacillus casei and type II collagen/glucosamine could significantly inhibit proinflammatory cytokines, such as TNF-α, IFN-γ, IL-17A, IL-12B, IL-12A, IL-6, IL-2, and IL-1β, and upregulate anti-inflammatory cytokines, including TGF-β, IL-10, and IL-4, in CD4+ T cells . In addiIn sum, probiotics can mitigate the development of OA by relieving pain, inhibiting inflammatory responses and restoring the imbalance in the intestinal microbiome. In addition, the synergistic effect of probiotics and other pharmaceuticals on the gut microbiota and OA is better than their separate application. To better treat OA, large sample size clinical studies of probiotics and OA are necessary.5.3Glucosamine sulfate and chondroitin sulfate are common medicines used to relieve the symptoms of OA in clinical therapy and are recommended by the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases; in contrast, chondroitin sulfate and glucosamine sulfate are not recommended for OA by the Osteoarthritis Research Society International . The int5.4Exercise has been proven to alleviate pain and ameliorate joint function in OA patients . In addi5.5To address diseases correlated with the intestinal microbiome, fecal microbiota transplantation (FMT) has been introduced, where the nature of FMT is to transfer a healthy subject’s feces into another’s distal gastrointestinal tract . Notably6Currently, the incidence rate and disability rate of OA are very high, which makes OA a crucial public health problem globally. The pathogenesis of OA needs to be fully elucidated to develop effective means for preventing and treating OA. The gut microbiome is one of the risk factors correlated with OA; however, the causal effect of the gut microbiome and OA is still controversial. As mentioned above, the gut microbiome interacts with risk factors for OA, such as obesity, estrogen, age, sex, diet, metabolic syndrome, genetics, inflammation, mechanical loading and exercise. In this manuscript, we have summarized the potential immune mechanisms of the intestinal microbiome during the onset and progression of knee osteoarthritis and relevant studies on the immunomodulation of the gut microbiome in the management of OA. To investigate the pathogenesis and uncover the immune mechanisms between OA and the gut microbiota, more research on the distribution and composition of the intestinal microbiome and immune cells in synovial fluids and synovial tissues is warranted. Moreover, the alterations in intestinal microflora in individuals with OA need further investigation to identify the specific pathogens involved in the immune responses. In addition, more research is required to identify the potential common pathways and the synergistic effect between prebiotics, probiotics, nutraceuticals and exercise in the immune modulation of the intestinal microflora. Due to the enormous development of metabolomics, transcriptomics and next-generation sequencing, future studies should include more novel interventions on immune cell modifications and gene regulation on specific gut microbiota related to OA, and it is likely that more studies will uncover the relationship between distinct cell subgroups and the gut microbiota in OA. Additionally, signals associated with enteric dysbacteriosis and immune modulation in OA patients will be identified, and novel therapies targeting the gut microbiota and immunomodulation will be proposed to prevent the progression of OA.CS and JM conceived the study. CS, XZ, and TG screened the literatures and wrote the review. JM reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.


Conclusions: Our study reported a high rate of antimicrobial prescriptions despite a limited number of documented or suspected bacterial infections among the large cohort of hospitalized COVID-19 patients.Background: The aim of this study was to evaluate the prevalence of bacterial infections and antimicrobial prescriptions in a large cohort of COVID-19 patients and to identify the independent predictors of infection and antibiotic prescription. Methods: All consecutive patients hospitalized for COVID-19 from March 2020 to May 2021 at 1 of the 17 centers participating in the study were included. All subjects showing a clinical presentation consistent with a bacterial infection with microbiological confirmation (documented infection), and/or a procalcitonin value >1 ng/mL (suspected infection) were considered as having a coinfection (if present at admission) or a superinfection . Results: During the study period, of the 1993 patients, 42 (2.1%) presented with a microbiologically documented infection, including 17 coinfections and 25 superinfections, and 267 (13.2%) a suspected infection. A total of 478 subjects (24.5%) received an antibacterial treatment other than macrolides. No independent predictors of confirmed or suspected bacterial infection were identified. On the contrary, being hospitalized during the second wave of the pandemic (OR 1.35, 95% CI 1.18–1.97,  After more than two years, the COVID-19 pandemic is still a global health threat, with a total of 765 million cases and 6.9 million deaths occurring up to April 2023 [It has been clearly described that the pandemic, particularly during the first waves, has deeply impacted almost every aspect of global healthcare, such as the rate of hospital admission for several diseases. Indeed, many studies reported during the first months of the COVID-19 pandemic a dramatic fall in the number of patients admitted for several clinical conditions, including myocardial infarction, sepsis, or chronic obstructive pulmonary disease ,4. The sAlthough the available data estimate a 5% prevalence of co-infection and 16% prevalence of superinfection, more than 65% of patients hospitalized for COVID-19 . MoreoveItaly is one of the countries with the highest rates of antibiotic consumption and antimicrobial resistance in Europe , with a We conducted a retrospective multicenter cohort study; all hospitalized patients admitted for COVID-19 to 1 of the 17 participating centers from March 2020 to May 2021 were evaluated for enrolment. The clinical centers were located in different cities of the Campania region in southern Italy; 15 out of 17 were Infectious Disease Units, while the remaining 2 were sub-intensive care Units; all these centers have collaborated in previous studies ,11. The patients were adults (≥18 years), hospitalized with a diagnosis of SARS-CoV-2 infection confirmed by a positive reverse transcriptase-polymerase chain reaction (RT-PCR) on a naso-oropharyngeal swab. The study period was from 28 February 2020 to 31 May 2021 ,11. PatiInfections were defined according to the CDC/NHSN Surveillance Definitions 2023 . FurtherA coinfection was defined as a microbiologically documented bacterial infection diagnosed within 48 h after hospital admission, while infections diagnosed after 48 h of hospital stay were considered superinfections. We identified three waves of the pandemic: the first wave included patients admitted from March to August 2020, the second was those hospitalized from September 2020 to February 2021, while the patients admitted from March 2021 onwards were included in the third wave. The outcomes evaluated included the prevalence of definite or suspected bacterial infections, as previously defined, and the prevalence of antimicrobial prescriptions. Furthermore, we aimed at identifying independent predictors of bacterial infections, as well as of antibiotic prescriptions. Since macrolides, particularly during the first wave of the pandemic, were prescribed for reasons not strictly related to their antimicrobial activity, this class was excluded from the analysis of antibiotic prescription. t-test or Mann–Whitney U test, as indicated; categorical variables were compared by the chi-squared test, using exact procedures if needed. To identify independent predictors of bacterial infections and of antimicrobial prescriptions, a multivariate analysis using logistic regression was performed; clinically meaningful variables, and variables associated with the evaluated outcomes at univariate analysis with a p value < 0.05 were included in the model. A p value < 0.05 was considered statistically significant. The analysis was performed using IBM SPSS v 21.0 .Continuous variables were summarized as mean and standard deviation, or median and interquartile range, and categorical variables as absolute and relative frequencies. For continuous variables, the differences were evaluated by Student’s The study was approved by the Ethics Committee of the University of Campania L. Vanvitelli, Naples (n°10877/2020). All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants included in the study.During the study period, a total of 1993 patients were enrolled, with a mean age of 62.2 ± 16 years; 61.5% of subjects were males. The demographic, clinical, and biochemical characteristics of the patients included are shown in S. aureus (16.7%) and Enterococcus spp. (21.4%) were commonly isolated strains, while Enterobacterales accounted for 70% of all isolated Gram-negative bacteria. A total of 17 (0.9%) out of the 1993 patients enrolled presented with a microbiologically documented bacterial infection at admission, while 25 (1.2%) acquired a bacterial infection during the hospital stay. The most common sites of infection included primary or CVC-related bloodstream infections (23.8% of cases) and urinary tract infections (35.7% of cases). Among Gram-positive bacteria, Additionally, 267 (13.4%) patients had a procalcitonin value ≥ 1 and were classified as having a suspected bacterial infection. Despite these findings, that is, a definite or suspected bacterial infection in 15.5% of cases, 416 patients (20.8%) received treatment with a macrolide and 478 (24%) with an antimicrobial of a different class. p = 0.028), diabetes , chronic kidney diseases , chronic liver diseases , and HIV infection . Regarding the biochemical features, a higher baseline creatinine value was observed among patients with bacterial infection (p = 0.001). The modalities of oxygen treatment were differently distributed in the groups (p = 0.001); in particular, a higher proportion of patients in the bacterial infection group received treatment with a nasal cannula or simple face mask (59.7 vs. 48.4%), while a lower proportion needed HFNC, CPAP, or NIV . Subjects with bacterial infections obviously received antibacterial treatments more frequently (p = 0.001); moreover, a higher rate of these patients was treated with corticosteroids , although no significant difference was observed in the prevalence of severe or critical disease (p = 0.19). No difference in hospital mortality was observed in the groups.As shown in p < 0.001), were mostly admitted during the second wave of the pandemic , and had a higher burden of comorbidities, as demonstrated by a higher median CCI , and a higher prevalence of diabetes  and chronic kidney disease  (p < 0.001) as well as in the proportion of patients with severe or critical disease  and the rate of subjects requiring HFNC, CPAP, NIV, or mechanical ventilation ; consequently, the prescription of corticosteroid treatment was more common in the first group . Finally, patients in the antibiotic treatment group presented with a higher white blood cell count at baseline (p < 0.001). When we focused our analysis on patients who received an antimicrobial prescription and stratified them according to the presence of a bacterial infection (p = 0.008), a higher median SOFA score (p < 0.001) and a higher rate of oxygen therapy delivered through HFNC, CPAP, NIV, or mechanical ventilation (p < 0.001).When stratifying patients according to treatment received, we found that patients who were treated with antibiotics were older  . Regardinfection , we founIn a multivariate logistic regression analysis, no independent predictors of confirmed or suspected bacterial infection were identified . p = 0.001), having a SOFA score ≥ 3 , a severe or critical disease , and a higher white blood cell count  were all independently related to having received an antimicrobial prescription  and mechanical ventilation  were the main predictors of bacterial infection . No indeDespite the low prevalence of microbiologically documented bacterial infections, in the present study, a significant proportion of patients received antimicrobial treatment during hospital stay. This finding is consistent with several reports in the literature. A systematic review and meta-analysis by Langford and co-workers , includiThe main strength of our paper is the large number of patients 1993) enrolled across the first three waves of the pandemic, for most of whom several demographic and clinical data were available. Furthermore, we evaluated meaningful clinical outcomes, including clinical severity of disease and in-hospital mortality. However, there are some limitations, including the retrospective study design and the definitions used to identify patients with bacterial infections. The most important limitation of our study is the low number of microbiologically documented infections, mainly due to the well-documented difficulties of collecting and analyzing microbiological samples in such a difficult-to-manage population, particularly during the first waves 93 enroll,29. In pThe present study reports a high rate of antimicrobial prescriptions despite a limited number of documented or suspected bacterial infections among a large cohort of hospitalized COVID-19 patients. These data underline the need to enhance antimicrobial stewardship programs during the different phases of health emergencies, such as in the pandemic, to reduce the inappropriate use of antimicrobials, the principal driver of the development of antimicrobial resistance .