Clostridioides difficile infection and the development and spread of antimicrobial resistance. This review aims to identify a correct classification of IAIs, guiding clinicians in the selection of the best antibiotic therapy in patients with IAIs.Adequately controlling the source of infection and prescribing appropriately antibiotic therapy are the cornerstones of the management of patients with intra-abdominal infections (IAIs). Correctly classifying patients with IAIs is crucial to assessing the severity of their clinical condition and deciding the strategy of the treatment, including a correct empiric antibiotic therapy. Best practices in prescribing antibiotics may impact patient outcomes and the cost of treatment, as well as the risk of “opportunistic” infections such as  Intra-abdominal infections (IAIs) are an important source of patient morbidity and may be associated with poor outcomes. Treatment of patients with IAIs has been described in the literature to have satisfactory results if the management is adequate. In many studies, inclusion criteria limit the enrolment of critically ill patients, while in observational studies enrolling patients with no restrictions in inclusion criteria the mortality rate is higher. The Complicated Intra-Abdominal Infection Observational (CIAO) study, the Complicated Intra-Abdominal Infection Observational Worldwide (CIAOW) study, and the World Society of Emergency Surgery (WSES) Complicated Intra-Abdominal Infections Score Study (WISS) study showed mortality rates of 7.5%, 10.5%, and 9.2%, respectively .Due to restrictive criteria, clinical trials usually over-represent patients who have perforated appendicitis, and patients enrolled in clinical trials have often a high likelihood of survival, because trial eligibility criteria usually restrict the inclusion of patients with serious comorbidities that can increase the death rate of patients .Patients with IAIs should be classified correctly and stratified into low-risk and high-risk groups . Many riCorrectly classifying patients with IAIs is crucial to assessing the severity of their clinical condition and deciding the strategy of the treatment, including an appropriate empiric antibiotic therapy.Adequate source control and appropriate antibiotic therapy are the cornerstones in the management of IAIs. There is general consensus that source control is pivotal in the management of IAIs, where both controlling the source of infection and controlling the ongoing contamination are very relevant to improving outcomes. Even if not definitively evaluated by randomised control trials, the increase in death and other adverse outcomes associated with inadequate source control demonstrates that it is of primary importance in treating patients with IAIs ,8.Clostridioides difficile and the spread of antibiotic resistance in the individual patient and the broader environment. There is evidence that inappropriate empiric antibiotic therapy is associated with treatment failure and poor patient outcomes [In patients with IAIs, antibiotic therapy is also important. It aims to eradicate the residual bacterial infection after source control. In addition, some uncomplicated IAIs (as in the case of some uncomplicated cases of acute appendicitis and acute cholecystitis) can now be treated only with antibiotic therapy. Best practices in prescribing antibiotics may influence patient outcomes and the cost of treatment, as well as the risk of “opportunistic” infections such as outcomes ,10,11,12The appropriateness of the initial empiric antibiotic therapy prescribed for IAIs is crucial, because at least 24–48 h is required to obtain standard microbiological data to target the antibiotic therapy. Core components of antibiotic prescription practices include the following: adequacy of empiric antibiotic therapy; timing of antibiotic therapy; optimisation of pharmacokinetic/pharmacodynamic (PK/PD) parameters; length of treatment; and reassessment of antibiotic therapy based on the microbiological results and susceptibility testing.In 2016, the WSES published the executive summary of a consensus conference held on July 23, 2016, in Dublin, Ireland, covering all aspects of the management of IAIs . The firThis review was designed to identify a correct classification of IAIs, guiding clinicians in the selection of the best antibiotic therapy for patients with IAIs.IAIs represent a heterogeneous group of infections of abdominal origin, ranging from simple acute appendicitis to more complex diffuse peritonitis. A universally accepted classification divides intra-abdominal infections into uncomplicated and complicated cases ,15.Uncomplicated IAIs are those infections that, originating from an abdominal organ, remain confined to the same organ, without extending to the peritoneum. In general, patients with uncomplicated IAIs can undergo only surgery or only antibiotic therapy (as in the case of the conservative treatment of uncomplicated acute appendicitis or acute cholecystitis) ,17,18. MComplicated IAIs are those infections that, originating from an abdominal organ, extend into the peritoneum, giving rise to peritonitis. Patients with complicated IAIs always require both antibiotic therapy and source control . NonethePatients with complicated IAIs are characterised as manifesting secondary peritonitis. Depending on the peritoneal extent of the infectious process, complicated IAIs are divided into localised and generalised.Complicated IAIs are localised when the extent of the infectious process is contained by the peritoneal defence mechanisms. They are common in patients with complicated diverticulitis and appendicitis that, although evolving into perforation, can contain the infectious process through an effective peritoneal reaction and with local defence mechanisms, forming a circumscribed secondary acute peritonitis.Complicated diffuse IAIs, on the other hand, represent the consequence of a massive contamination of the entire peritoneal cavity, following the inability of the patient’s peritoneal defence mechanisms to limit the extent of the intra-abdominal infectious process, forming a secondary acute diffuse peritonitis.In patients with complicated IAIs, when source control is complete, a short course (3–5 days) of postoperative therapy is generally suggested. This has been confirmed by a prospective control trial (STOP-IT) published in 2015 . The stuPatients who have ongoing signs of infection or systemic signs of inflammation beyond 5–7 days of antibiotic treatment should warrant a diagnostic investigation to address an ongoing source of infection or failure of antibiotic therapy, and to determine whether a re-laparotomy is necessary.The limit of this classification is that it does not really describe the complexity of the patient and can create confusion by mixing elements of the extension of the infectious process and severity of the disease expression . On the Finally, patients with complicated IAIs may be characterised as manifesting tertiary peritonitis. There is much less agreement regarding what is meant by tertiary peritonitis. It is typical of critically ill or immunocompromised patients and is often associated with multidrug-resistant organisms (MDROs). It is typically associated with poor patient outcomes and is generally considered to be a distinct form of peritonitis ,24,25. TA classification according to the place where the patient contracts the infection divides the IAIs into community-acquired (CA-IAIs), if acquired in the community, or hospital-acquired (HA-IAIs), if acquired in a hospital or in health residences.“Healthcare-associated infection” (HCAIs) is a commonly used term for describing infections acquired during the course of receiving healthcare, including not only hospital-acquired infections but also infections in patients living in long-term care facilities, recently hospitalised, or undergoing recent aggressive medical therapies. However, in the setting of HCAIs, there are few data regarding the concept of HCAIs as opposed to hospital-acquired infections .Differentiating patients with CA-IAIs from patients with HA-IAIs allows the identification of patients with increased likelihood of IAIs caused by MDROs.Unlike CA-IAIs, HA-IAIs show a poorer prognosis and require more aggressive antibiotic therapy. HA-IAIs usually occur after a period of hospitalisation—often after surgery, and in patients already treated with antibiotic therapy. From a microbiological point of view, CA-IAIs are characterised by the presence of bacteria usually residing in the gastrointestinal tract, and are therefore generally predictable ,2. HA-IACandida spp. infections [Among patients with HA-IAIs, those with postoperative peritonitis may be associated with increased mortality due to underlying patient comorbidity, atypical presentation due to non-specific clinical signs, and risk factors for acquiring MDROs and fections ,30,31.Bacteroides fragilis  [Escherichia coli [Antibiotics for empiric treatment of CA-IAIs should cover enteric Gram-negative aerobic and facultative bacteria, enteric Gram-positive streptococci, and obligate anaerobic bacilli such as r colon) . The moshia coli ,2.Enterobacterales resistant to amoxicillin/clavulanate observed in community-acquired infections, caution should be taken in using amoxicillin/clavulanate in settings with a high local rate of resistance to this antibiotic.Due to the increasing prevalence of Enterobacterales remain susceptible to piperacillin/tazobactam. Its broad-spectrum activity makes it an attractive option in the management of CA-IAIs, especially in critically ill patients or in patients with CA-IAIs with other risk factors of adverse outcomes—including advanced age (70 years of age or older); presence of malignant disease; major compromise of cardiovascular, hepatic, or renal function; and hypoalbuminemia [However, most uminemia .E. coli and other Enterobacterales in CA-IAIs remain susceptible to third-generation cephalosporins. Among third-generation cephalosporins, cefotaxime or ceftriaxone in association with metronidazole may be options for empiric therapy of CA-IAIs in patients with no risk factors for ESBLs. Cefepime is a fourth-generation cephalosporin, with broader-spectrum activity than ceftriaxone. It is poorly hydrolysed by AmpC beta-lactamase, allowing it to be effective against AmpC-producing organisms [Most isolates of rganisms . For empE. coli and other Enterobacterales has limited the use of fluoroquinolones for empiric treatment of CA-IAIs, and they are generally suggested in association with metronidazole and only in non-critically ill patients with allergy to beta-lactam agents.In recent years, fluoroquinolones have been widely used in the treatment of IAIs due to their activity against aerobic Gram-negative bacteria and tissue penetration. The worldwide increase in resistance among Enterobacterales, which are prevalent in hospital-acquired infections but are also observed in community-acquired infections [In the context of complicated IAIs, the main resistance burden is posed by ESBL-producing fections ,34. ESBLfections ,36. Mostfections , non-susfections ,39.Enterobacterales may be components of the infection.ESBLs generally should not be covered in patients with CA-IAIs with no signs of severity or without risk of treatment failure, except in regions with particular local epidemiological conditions, where there is a high likelihood that ESBL-producing Enterobacterales in CA-IAIs include recent exposure to antibiotics  and known colonisation with ESBL producing Enterobacterales. These aspects should always be considered in stratifying patients with CA-IAIs to prescribe an adequate empiric therapy.Risk factors of ESBL-producing Enterobacterales. Group 1 carbapenems include ertapenem—a once-a-day carbapenem sharing the same activity of Group 2 carbapenems against ESBL-producing Enterobacterales. However, it is not active against Pseudomonas aeruginosa and enterococci [Carbapenems have been considered the empiric antibiotics of choice for treating patients with ESBL-producing erococci . Group 2However, in order to avoid excessive carbapenem use, carbapenem-sparing strategies using other antibiotics—such as piperacillin/tazobactam, an aminoglycoside agent, tigecycline, or eravacycline—should be considered.Enterobacterales has been a debated issue. Gram-negative bacteria have the ability to concomitantly produce multiple ESBLs as well as AmpC beta-lactamases and can possess other mechanisms of resistance limiting the activity of piperacillin/tazobactam [The significance of piperacillin/tazobactam for treating ESBL-producing zobactam . On the zobactam .Enterobacterales bloodstream infections [Enterobacterales [An RCT conducted in patients with ESBL-producing fections  demonstrcterales , it may cterales .Enterobacterales, and act synergistically against certain Gram-positive bacteria.Aminoglycosides have in vitro activity against aerobic Gram-negative bacteria, including ESBL-producing Because of their serious toxic side effects, including nephrotoxicity and ototoxicity, some authors do not recommend aminoglycosides for the routine empiric treatment of IAIs . They maP. aeruginosa or certain Enterobacterales, including Proteus spp. and Serratia spp. Excess mortality was observed in patients treated with tigecycline when compared with other antibiotics [Tigecycline remains a useful option for treating patients with complicated IAIs, due to its favourable activity against anaerobic organisms, enterococci, and ESBLs . It has ibiotics . Study-libiotics . TigecycEnterobacterales—and anaerobic bacteria. Like tigecycline, it is inactive against P. aeruginosa [Eravacycline is an antibiotic that is structurally similar to tigecycline. Eravacycline demonstrates broad-spectrum activity against Gram-positive, Gram-negative—including ESBLs-producing ruginosa . EravacyEnterobacterales [Enterobacterales (CRE), ceftazidime/avibactam should be reserved for the treatment of CRE.Finally, ceftolozane/tazobactam and ceftazidime/avibactam have shown efficacy in treating patients with IAIs caused by ESBL-producing cterales ,51. Theycterales . In settEnterobacterales represent a heterogeneous group of bacteria with more potential mechanisms of antibiotic resistance. They are generally divided into carbapenemase-producing Enterobacterales and non-carbapenemase-producing Enterobacterales. Carbapenemases hydrolyse penicillins, all cephalosporins, beta-lactamase inhibitors, and even carbapenems. The most widespread carbapenemases are Klebsiella pneumoniae carbapenemases [Among Gram-negative bacteria, carbapenem-resistant enemases ,53,54,55P. aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii. Non-fermenting Gram-negative bacteria are intrinsically resistant to many antibiotics; moreover, they can acquire additional resistance to other important antibiotic agents. These mechanisms may be present simultaneously and can confer resistance to different classes of antibiotics.Alarming rates of resistance to many antibiotics in health facilities worldwide have been reported for non-fermenting Gram-negative bacteria, including P. aeruginosa, including membrane permeability defects, expression of efflux pumps, and production of antibiotic-hydrolysing enzymes such as AmpC beta-lactamases or carbapenemases. The most prevalent carbapenemases in P. aeruginosa are MBLs, such as Verona integron-encoded metallo-β-lactamase (VIM) and imipenemase (IMP) types.Various mechanisms of resistance have been identified in K. pneumoniae infections [In recent years, several new antibiotics with predominant activity against Gram-negative bacteria have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Meropenem/vaborbactam, ceftazidime/avibactam, and imipenem/cilastatin/relebactam are the options for carbapenemase-producing fections ,63,64,65The lack of in vitro activity of ceftazidime/avibactam against metallo-beta-lactamase (MBL) and the observation that many MBL-producing infections can coproduce other beta-lactamases—including ESBLs, AmpC, and OXA-48—suggest a potential effect of combining ceftazidime/avibactam with aztreonam, which is not hydrolysed by MBLs ,67,68.Enterobacterales. It has no activity against anaerobic bacteria.Ceftazidime/avibactam is the preferred treatment option for OXA-48-like-producing Enterobacterales.Ceftazidime/avibactam in combination with aztreonam or cefiderocol  as monotEnterobacterales.Due to its epithelial lining fluid penetration, meropenem-vaborbactam should be used as first therapeutic choice in patients with ventilator-associated pneumonia due to KPC-producing P. aeruginosa, including MBLs. Cefiderocol is also effective in vitro against extreme drug resistant A. baumannii. Despite these promising early data, a recent clinical trial did not support the higher effectiveness of cefiderocol in the patient subset with A. baumannii infections [Cefiderocol appears to be effective in vitro against all resistance phenotypes of fections . Therefofections .Local epidemiological data for selecting an adequate empiric antibiotic therapy in patients with HA-IAIs at risk of infection with drug-resistant Gram-negative bacteria can be useful to define the appropriate antibiotic approach.E. coli and anaerobes [Enterococcus isolation was not found to be an independent risk factor for the composite outcomes in non-critically ill patients in a post hoc analysis of the STOP-IT trial database [Among Gram-positive bacteria, the pathogenicity of enterococci in IAIs has been a debated issue ,72,73,74naerobes ,76,77. Sdatabase .Enterococcus faecalis are susceptible to ampicillin. In patients with IAIs, Enterococcus faecium is increasingly encountered—particularly in patients with HA-IAIs. In contrast to E. faecalis, nearly all strains of E. faecium are resistant to ampicillin [E. faecium includes vancomycin or other glycopeptides, such as teicoplanin. The incidence of vancomycin-resistant E. faecium (VRE) is increasing. The acquisition of glycopeptide resistance by enterococci can seriously affect the treatment and control of these organisms in patients with hospital-acquired infections [Most strains of picillin ,79. Firsfections . LinezolStaphylococcus aureus is not common in patients with IAIs. It is rarely isolated in patients with CA-IAIs, but it may be found to some extent in patients with HA-IAIs and may be resistant to methicillin (MRSA). Patients with HA-IAIs, those colonised with MRSA, and those with multiple healthcare-associated risk factors for MRSA colonisation—including previous hospitalisation or surgery—and significant recent exposure to antibiotics may be considered at risk of infection by MRSA. Glycopeptides such as vancomycin may be used for the treatment of IAIs to provide therapy for suspected or proven infections caused by vancomycin-susceptible MRSA. S. aureus strains with increased resistance to vancomycin—known as vancomycin-intermediate-resistant S. aureus (VISA), exhibiting an MIC of 4–8 µg/mL, and S. aureus completely resistant to vancomycin (VRSA), exhibiting an MIC ≥ 16 µg/mL—are increasing [creasing . LinezolGrading of the clinical severity of patients with IAIs has been well described by the sepsis definitions . A prospThe data from the WISS study  showed tJournal of the American Medical Association (JAMA) [In February 2016, a proposal for new definitions for sepsis (Sepsis-3) was published in the n (JAMA) . These dn (JAMA) ,84. The Human immune responses to an infectious focus can vary greatly from individual to individual. Some individuals greatly overreact to infection, producing a massive cytokine storm leading to multi-organ failure. On the other hand, some individuals have little response to the same infection. Identifying patients overreacting to the infection as soon as possible and correcting the underlying dysfunction may improve patient outcomes ,86. An iThese findings have been confirmed in a recent meta-analysis demonstrating reduced mortality and significantly shorter hospital lengths of stay, with corresponding reductions in hospital costs, in patients receiving early appropriate versus inappropriate antibiotic therapy for severe bacterial infection .The reduction in mortality associated with early administration of antibiotics is strongest in patients with septic shock. Studies have reported a strong association between time to antibiotics and mortality in patients with septic shock, but a weaker association in patients with sepsis but without septic shock.A recent review suggests that a reasonable timeframe would be no later than 3–5 h after the onset of infection, but immediately for patients with septic shock . Based oThe 2021 Surviving Sepsis Campaign guidelines recommend the immediate administration of antibiotics in patients with possible septic shock or a high likelihood of sepsis—ideally within 1 h from the time when sepsis was first recognised. In patients with possible sepsis without shock, the guidelines suggest a time-limited course of rapid investigation and administering antibiotics within 3 h of recognition .Considering the spread of MDROs in many regions of the world and the association between delaying appropriate antibiotic therapy and worse outcomes in patients with sepsis and septic shock, the initial administration of a broad-spectrum regimen including at least one effective agent active against the suspected or proven offending organism is crucial.Antibiotic de-escalation is now considered a key point of antimicrobial stewardship programs in intensive care units (ICUs) . HoweverMontravers et al.  evaluateThe “dilution effect” should be considered for hydrophilic antibiotics—including beta-lactam agents, aminoglycosides, and glycopeptides—that selectively distribute to the extracellular space . Low plaHigher than standard loading doses of beta-lactam agents, aminoglycosides, fosfomycin, or glycopeptides should be administered to ensure optimal antibiotic concentration at the infection site in patients with sepsis or septic shock.Once appropriate initial loading is administered, it is crucial to reassess the antibiotic regimen daily, because pathophysiological changes may occur, significantly affecting antibiotic distribution in critically ill patients. Lower dosages of renally excreted antibiotics should be administered in patients with impaired renal function, while higher dosages of renally excreted antibiotics are needed for optimal exposure in patients with glomerular hyperfiltration. In critically ill patients, plasma creatinine is an unreliable marker of renal function .Adequate dosing is associated with the concept of time-dependent versus concentration-dependent killing. Beta-lactam agents have time-dependent activity, exerting optimal activity when antibiotic concentrations are maintained above the minimum inhibitory concentration (MIC) . For betThese results should not be generalisable to critically ill patients and patients with infections caused by less susceptible bacteria with high MIC values, where prolonged infusion with a beta-lactam agent seems to have more advantages than in patients with infections caused by sensitive bacteria ,98.Although there is insufficient evidence to reach definitive strong conclusions, two meta-analyses have demonstrated reduced short-term mortality with prolonged infusion of beta-lactam agents in critically ill patients ,100. No Interestingly, a recent meta-analysis evaluating prolonged versus intermittent beta-lactam antibiotics in patients with sepsis or septic shock  showed fOn the other hand, antibiotics such as aminoglycosides have concentration-dependent activity and should be administered in a once-daily manner (or with the least possible number of daily administrations) in order to achieve high peak plasma concentrations . NephrotAnother group of patients at high risk of treatment failure are immunocompromised patients. Immunosuppressive status includes congenital conditions such as T- or B-cell defects and macrophage dysfunctions, as well as acquired conditions such as infections with human immunodeficiency virus (HIV) developing acquired immunodeficiency syndrome, haematological malignancies or solid malignancies needing chemotherapy, and solid organ transplantation patients or those with inflammatory disease/rheumatologic disease needing immunomodulatory agents . The manThe anatomical extent of the infection, the presumed pathogens involved, the risk factors for major resistance patterns, and the patient’s clinical condition should be assessed independently so as to classify patients. This allows the stratification of patients with IAIs, guiding physicians in providing the appropriate antibiotic strategy.To assess the anatomical extent of the infection, physicians should always determine whether the IAI is complicated or uncomplicated. Unlike patients with complicated IAIs, who always require both antibiotic therapy and source control, patients with uncomplicated IAIs do not require postoperative antibiotics when source control is managed adequately.Candida spp. infections.To assess the presumed pathogens involved and any risk factors for major resistance patterns, physicians should always determine whether the IAI is community-acquired or hospital-acquired. HA-IAIs often involve multidrug-resistant and unpredictable bacteria, in terms of both the bacterial species involved and their sensitivity to antibiotics. Among patients with HA-IAIs, those with postoperative peritonitis may be associated with increased mortality due to underlying patient comorbidity, atypical presentation due to non-specific clinical signs, and risk factors for acquiring MDROs and In order to assess the grading of the clinical severity of patients, physicians should always determine the patients’ sepsis condition. Identifying patients overreacting to the infection as soon as possible and administering antibiotics may improve patient outcomes, especially in patients with septic shock. The 2021 Surviving Sepsis Campaign guidelines recommend the immediate administration of antibiotics in patients with possible septic shock or a high likelihood of sepsis—ideally within 1 h from the time when sepsis was first recognised. In patients with possible sepsis without shock, the guidelines suggest a time-limited course of rapid investigation and administering antibiotics within 3 h of recognition .In Abdominal tuberculosis (TB) is an increasingly common disease posing diagnostic challenges because of the non-specific features of the disease. Abdominal TB is a type of extrapulmonary tuberculosis that involves the abdominal organs, such as the intestines, peritoneum, and abdominal lymph nodes. It can occur either in isolation or along with a primary focus (such as the lungs) in patients with disseminated tuberculosis. This review does not refer to abdominal TB, which will be considered individually in an upcoming review.


Cancer of the skin is by far the most common of all cancers. Although the incidence of melanoma is relatively low among skin cancers, it can account for a high number of skin cancer deaths. Since the start of deeper insight into the mechanisms of melanoma tumorigenesis and their strong interaction with the immune system, the development of new therapeutical strategies has been continuously rising. The high number of melanoma cell mutations provides a diverse set of antigens that the immune system can recognize and use to distinguish tumor cells from normal cells. Peptide-based synthetic anti-tumor vaccines are based on tumor antigens that elicit an immune response due to antigen-presenting cells (APCs). Although targeting APCs with peptide antigens is the most important assumption for vaccine development, peptide antigens alone are poorly immunogenic. The immunogenicity of peptide antigens can be improved not only by synthetic modifications but also by the assistance of adjuvants and/or delivery systems. The current review summarizes the different chemical approaches for the development of effective peptide-based vaccines for the immunotherapeutic treatment of advanced melanoma. Melanoma is developed by the malignant transformation of melanocytes, which are melanin-producing, neural crest-derived cells. It is formed either by the dysfunction of dysplastic nevi or a single melanocyte . MelanocDespite being relatively rare among skin cancers , malignant melanoma is very aggressive and was responsible for nearly as many deaths as all other non-melanoma skin cancer types in 2020 . AlthougSignificant developments have been made in the treatment of melanoma during the last decade. With the improvement of immunotherapy and targeted molecular therapies, the life expectancy of distant and metastatic melanoma has considerably risen . DespiteIn 1909, Paul Ehrlich introduced the concept of immune surveillance, which plays a pivotal role in the prevention and repression of tumor progression ,7. The rCancer cells are derived from normal cells that undergo changes leading to uncontrolled proliferation. However, the immune system is not able to distinguish between them, hence their recognition and elimination are minimal ,11. TumoImmunoediting can be divided into three phases: elimination (recognition of the tumor by the adaptive immune system), equilibrium , and escape (phase of cancer progression that results in poorly immunogenic tumors and during which metastases are developed).elimination phase (which agrees with the concept of immunosurveillance), tumor cells can be successfully recognized and eradicated by innate and adaptive immune system components. Transformed cells initiate stromal remodeling that results in local tissue disruption, which is recognized as a danger signal by natural killer (NK) cells, T cells, and macrophages. These adaptive immune system cells produce cytokines, such as interferon-γ (IFN-γ) and other interleukins (ILs), to trigger extrinsic tumor suppressive mechanisms which result in the death of tumor cells . F. F93]. F peptide . The Cys linkers . First, peptide) b. The trreaction . In anotreaction . TRP-2 peptide via its N-terminus using amide bond formation. This chimera peptide was then attached to cholesterol using a succinic acid spacer. This structure could form a micelle, which produces a high immune response [To simplify these constructs, the antigen peptide can also be modified to get a derivative that can self-assemble into a nanocarrier. Human gp100(25–33) epitope peptide was modified at its hyrin IX . This amN-terminus of the antigen peptide, and the conjugate transformed into a hydrogel when a few drops of salt were added [C-terminus of the TRP-2 antigen does not result in a self-assembling peptide. However, the octaarginine-elongated epitope peptide with the CpG (as a negatively charged molecule) spontaneously forms nanocomplexes via electrostatic interaction. This vaccine activated DCs and elicited comparable therapeutic antitumor efficiency [Not only hydrophobic moieties may induce self-assembly of the antigen construct, but peptides also tend to form nanostructures. For instance, nanoscale structure formation was observed in the case of the peptide KKFKFEFEF in an aqueous solution coupled to the TRP-2 antigen via a disulfide bridge. For conjugation, a Cys was introduced to the re added . A similficiency .N-terminus and conjugated with an amphiphile diacyl glycerol-PEG tail using maleimide-thiol chemistry. During further optimization of this construct, the diacyl glycerol moiety was replaced by an albumin binding cyclic peptide and E-vitamin (α-tocopherol). These novel analogs had the same efficacy [N-terminus (extra Cys or 5-azidopentanoyl group) for the conjugation reaction. Evans Blue (EB) and its derivatives can bind to serum albumin and may be used to design albumin binding antigen constructs. When a melanoma-associated subunit Ag, TRP-2(180–188), was attached to a maleimide-functionalized EB derivative, the peptide was extended with a Cys at its N-terminus. This construct formed self-assembled amphiphilic nanoparticles in an aqueous solution and was efficiently accumulated in lymph nodes via albumin binding, which significantly prohibited tumor growth [Antigen efficacy may be improved by binding to endogenous proteins, thus increasing its half-life and targeting it to lymph nodes. A systematic study identified diacyl glycerol as an ideal binding partner of albumin . Modificefficacy . Three cr growth .in vivo. One possible explanation could be its poor presentation by MHC I, which is associated with low cellular uptake. Although the internalization of antigens into DCs may be enhanced by nanoparticles, their fate is often degradation in endolysosomes. To avoid this unwanted degradation, the use of carriers with pH-dependent membrane destabilizing activity may solve the problem and promote an immune response. When cell-penetrating peptides (CPP) were conjugated to gp100(20–38) antigen peptide, the immunogenicity was increased in vivo by as much as 25-fold. Similar to albumin binders, the CPP has resulted in increased accumulation in lymph nodes. In addition to the different CPPs, the influence of structure and conjugation mode was also studied. It turned out that neither the connection of the antigen and the CPP (C-N or C-C terminus), nor the type of the covalent linkage  influences the activity of the constructs; all of them had the same behavior [N-terminus by decalysine. The cellular uptake may be enhanced by conjugation with a cell-penetrating peptide as well. A cell-penetrating peptide variant of the protein ZEBRA was conjugated with epitope peptides via an amide bond. Using the proper adjuvant, these conjugates showed potent antitumor immunity [There may be several reasons why a peptide antigen may not be efficient enough behavior . In anotbehavior . To reacimmunity .These examples show that although immune recognition is very sensitive to the chemical structure, there are possibilities for chemical modification and thus improvement of the epitope peptides. One bottleneck of this strategy is its unique and often unpredictable effect. There is not a generally applicable modification, but there are possibilities that may be tried for better efficacy.+ and CD4+ T cells to induce an immune response. Peptide antigens correlating with MHC I and CD8+ T cells are typically 8–10 amino acids long, while antigens for MHC II receptors and CD4+ T cells are usually 13–18 amino acids long [The main requirement for antigens is their strong potency for TLRs or MHC receptors to promote the activation of CD8ids long . As thesids long .Alum), a continuously growing number of adjuvants have been reported in parallel with the development of vaccines. Various adjuvants have been evaluated in preclinical, as well as clinical trials to overcome the immune tolerance of peptide vaccines. Here, we briefly list the main adjuvant groups and their most prominent representatives, which have been used in preclinical or clinical trials. In general, they can be classified into two main groups: molecular adjuvants and immunostimulating complexes [Since the license for human usage of the first adjuvant, aluminum salts  was detected after the immunization, and the vaccine-induced spleen lymphocytes have significant antitumor activity [TM ISA-51 alone or in combination with TLR 7 agonist imiquimod were used. It was found that CD8+ T cell responses were stronger in DMSO-containing groups alone or in combination with imiquimod , compared to MontanideTM ISA-51 involved groups alone or in combination with imiquimod  [Freund’s adjuvants exist in completed (CFA or FCA) or incomplete (IFA or FIA) forms; they are popular delivery methods for various peptides ,128. IFAd humans ,130,131.d humans . They fad humans . Chu et activity . Meneveactively) .+ T cell response, especially to NY-ESO-1, while the Arm A construct elicited CD8+ T cell responses against MelanA/MART1 compared to Arm A against both Melan-A/MART-1 and NY-ESO-1. The determination of the deeper molecular mechanism evoked by the vaccines, namely the correlation of HLA-type and cellular immune responses, is in progress [Poly-ICLC is a double-stranded RNA (dsRNA) therapeutic compound with a combined immunostimulatory effect. It is composed of synthetic polyinosinic-polycytidylic acid (poly-IC) and two stabilizer compounds , such as poly-L-lysine (PLL) and carboxymethylcellulose (CMC). Poly-IC is an immune stimulator with multiple effects; due to its pathogen-associated molecular pattern (PAMP), which mimics viral dsRNA properties, it can activate TLR 3 and cytoplasmic RNA helicases . In Phasprogress .+ T cell-generating ability [in vivo and during clinical trials, it has been established that the most promising results might be obtained by the application of different adjuvant combinations. One intensively developed group of adjuvants is using immunostimulating complexes (ISCOMs), which are cargo-like nanoparticles with approx. 40 nm size, and obtained a combination of antigens, cholesterol, phospholipid and the Quillaja saponaria saponin. This combination results in a hydrophobic interaction between the lipids and the antigen [Not only synthetic molecules, but often immunostimulatory proteins are also used in combination with antigens to boost the immune system. Since 1985, when granulocyte-macrophage colony-stimulating factor (GM-CSF) was identified, intensive studies have been driven to establish its mechanism of action. GM-CSF is frequently used for peptide vaccines due to its increased antibody production and CD8 ability . Based o antigen .In vaccines, the delivery system generally means different kinds of nanoparticles, which should protect the peptide vaccine from enzymatic degradation and also have depo-like effects; they ensure a continuous and sustained level of antigen at the injection site, thereby increasing the immune response. Due to their specific and different chemical and physiological properties, nanoparticles can influence their interaction with the target cells. The interaction can be easily optimized by the modification of the surface of nanoparticles  ,140; or + T cells in B16F10 melanoma tumor-bearing mice [8-conjugated SIINFEKL, which is an MHC I restricted melanoma antigen), CpG, and 1-methyl-tryptophan (1-MT), which is the inhibitor of the immune checkpoint indoleamine-2,3-dioxygenase (IDO). The construct can not only be effectively taken up by DCs and promote their maturation but also result in a CD8+ T cell response against B16-OVA tumor cells in vitro. Moreover, the vaccine significantly increased the recruitment of CD8+ and CD4+ T cells and inhibited tumor growth in tumor-bearing mice [Liposomes can be characterized by their lipid composition, surface charge, and size. All parameters can be easily changed by combining different lipids or by chemical modification to alter the surface charge  . Chemicaing mice . In anoting mice .natural  or synthetic , poly-lactic acid (PLA), poly-ethylene glycol (PEG), poly-vinyl alcohol (PVA), poly-acrylic acid (PAA), poly-acrylamide (PAAm), etc.), and their combination . Due to their highly porous construction, they form an extracellular matrix (ECM)-like structure that provides a stable repository for immunotherapy agents without damaging their biological activity [in situ gelation method, and many of them have injectable sol-gel properties [Hydrogels are another type of delivery carriers that can be classified based on their origin: activity . The firactivity . Hydrogeactivity ,164. Theactivity . Due to activity . Nowadayoperties ,168.60 (mellitin and RADA8) system has a direct antitumor effect and induces DC maturation. This construct was further developed, resulting in the MRK system, in which the hydrogel scaffold was composed of mellitin and RADA6. This MR52 hydrogel scaffold was loaded with a drug molecule . It was found that MRK elicits a strong antitumor immune response and offers potent therapeutic efficacy for melanoma [Peptide-based hydrogels can be successfully used for cancer immunotherapy, they can deliver cytokines, anticancer vaccines, checkpoint inhibitors, and chimeric antigen receptor (CAR)-T cells . Wakabaymelanoma .in vivo model [Since the development of the multiple antigen peptide system in 1988, which was a dendrimer structure from lysine monomers , a serievo model . An excevo model ,177.180SVYDFFVWL188) was complexed to the co-assembled nanoparticle, which was formed by conjugation of low-molecular-weight short-chain poly(ethylene glycol)  and pyropheophorbide-A (PPa) nanoconjugates, by hydrophobic interaction. This antigen-delivery nanoplatform is suitable for immunotherapy of melanoma through stimulation of DCs. It was found that the nanoplatform can itself, without any adjuvants, effectively be taken up by DCs and can cause their maturation in vitro. The nanoparticle pulsed DCs were injected into B16-F10 bearing C57BL/6 mice, resulting in a significant antitumor immune response with delayed tumor growth. Moreover, the nanoparticle has a self-chelating ability; therefore, the migration process and the biodistribution of the construct can be easily tracked by, e.g., PET imaging [in vivo melanoma models, self-adjuvant lipopeptide vaccine micelles efficiently prevented tumor growth, as well as tumorigenesis [Polymeric micelles are thermodynamically stable colloidal solutions that are generally formed by the self-assembly of amphiphilic block copolymers. Just as the other nanoparticle types, micelles can also enhance drug stability and efficacy by maintaining their delayed release. Micelles can be targeted to tumor cells; therefore, they can significantly reduce the toxicity of drugs . Polymerigenesis . Accordiigenesis . Intensiigenesis .Due to their unique optical, physical, chemical, electronic, and magnetic properties, inorganic nanoparticles have been widely used for cancer diagnosis and treatment. Due to their biostability, biocompatibility, immunogenicity, and safety, some of them are used in preclinical experiments as vaccine delivery systems. The most commonly used inorganic nanoparticles against melanoma are gold and aluminum-based nanoparticles .+ T cells, resulting in the inhibition of tumor growth, as well as metastasis in both B16-F10 prophylactic and lung metastasis models [+ T cell response with the association of a significantly increased number of tumor infiltrating CD8+ T cells. The vaccinated B16-OVA-bearing mice showed slower tumor progression with a prolonged survival rate [A DC-specific antibody-modified liposome-coated gold nanoparticle was introduced by Liang et al. for targeted delivery of MPLA adjuvant and TRP-2(180–188) melanoma antigen peptide. The construct, due to its efficient DC stimulatory and maturation ability, successfully activated CD8s models . To avoival rate . Anotherval rate .+ T cell responses and prolong the survival of B16-OVA-bearing mice [2 spacer, in the presence of CpG ODN 1826 (5′-tccatgacgttcctgacgtt-3′). The obtained results suggested that ANLs can elicit a double response, namely, they promoted the presentation of MHC I epitopes in APC and simultaneously activate and induce the proliferation of CD8+ T cells. Based on the in vivo data obtained after vaccination of B16-OVA and B16F10 mice, significantly decreased tumor growth and prolonged survival were observed in both models [As we discussed above, aluminum adjuvant has been used for more than 100 years and has an irreplaceable ability to stimulate the humoral immune response . The ideing mice . These ih models .in vitro, this effect was diminished in vivo. The increasing number of epitopes per VLP subunit did not affect the activation of T cells in vivo. The functionalization of two copies of gp100(25–33) consisting of VPL surface by dimannosylation significantly enhanced the uptake by APCs due to the mannose-specific internalization way and resulted in delayed tumor growth and enhanced mouse survival [KAVYNFATMGGCK(N3)-NH2, where the sequence corresponding to p33 is bold) was carried out by a Cu-free click reaction. The results obtained in vivo showed that formulating the CuMVTT-p33 nano-vaccine with MCT was more potent in blocking tumor growth compared to using the nanovaccine alone. The CuMVTT-p33 vaccine formulated with MCT adjuvant enhanced the level of infiltrating of CD8+ and p33-specific CTLs into the tumor, and the production of IFN-γ in B16F10p33 melanoma model [+ T cell response, but the best therapeutic effect was achieved with the mixed epitope, including VLP. Due to the Cu-free click chemistry, which is a non-toxic bio-orthogonal linkage method, the developed peptide-VLP platform has been ready for clinical translation of neoantigen-based personalized immunotherapy [Viral capsid proteins are able to self-assemble into particle structures that closely resemble the natural virus from which they are derived. Virus-like particles (VLPs) are unable to replicate and are non-infectious, but they can efficiently deliver antigens to APCs, which are cross-presented in association with both MHC class I and class II, eliciting humoral and cellular immune responses, respectively . Cheng asurvival . In anotma model . An exceotherapy . The maiclinicaltrials.gov website (accessed on 11 December 2022). The table includes studies that were last updated in 2017 or later. The high number of clinical trials related to peptide-based immunotherapies targeting melanoma also displays the relevance of peptide vaccines. Among the 3019 studies  that cliData from clinical trials show that only small successes are achieved with peptide-based vaccines, but a breakthrough is still a long way off. Several processes may play a role, either together or separately, in the background of this failure. In most of the clinical trials, multiepitope vaccines have been used for the treatment of melanoma, which can reduce the risk of immune escape . UnfortuThe immune escape of tumor cells is the other critical issue hindering the efficacy of peptide-based vaccines. Tran and coworkers established that loss of HLA site heterozygosity strongly limits clinical responses to either peptide-based vaccines or adoptive T cell therapies . In addiAnother possible reason for ineffective melanoma treatment is the phenomenon of epitope spreading. This process can be induced by either vaccination, applying immune checkpoint inhibitors, and/or adoptive T cell therapy . In all Last, but not least, the following parameters must be kept in mind when trying to find a way to maximize the effectiveness of the vaccination: (i) What is the general state of health of the patient? (ii) Which tumor stage (primary or metastatic) can be determined? (iii) Can we identify prognostic factors or predictive biomarkers? .Considering the above-described reasons, the combination of tumor vaccines with other traditional therapies, such as chemoradiotherapy and targeted therapy, might be the solution to the ineffectiveness of melanoma treatment.Cancer vaccines, especially peptide-based vaccines, have emerged and developed rapidly in the last few decades. They are based on tumor antigens that can activate and maturate DCs; therefore, targeting DCs is one of the most important goals for vaccine development, which can be achieved by several kinds of chemical alterations of possible antigens, such as modification of their structure, using conjugation strategies, or encapsulation into nanoparticles. Peptide-based vaccines are produced almost exclusively by synthetic methods and can be fully and accurately characterized as chemical entities. Due to synthetic approaches, the production of peptide vaccines is simple, easily variable, and reproducible. Despite the synthetic advantages, peptide antigens are poorly immunogenic themselves; the use of adjuvants is essential for eliciting a high-frequency immune response. Unfortunately, adjuvants alone cannot increase the immunogenicity of peptide antigens; the use of delivery systems is usually required to achieve the desired response. The diverse possibilities to chemically modify the peptide structure; the versatile conjugation methods, along with the numerous possibilities of adjuvant usage and delivery options, make the topic of anti-cancer peptide vaccines a dynamic and constantly developing field. Moreover, the continuously increasing number of clinical trials related to peptide-based immunotherapies targeting melanoma unequivocally displays the relevance of peptide vaccines.


Enterobacterales (CRE) has been observed. This presents a substantial challenge in the treatment of CRE-infected individuals. Bacterial plasmids include the genes for carbapenem resistance, which can also spread to other bacteria to make them resistant. The incidence of CRE is rising significantly despite the efforts of health authorities, clinicians, and scientists. Many genotypic and phenotypic techniques are available to identify CRE. However, effective identification requires the integration of two or more methods. Whole genome sequencing (WGS), an advanced molecular approach, helps identify new strains of CRE and screening of the patient population; however, WGS is challenging to apply in clinical settings due to the complexity and high expense involved with this technique. The current review highlights the molecular mechanism of development of Carbapenem resistance, the epidemiology of CRE infections, spread of CRE, treatment options, and the phenotypic/genotypic characterisation of CRE. The potential of microorganisms to acquire resistance against Carbapenems remains high, which can lead to even more susceptible drugs such as colistin and polymyxins. Hence, the current study recommends running the antibiotic stewardship programs at an institutional level to control the use of antibiotics and to reduce the spread of CRE worldwide.Improper use of antimicrobials has resulted in the emergence of antimicrobial resistance (AMR), including multi-drug resistance (MDR) among bacteria. Recently, a sudden increase in Carbapenem-resistant  Acinetobacter baumannii (CRAB), and Carbapenem-resistant Pseudomonas aeruginosa (CRPA) as priority AMR pathogens that pose significant threats to human health [One of the primary reasons for the emergence of antibiotic resistance (AMR) worldwide is over-the-counter availability of antibiotics. With the incidences rising alarmingly, AMR poses severe challenges to the general public and the medical fraternity. AMR accounts for a significant proportion of the global morbidity and mortality rates associated with bacterial infections. The most important contributor to multi-drug resistance (MDR) is Gram-negative bacteria. Recently, MDR focus has been placed on Carbapenem-resistant Gram-negative bacteria. The World Health Organization (WHO) lists CRE, Carbapenem-resistant n health .Enterobacterales are Gram-negative facultative anaerobes that cause a broad spectrum of severe infections such as septicemia, community/hospital/ventilator-acquired infections, complex urinary tract infections (cUTIs), and intra-abdominal infections [Enterobacterales. Widespread use of Carbapenem against these organisms has led to the emergence of carbapenemase-producing Enterobacterales (CPE), whose infections are challenging to treat with the other drug [fections ,3,4. Becher drug . Carbapeher drug .There are very few treatment options for CRE infections, making this issue even worse. With an increasing incidence of CRE infections and lack of treatment modalities for CRE infections, it becomes imperative to analyse the molecular mechanisms of MDR developed by CRE. This review focus is to discuss the prevalence of CRE, the molecular mechanism of MDR developed by CRE, modes of transmission of CRE, the genetics involved, diagnostics, and treatment modalities available to manage CRE infections.AmpC cephalosporinase and ESBL [Klebsiella pneumoniae carbapenemase (KPC), Class-B metallo-β-lactamases (MBLs), and Class-D oxacillinases (OXA)-type enzymes. The enzymes from different classes exhibit differential inhibitory effects on carbapenem. The beta-lactamases are further classified into 4 classes  is the most frequently detected Class A carbapenemase, and it is currently the most common in the United States [Enterobacterales. This category includes carbapenemases such as Serratia marcescens enzyme (SME), imipenem-hydrolyzing beta-lactamase, non-metallo-carbapenemase of class A (NMC-A), and Guyana extended-spectrum beta-lactamase (GES). But aztreonam and ion chelators such as ethylenediaminetetraacetic acid (EDTA) or dipicolinic acid (DPA) block class B carbapenemases, which helps treat or identify these microorganisms [Class B carbapenemases, also called Metallo beta-lactamases (MBLs), have at least one zinc ion to break down the beta-lactam ring. Class A and D carbapenemases have a serine amino acid at their active site. d States . Penicilrganisms .Acinetobacter spp., in particular, OXA-51, which is chromosomally expressed in Acinetobacter baumannii and may confer carbapenem resistance. Notably, although they are found in Acinetobacter spp., OXA-48-like enzymes are the only subgroup with an actual prevalence in the Enterobacterales [Additionally, when no additional resistance mechanisms are exhibited, MBLs are resistant to beta-lactamase inhibitors but remain susceptible to ATM. The Verona integron-encoded MBL (VIM), the New Delhi MBL (NDM), and imipenemase are some of the MBLs with the greatest clinical significance. Due to the many sequence variants that have been discovered within this group, class D carbapenemases (sometimes referred to as oxacillinases or OXAs) comprise the largest class of carbapenemases. Class D carbapenemases differ from class A and class B in that they are resistant to beta-lactamase inhibitors and EDTA, have only modest hydrolytic activity against carbapenemases, and have no impact on extended-spectrum cephalosporins. The majority of Class D carbapenemases have been discovered in cterales ,8.The growing use of carbapenem has caused more selection for CRE genes, consequently accelerating the emergence of CRE . The rismcr 1-5) carried on the plasmids are transferable between bacteria, thereby spreading the colistin resistance [However, there are concerns about the effectiveness, adverse effects, and increasing resistance against these antibiotics . Some CRsistance ,17,18,19sistance . There isistance .Combination therapy serves the purpose in treating several complex infectious diseases. Combination therapy has been shown to increase the efficacy of treatment, but it also increases the side effects as well as the probability of death ,21. SpecEnterobacterales carry the resistance genes on their plasmids that express the most important carbapenemases, i.e., KPC and the New Delhi Metallo-β-lactamase (NDM). KPC has been widely reported in the United States, southern Europe, Israel, and China, while NDM has been reported in northern Europe, the UK, and India [The carbapenem resistance is carried out by the genes present on the plasmids of the bacteria. These genes encode for β-lactamases . The carnd India ,25,26. Knd India . The thiFrom a U.S. study conducted in 2016, 48 states reported the presence of CRE resulting from KPC, 25 states reported NDM, 19 states reported OXA-48, and 6 states reported VIM . CurrentThe reports of the prevalence of CRE infections come from studies conducted in different countries. In an extensive study involving CRE cases from 7 states in the U.S., CRE incidences were reported to be 2.93 per every 100,000 individuals in the U.S. . DespiteA study conducted in a large university hospital in Bangkok, Thailand, reported a CRE prevalence of 1.4% between 2009 and 2011 . In a reCRE have significant potential to result in hospital outbreaks; some of which have been reported in hospitals in European countries ,46,47,48CRE colonise the digestive tract, which serves as their reservoir. CRE colonisation later leads to severe infections such as pneumonia, followed by UTIs, septicaemias, cutaneous infections, and surgical site infections . Long-teEnterobacterales, Pseudomonas, and Acinetobacter [Enterobacterales species, conferring resistance against carbapenem [A recent systematic review reported the role of the hospital environments, such as wastewater drainage. It sinks to act as a reservoir of CRE. This study identified 32 CRE outbreaks associated with wastewater reservoirs of CRE such as tobacter . Severalrbapenem ,60,61. TResearch has narrowed the predisposing factors for a patient contracting CRE to the following: overnight stay in a medical facility; chemotherapy; previous history of CRE infection; and epidemiological contact with a known carrier—all within one year . Other iStrict measures should be taken to maintain a closed sterile environment for the hospitalised patients and adequate steps should be taken to avoid contamination. Different CRE infection control measures proposed by the ECDC are screening for rectal CRE carriage on hospital admission, hand hygiene, patient isolation, antibiotic restriction, awareness, and education imparted to the patients and the staff . A recenAntimicrobial stewardship should also be followed along with infection control measures to control or prevent the rise in CRE. Reduced carbapenem use has been shown to be effective for CRE control .E. coli are reported to be transmitted through animal sources to humans [E. coli in the healthy population. Such patients carry the CRE in the intestinal flora and serve as potential candidates to spread CRE to other patients. Several studies have reported the contamination of food items such as chicken, meat, and vegetables [The CRE have excellent potential to spread in the community. The CRE can be transferred to humans through the food chain . The exogenous pathogenic o humans . Feco-orgetables ,72,73,74getables ,76,77,78For communities, CRE acquisition is either through horizontal transmissions or carbapenem-susceptible strain-associated resistance . The lacA need to evaluate the interactions between the community and hospital settings on the acquisition of CRE is an approach that is proposed to enhance infection control. In a recent study, the author believes that the emergence of carbapenem-resistance strains is also common in agricultural and environmental settings . CRE wasRapid detection and isolation of carriers on admission has been shown to significantly reduce the rates of hospital transmission of CRE infections . The samDevelopment and implementation of control and management options for CRE infection greatly rely on efficient and reliable laboratory detection and identification of CRE. However, the detection of CRE becomes challenging because of the heterogeneous expression of the CRE resistance genes and the multitude of pathways/mechanisms involved in conferring resistance to the bacteria . DiffereEnterobacterales isolates carry the antibiotic resistance gene blaKPC but can be acted upon by ertapenem, meropenem, and imipenem [Enterobacterales isolates are suspected to be producing carbapenemase by carbapenem breakpoints, they should be further confirmed by molecular techniques and others such as MHT and the Carba NP test [This is among the most typical and first-line methods for detecting carbapenemases. Several indicators could be utilised for this, but ertapenem is generally considered the most sensitive. MIC breakpoints are also used to confirm the sensitivity tests. These testing methods show high variability in detecting carbapenem resistance. The breakpoints suggested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) are helpful in predicting carbapenemase-resistant Gram-negative bacteria, but by this method, some bacteria remain susceptible to many carbapenems. For instance, some imipenem ,91,92. Timipenem . KPC is imipenem . The CLS NP test . Althoug NP test . The advAmpC [These methods rely on the inhibitors such as boronic acids for KPC detection, Ethylenediaminetetraacetic acid (EDTA) or dipicolinic acid for MBL, avibactam (NXL104) for OXA-48, clavulanate for ESBL, and cloxacillin for AmpC . These mAmpC .The Mastdiscs ID inhibitor combination detection disks (MDI) method detects carbapenemase by calculating the inhibition zones of the disks containing the enzyme inhibitors. Reports suggest that this method is insufficient to differentiate between OXA-48-type genes and different MBL genes such as NDM, VIM, and IMP . The RosThese methods are considered one of the optimal methods to screen for CRE . They arEnterobacterales isolates. MHT is used to detect resistance by quantifying the concentration of carbapenem, which is broken down by carbapenemase produced by resistant organisms, allowing carbapenem-susceptible Escherichia coli to survive. It is sensitive to detecting VIM, IMP, and OXA-48 [The CLSI for many years suggested the MHT. This growth-based approach was widely used to detect carbapenemase producers, until 2018, when it was removed from the CLSI M100 owing to various drawbacks and better alternatives. The MHT is a good assay in general, although it has very low sensitivity for MBLs and excellent sensitivity for class A carbapenemases  and class D carbapenemases . This ted OXA-48 . The majd OXA-48 . The turd OXA-48 .Enterobacterales and Pseudomonas spp. Imipenem is hydrolysed by carbapenemase, causing changes in pH, which are detected by colour changes in phenol red, a pH indicator. This technique is fast, cost-effective, sensitive, and specific as compared to several other phenotypic methods [The Carba NP employs colorimetric analysis for the rapid detection (≤2 h) of CPE. It is one of the standard phenotypic techniques recommended by the CLSI and was first presented in 2012 as a rapid test for carbapenemase identification in both  methods . Carba N methods . Therefo methods . OXA-48-like carbapenemases in the Enterobacterales have limited sensitivity [Enterobacterales, another variation of the CIM, known as the eCIM, was added to the CLSI recommendations in 2018. It should be used in combination with the mCIM [This method was first described in 2015. CIM detects the rate of carbapenem inactivation by the carbapenemase-producing organisms. Bacterial colonies are incubated in water suspended with carbapenem disks  and incubated. Then the discs are placed in carbapenem-sensitive E. coli strain plates with Mueller–Hinton agar (MHA). A small zone of inhibition is indicative of a carbapenemase-producing organism. This method of detection of CRE is widely utilised due to its specificity, sensitivity, and cost-effectiveness. Studies have shown CIM to be more accurate than other phenotypic tests, such as Carba NP and MHT, for detecting CRE. CIM also detects OXA-48 with high sensitivity . This tesitivity ,110. DueNumerous IC assays rely on antigen–antibody interaction to detect carbapenemase antigens. Commercial kits (KPC-k-set and OXA-48 K-set tests) combine nanotechnology principles and antigen–antibody interaction to detect OXA-48-like enzymes. These kits use gold nanoparticles cross-linked to nitrocellulose membranes . These tMatrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) is based on the analytical detection of various chemical products of the pathogens based on their molecular weight. It is now used to detect β-lactamase activity to detect Gram-negative bacteria . The proDNA-based techniques are more reliable and accurate than other techniques to detect CRE . DNA-bas® Blood Culture Identification Panel from BioMerieux. An automated multiplex PCR technique called Filmarray® can quickly identify 24 pathogens from positive blood cultures, including three resistance mechanisms  and 8 Gram-positive, 11 Gram-negative, and 5 yeast species [The first molecular test that received FDA approval was the BioFire Filmarray species .® Gram-negative Blood Culture Test is another FDA-cleared panel that can identify the ESBL CTX-M and five common carbapenemases  from positive blood cultures in as few as two hours. The Verigene® test has shown outstanding efficacy in multicentre investigations for identifying carbapenemase genes [The Nanosphere Verigenese genes .® Carba-R (Cepheid), which is also an FDA-approved method. This test offers the benefit of assessing samples from resistant cultures directly from rectal swab specimens, blood, urine or sputum in just 48 min [The KPC, NDM, VIM, IMP, and OXA-48 families of carbapenemase-producing organisms have 91 gene targets that may be detected using the real-time PCR test Xpertt 48 min . Severalt 48 min .K. pneumoniae, E. coli, Citrobacter freundii, P. aeruginosa, Enterobacter cloacae, and A. baumannii [PCR-based methods amplify the signature sequences present on the chromosomal DNA of the bacteria for their identification. PCR-based methods are employed to confirm the results of phenotype-based methods. PCR can be performed on the DNA isolated from bacterial cultures or directly from clinical samples . Numerous PCR-based detection methods have been developed to identify CRE . Many muaumannii .By amplifying and tracking the bacterial DNA sequences in real-time, multiplex real-time PCR identifies the bacterium. Due to the quick turnaround times of detection, which can be as quick as an hour, it has grown in popularity in recent years. The sensitivity range for PCR-based techniques is between 97 and 100% . These PThe principle behind microarray is the hybridisation of the target sample with the probes. Multiple samples are detected by microarray in a single reaction. The probes on the chip hybridise with the bacterial nucleotide sequences before the chip is checked for hybridisation. Numerous bacterial species have been reported to be easy to identify using microarrays . MultiplTo analyse the complete DNA sequence of the bacteria, WGS uses the next-generation sequencing (NGS) method. In a short time, WGS analyses the entire bacterial genome. Numerous epidemiological research has made use of it ,120. WGSAntimicrobials are used more frequently due to improvements in medication discovery and delivery. Due to selection pressure, AMR/MDR bacteria have emerged due to the unprecedented use of antibiotics. Public authorities should also raise knowledge regarding the development of AMR and MDR among medical professionals and the local population. Only after a licensed doctor has prescribed a legitimate prescription should antibiotics be given out. Since CRE can vertically transfer the genetic materials carrying the resistance genes to other bacteria, hospitals should take the necessary precautions to guarantee effective containment and disinfection of the potential sources of contamination or infection. Even though there are several genotypic and phenotypic screening methods for CRE, there is still lack of tests or methods for screening all the many carbapenemase-producing species. The robust techniques to identify the CRE efficiently with high sensitivity are expensive. Therefore, there is a need to develop a cost-effective and sensitive technique to detect and identify all the different types of carbapenemase-producing organisms, especially the CRE.Although the World Health Organization has identified CRE as a priority pathogen for prioritising new drug development and initiated the development of new drugs such as zidebactam, taniborbactam, nacubactam, etc., it is mandatory to control the use of drugs at the end-user level. There should be antibiotic stewardship programs at the institutional level that monitor the use of antibiotics. The proper and timely use of available antibiotics may help to stop the emergence of high AMR rates.


DNA- and RNA-binding proteins (DRBPs) typically possess multiple functions to bind both DNA and RNA and regulate gene expression from more than one level. They are controllers for post-transcriptional processes, such as splicing, polyadenylation, transportation, translation, and degradation of RNA transcripts in eukaryotic organisms, as well as regulators on the transcriptional level. Although DRBPs are reported to play critical roles in various developmental processes and diseases, it is still unclear how they work with DNAs and RNAs simultaneously and regulate genes at the transcriptional and post-transcriptional levels. To investigate the functional mechanism of DRBPs, we collected data from a variety of databases and literature and identified 118 DRBPs, which function as both transcription factors (TFs) and splicing factors (SFs), thus called DRBP-SF. Extensive investigations were conducted on four DRBP-SFs that were highly expressed in chronic myeloid leukemia (CML), heterogeneous nuclear ribonucleoprotein K (HNRNPK), heterogeneous nuclear ribonucleoprotein L (HNRNPL), non-POU domain–containing octamer–binding protein (NONO), and TAR DNA-binding protein 43 (TARDBP). By integrating and analyzing ChIP-seq, CLIP-seq, RNA-seq, and shRNA-seq data in K562 using binding and expression target analysis and Statistical Utility for RBP Functions, we discovered a two-layer regulatory network system centered on these four DRBP-SFs and proposed three possible regulatory models where DRBP-SFs can connect transcriptional and alternative splicing regulatory networks cooperatively in CML. The exploration of the identified DRBP-SFs provides new ideas for studying DRBP and regulatory networks, holding promise for further mechanistic discoveries of the two-layer gene regulatory system that may play critical roles in the occurrence and development of CML. Nucleic acid–binding proteins (NBPs), including DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), can regulate genes by interacting with DNAs or RNAs. DBPs and RBPs were considered to be functionally distinctive and were studied independently. However, this is an outdated concept as there is an increasing number of evidence suggesting that there are no well-defined differences between DBPs and RBPs, whereas many proteins are capable of interacting with both nucleic acids. These proteins are called DNA- and RNA-binding proteins (DRBPs) . DRBPs tin vivo  not a single experimental technique is available for directly identifying DRBPs , 2) currin vivo . Ultraviin vivo . The strhttps://rbpbase.shiny.embl.de/) integrates datasets from high-throughput RNA–binding protein (RBP) detection studies. RNA-binding proteins database (RBPDB)  and their DNA-binding motifs . AnimalT (RBPDB)  is a dat (RBPDB)  and ATtR (RBPDB)  are also (RBPDB) .Although DRBPs are reported to play critical roles in various developmental processes and diseases, it is still unclear how they work with DNAs and RNAs simultaneously and regulate genes at both transcriptional and post-transcriptional levels. To tackle this question, we collected data from a variety of databases and literature mentioned above and identified DRBPs as well as investigated the functional mechanism of DRBPs . FunctioAs of September 2021, we collected 995 DRBPs  from thrWe collected a total of 545 SFs  from thep-value < 0.01 were considered significantly enriched with DRBPs. Venn diagram was plotted to show the number of overlapping genes using the jvenn tool (Gene Ontology (GO) annotation enrichment test was used to explore the functional roles of DRBPs in terms of biological process (BP), cellular component (CC), and molecular function (MF). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to search metabolic pathways that DRBPs involved. GO and KEGG analyses were performed using the R package clusterProfiler v4.2.1 . GO termenn tool .via UCSC Xena  to analyze gene correlation for differentially expressed genes and performed principal component analysis dimensionality reduction on two datasets called “Cells-Leukemia cell line (CML)” and “Whole Blood” , heterogeneous nuclear ribonucleoprotein L (HNRNPL), non-POU domain-containing octamer-binding protein (NONO), and TAR DNA-binding protein 43 (TARDBP). And the IDs of their ChIP-seq data are ENCFF505RNR, ENCFF854WAP, NCFF211TTD and ENCFF564QOL, respectively. The information of differential expression was the CML differential expression files obtained in the previous step. The four ChIP-seq data were successively input into BETA V1.0.7, and the target genes of each DRBP-SF were inferred in combination with the differential expression data. We used the default parameters, except that the threshold of the Kolmogorov–Smirnov test was set to 0.05.To have an integral understanding of the specific roles of these four DRBP-SFs in AS, we selected four AS events in the SURF database, exon skipping (ES), alternative 3′ (A3SS) or 5′ (A5SS) splicing, and intron retention (RI) to extract splicing regulatory networks. The results of BETA and SURF were imported into Cytoscape v3.9.0  to visuaTo identify co-regulators of DRBP-SF, we searched protein–protein interactions (PPIs) between DRBP-SFs and TFs/SFs in the String database . We impoin vitro DNA-binding motif of HNRNPK has not been experimentally validated; thus, the top 3 de novo motifs with the smallest p-value from MEME  data (HNRNPK: ENCSR268ETU) were adjusted to 100 bp. Bedtools getfasta was used to convert the peak coordinates into RNA sequence by taking strand information into consideration. Then, MEME-chIP was used for motif discovery. RNA-binding motifs discovered by MEME-chIP were compared with motifs of HNRNPK downloaded from the database CISBP-RNA by Tomtom , with a database . CISBP-RFrom various resources listed in In addition, KEGG analysis revealed that DRBPs are enriched in many cancer-related pathways , includiTo clarify the relationship between DRBP-SFs and cancer, RNA-seq data from CML patients and healthy donors were compared. The results showed that approximately 73% (86 out of 118) of DRBP-SFs were significantly differentially expressed . Some ofBy integrating and analyzing the ChIP-seq, CLIP-seq, RNA-seq, and shRNA-seq data using BETA and SURF, we constructed a two-layer regulatory network system controlled by HNRNPK, HNRNPL, NONO, and TARDBP . In the HNRNPK, HNRNPL, and TARDBP could regulate their own splicing, as well as the splicing activities of each other. For example, HNRNPK can regulate the splicing of HNRNPL; HNRNPL can regulate the splicing of HNRNPK; NONO can regulate the splicing of TARDBP and HNRNPK; while TARDBP can regulate the splicing of HNRNPL and HNRNPK. Besides, the expression of these four genes in CML had a high correlation in a GEPIA analysis  Supplem.DRBP-SFs may regulate the same genes directly over the transcriptional and splicing levels as TFs and SFs, respectively . The numThe results of dimensionality reduction analysis of the two-level co-regulated genes in CML and whole blood sample are shown in de novo DNA motifs with the smallest p-value as the most likely DNA-binding motifs of HNRNPK , which was regulated by these three DRBPs at the splicing level. SAFB binds to the scaffold–matrix attachment region (S–MAR) DNA and forms a molecular assembly point to allow the formation of a “transcriptosomal” complex coupling transcription and RNA processing . In contHNRNPK splicing regulatory target genes, which were TFs, as well as HNRNPK and SAFB transcriptional regulatory target genes, were selected to construct a regulatory network of regulatory model III . HNRNPK To validate the proposed models, we used text mining to search literatures in PubMed using keywords “DRBP-SF & target gene,” “DRBP-SF & transcription–splice,” and “DRBP-SF & co-transcriptional splicing,” where “DRBP-SF” is one of HNRNPK, HNRNPL, NONO, and TARDBP and “target gene” iterates all targets controlled by these four DRBP-SFs in three models connecting both transcriptional and AS regulatory networks. Approximately 600 articles that matched the aforementioned keywords were manually screened and reviewed. However, due to the complexity of biological transcription and post-transcription mechanisms, limitations of the current understanding of DBPs and RBPs, and the lack of technology for DRBP study, these studies mainly focus on PPIs and expression changes of the proteins rather than the transcriptional or AS regulation networks of these DRBP-SFs on their target genes. Nevertheless, we found some evidence that support our models.For model I, it has been confirmed by many studies that AS is coupled with transcription that permits the sequential recognition of emerging splicing signals by the splicing machinery . This phIn this study, we investigated a class of DRBPs that also functioned as SFs, called DRBP-SFs. These proteins play critical roles in regulating gene expression at both the transcriptional and splicing levels with the capabilities to bind both DNAs and RNAs. By using BETA and SURF to construct regulatory networks in CML, we discovered a two-layer regulatory network system, connecting transcriptional and splicing regulatory networks through DRBP-SFs. Three transcriptional and splicing co-regulatory models were proposed by investigating the two-layer regulatory network system controlled by four DRBP-SFs, namely, HNRNPK, HNRNPL, NONO, and TARDBP. In model I, there are some genes directly regulated at the transcriptional and splicing levels by the same DRBP-SFs that function as TFs and SFs simultaneously, which might be involved in co-transcriptional splicing for rapid expression. In models II and III, DRBP-SFs dually control transcriptional and splicing networks through direct and indirect mechanisms, respectively, in which they collaborate with their own targets at one regulatory level and regulate other targets at the other regulatory level. Our results provide supporting evidence for understanding the dual role of DRBP-SFs in transcriptional control and AS.Through motif analysis, we further explored how HNRNPK binds to its two-level co-regulated genes in model I and found that HNRNPK directly binds to its promoters in most cases while indirectly binding to its pre-mRNA through other SFs in most cases; moreover, in a small number of cases, HNRNPK directly binds to its pre-mRNA targets. Further, we speculate that in regulatory model I, HNRNPK regulates transcription and splicing in a synergistic rather than a competitive manner. Because HNRNPK has multiple DNA and RNA binding domains, DNA and RNA are possible to bind HNRNPK simultaneously . This coThe four DRBP-SFs have been reported to play vital roles in cancers and other important BPs. HNRNPK regulates a wide range of BPs and disease pathogenesis, which is central to many cellular events, including long noncoding RNA (lncRNA) regulation, cancer development, and bone homeostasis . HNRNPL This study has several limitations. The DNA- and RNA-binding data of DRBP-SFs are from different sources, and ChIP-seq and eCLIP experiments were performed by different laboratories. Furthermore, we still lack an experimental technique that can investigate how DRBP-SFs bind to DNA and RNA at the same time. Due to the limitations of the current technologies and followed bioinformatics analysis methods, the transcription and splicing networks may not be able to truly, accurately, and completely reflect the actual situation in cells. The three regulatory models are worthy of validation in more cells and DRBP-SFs. Besides, although we observed that HNRNPK binds to DNA directly and to RNA indirectly on many target genes in regulatory model I, it requires more interaction data to support the finding. For regulatory models II and III, whether the DRBP-SFs and the coordinated and regulated TFs–SFs possess direct physical interaction would also require further experimental verification.In conclusion, DRBP-SFs are key players in transcriptional and post-transcriptional events. The combination of versatility of their DNA- and RNA-binding domains and their structural flexibility enables DRBP-SFs to control the metabolism of a large array of transcripts. The DRBP-SF regulatory networks we have constructed here suggested a novel two-layer regulatory system on both transcriptional and splicing levels where DRBP-SFs are demonstrated to co-regulate DNA and RNA in conjunction. For this, three regulatory models were proposed with supporting evidence. This study can provide new ideas for further mechanistic research on DRBP-SFs and their two-layer gene regulatory systems that may play critical roles in cancer.


Magnetic Resonance Image-guided High Intensity Focused Ultrasound (MR-HIFU) is a non-invasive treatment option for palliative patients with painful bone metastases. Early evidence suggests that MR-HIFU is associated with similar overall treatment response, but more rapid pain palliation compared to external beam radiotherapy (EBRT). This modelling study aimed to assess the cost-effectiveness of MR-HIFU as an alternative treatment option for painful bone metastases from the perspective of the German Statutory Health Insurance (SHI).A microsimulation model with lifelong time horizon and one-month cycle length was developed. To calculate the incremental cost-effectiveness ratio (ICER), strategy A  was compared to strategy B  for patients with bone metastases due to breast, prostate, or lung cancer. Input parameters used for the model were extracted from the literature. Results were expressed as EUR per quality-adjusted life years (QALYs) and EUR per pain response . Deterministic and probabilistic sensitivity analyses (PSA) were performed to test the robustness of results, and a value of information analysis was conducted.Compared to strategy B, strategy A resulted in additional costs (EUR 399) and benefits (0.02 QALYs and 0.95 months with pain response). In the base case, the resulting ICERs (strategy A vs. strategy B) are EUR 19,845/QALY and EUR 421 per pain response. Offering all patients MR-HIFU as first-line treatment would increase the ICER by 50% . PSA showed that at a  willingness to pay of EUR 20,000/QALY, the probability of MR-HIFU being cost-effective was 52%. The expected value of perfect information (EVPI) for the benefit population in Germany is approximately EUR 190 Mio.Although there is considerable uncertainty, the results demonstrate that introducing MR-HIFU as a treatment alternative for painful bone metastases might be cost-effective for the German SHI. The high EVPI indicate that further studies to reduce uncertainty would be worthwhile. Bone metastases occur in 65% of patients with advanced solid cancer, particularly originating from malignancies of the lung, prostate, and breast. For these patients, pain is a common and devastating symptom affecting both quality of life and functionality –3. OpioiApproximately 60-70% of patients initially respond to EBRT over the course of four weeks following treatment . HoweverMagnetic Resonance Image-guided High Intensity Focused Ultrasound (MR-HIFU) is a non-invasive treatment modality that may substantially improve pain palliation and can be offered as first-line treatment or after prior radiation . A randoAn early assessment of the cost-effectiveness of adding MR-HIFU as first-line treatment or after prior radiation compared to EBRT can provide an appraisal of the potential value of this new technology . This economic modelling study assessed the cost-effectiveness of MR-HIFU as treatment alternative for the palliative treatment of patients with bone metastases in comparison to the current standard of care , from the perspective of the Statutory Health Insurance (SHI) in Germany.To reflect the clinical and economic consequences of MR-HIFU and EBRT for the treatment of bone metastases, we developed a patient-level simulation model (software TreeAge Pro 2019) with a lifetime horizon and a one-month cycle length. The cycle length was chosen because retreatment of patients with painful bone metastases can be considered after one month of persistent pain . The anaPatients entering the model were assumed to be male and female adults with non-vertebral painful bone metastases originating from lung cancer, prostate cancer, or breast cancer in an even distribution. The model population reflected that over 80% of bone metastases from solid tumors arise from cancers of the breast, prostate, or lung . In the For the main comparison, two strategies were outlined. Strategy A was defined as MR-HIFU either as a first-line treatment (about 60%) or as retreatment option after failed EBRT (about 40%). In strategy A, not all patients received MR-HIFU as first line-treatment because in a realistic scenario MR-HIFU is unlikely to replace EBRT completely as first-line treatment. Patients who receive EBRT as a first-line treatment were assumed to be re-treated with MR-HIFU in case of persistent pain or only partial pain relief. The proportions were chosen to confer more internal consistency with the trial informing data on MR-HIFU effectiveness . StrategThe patient-level simulation model reflected the clinical course that may follow palliative treatments with MR-HIFU or EBRT: i. complete pain relief (pain score of zero in the NRS), ii. partial pain relief , iii. persistent pain, iv. retreatment in case of persistent pain or pain relapse and v. death. In addition, the risk of suffering a pathological fracture was considered as an event that could occur in any health state except death, because of its economic consequences and potential impact on quality of life. via PubMed) were performed to identify adequate input parameters . Studies were selected with regard to methodological quality and representativeness for the German context. Input parameters are reported in Several systematic literature searches in Medline  . The retCancer-specific overall survival (OS) was obtained from a Danish population-based cohort study that included 17,251 patients with bone metastases . In thatHealth state utility values were taken from a time trade-off study from Matza et al, which elicited utility values for patients with bone metastases and skeletal-related events  from 187 participants living in the United Kingdom (UK) and Canada . The incUtilities were subtracted due to adverse events related to treatment/retreatment and pathological fractures. Common adverse events associated with EBRT are nausea and vomiting for two weeks following treatment . ReporteCosts of MR-HIFU included one overnight stay at the hospital, general anesthesia and one post-treatment MRI. An additional pre-treatment out-patient MRI was considered in case MR-HIFU was performed as first-line treatment. Depending on the general condition of the patient and the total dose required, in Germany, EBRT is performed as in- or out-patient treatment. Published cost-of-illness studies and surveys indicate that the proportion of out-patient treatments in Germany is 50-60% , 23, 27 According to the perspective of the SHI, direct medical costs related to EBRT and MR-HIFU were based on the German Physicians’ Fee Schedule 2022 (Einheitlicher Bewertungsmaßtab) for out-patient procedures , and theIn line with similar models, for all health states except for complete pain relief, costs with pain medication were estimated considering oral oxycodone as a reference medication , 31. ForCosts associated with a pathological fracture were extracted from a retrospective cost-of-illness study based on German claims data including 2434 patients with bone metastases and solid tumours . These cTo compare the alternatives, the incremental cost-effectiveness ratio (ICER) was calculated as cost per pain response  and cost per QALY. Because survival after diagnosis of bone metastasis varies by cancer type, in subgroup analyses, the ICER was calculated for each primary cancer diagnosis . Costs and benefits were discounted at a 3% annual rate .To validate the model, we consulted experts on the adequacy of input data and the conceptual appropriateness of the model. Technical accuracy was checked regarding data entry and programming errors. For cross model validation, we compared our assumptions to those in similar models. We report the validation efforts in detail in the In deterministic sensitivity analyses (DSA) all input parameters were varied, except for the cancer-specific mortality rates. Structural sensitivity analyses were performed to calculate the ICER considering different scenarios: i. all patients receiving MR-HIFU as first-line treatment in strategy A, ii. alternative retreatment rates in strategy A , iii. a cost-covering lump-sum for MR-HIFU, iv. a range of proportions of single-fraction EBRT (in both strategies), v. a range of proportions for out-patient EBRT (in both strategies). The cost-covering lump sum was taken from a recent time-driven activity-based costing study prospectively conducted at an university hospital from the hospital perspective . The cosA probabilistic sensitivity analysis (PSA) was conducted to test the robustness of the results. Because there is no commonly accepted willingness-to-pay threshold for Germany, the probability of strategy A being cost-effective was assessed for different levels of willingness-to-pay (WTP)  .A VOI analysis was conducted to estimate the value of collecting additional evidence  for reducing uncertainty of the analysis . While tCompared to strategy B , strategy A (with MR-HIFU) resulted in slightly higher costs (EUR 399) and more benefits (0.02 QALYs and 0.95 months with pain response), with ICERs of EUR 19,845 per QALY and EUR 421 per month with pain response. Limiting the analysis to cancer-subgroups, strategy A resulted in increased costs and more benefits . In DSA, the variables with the highest impact on the ICER were the effectiveness of MR-HIFU for complete pain relief, the retreatment rate in strategy A, the MR-HIFU treatment costs, and EBRT costs in that order. In the In structural sensitivity analyses, applying alternative retreatment rates in strategy A resulted in similar results as in the base case . Higher retreatment rates in strategy A resulted in higher ICERs, meaning that the more often retreatments were performed, the lesser cost-effective strategy A was. Moreover, offering MR-HIFU as first-line treatment to all patients at strategy A resulted in higher additional costs (EUR 721 vs. 364 in the base case) and slightly more QALYs (0.023 vs. 0.020 in the base case). The resulting ICER in this scenario  is 50% increased compared to the base case. Furthermore, structural sensitivity analyses assuming less costly EBRT practices  resulted in higher ICERs . Complete results are provided in In PSA, the iterations spread across the four quadrants of the cost-effectiveness plane  evidence and set the maximum amount that should be applied into additional research to reduce uncertainty of the analysis. The parameters with the highest EVPPI per person were MR-HIFU costs  and the fracture rate following MR-HIFU . Further relevant parameter sets worthy of collecting further information were: QALY values , proportion of single-fraction and of out-patient EBRT jointly  and effectiveness of MR-HIFU on pain palliation , as detailed on the In comparison to EBRT, the MR-HIFU strategy resulted in higher costs and more benefits (QALYs and months of pain response) for patients with bone metastases. The overall results were confirmed in subgroup analyses for breast cancer, prostate cancer, and lung cancer. Patients with bone metastases due to lung cancer had the lowest lifetime (cumulative) costs and benefits, probably because 90% of these patients died within the first year. Because the added benefit of MR-HIFU is short-term  and the most impactful additional costs are long-term , strategy A was more cost-effective for this subgroup of patients with poorer prognosis. While the German SHI cannot expect savings with MR-HIFU, the cost-effectiveness was similar to various medical interventions for bone metastases .Due to a lack of appropriate data  the model had to be based on different clinical studies several assumptions. As a result, at a WTP of 20,000 EUR/QALY, the probability of MR-HIFU being cost-effective is 52%, whereas for a WTP of 60,000 EUR/QALY the probability is 69%. In Germany, there is not a commonly accepted WTP threshold to determine reimbursement decisions. For WTP thresholds higher than 20,000 EUR/QALY, the potential cost-effectiveness might justify investments in infrastructure installation. Moreover, early economic models are useful to explore (i) MR-HIFU’s role in the clinical management of bone metastases and (ii) the potential value of further research , 41.Because the role of MR-HIFU in the clinical management of painful bone metastases is still incipient, we explored several alternative scenarios in structural sensitivity analyses. For example, it was detectable that higher retreatment rates at strategy A tend to increase the ICER. Furthermore, a scenario with MR-HIFU being offered as first-line treatment for all patients increased the ICER , due to a higher increase in costs despite a slight increase in QALYs. Although not considered in our calculations, some case series indicate that patients without prior radiation might respond better to MR-HIFU than those with prior radiation , 42. TheRepeated irradiations from EBRT are limited due to normal tissue tolerance , 19. In The high populational EVPI (approximately EUR 180 Mio.) indicates that further studies would be worthwhile for reducing uncertainty . MoreoveIn addition to the primary goal of pain palliation, a technology’s ability to induce local tumor control may contribute to the prevention of pathological fractures . CurrentSome limitations should be acknowledged. Firstly, choosing multi-fraction EBRT as the preferred comparator for our model may limit the generalizability of the results to other settings . The preSecondly, costs with transportation to out-patient radiotherapy treatment are partially reimbursed but were excluded from our analysis due to lack of data. Nevertheless, transportation costs were expected to be very low , hence the impact on model outputs are likely to be negligible. Thirdly, cancer patients can opt for rehabilitation after treatment with EBRT or MR-HIFU. Costs due to rehabilitation were not included in the analysis, because these costs are expected to incur in both groups. Moreover, costs with oncologic rehabilitation are commonly reimbursed by the German Pension Insurance.Finally, although the clinical impact of adverse events on QALYs were accounted for, the corresponding costs associated with diagnosing and treating adverse events were not included. For example, 40% of patients treated with EBRT need symptomatic medication for nausea and vomiting for the first two weeks . HoweverSimilar to our model, a previous cost-effectiveness Markov model from the US showed that MR-HIFU results in both additional costs and QALYs, yielding an ICER of $54,160 per QALY . HoweverIn summary, for patients with bone metastases the MR-HIFU-based strategy resulted in moderately higher costs and benefits in terms of both QALY (which accounted for adverse events of both treatments) and pain response compared to EBRT alone. Although there is still considerable uncertainty around the model results, this analysis can inform research prioritization, support decisions about reimbursement, and investments in infrastructure installation. Once further evidence is available, an updated economic modelling study would be opportune.The original contributions presented in the study are included in the article/Conceptualization and methodology, JG, SS, DM. Selection of input data: JG, SY, DA-B. Data imputation: JG. Verification: DM, AS. Face validation: DA-B, CB, HG, SY, CBo, HV. Analysis, JG, DM. Resources and funding acquisition: SS, HV. Writing – original draft preparation: JG, DM. Writing – review and editing, JG, SY, HG, GB, DA-B, CBa, CBo, HV, AS, SS, DM. Supervision and project administration: SS, DM. All authors contributed to the article and approved the submitted version.This project has received funding from the European Union’s horizon 2020 research and innovation programme under grant agreement No 825859.Author SY is employed part-time by Profound Medical GmbH.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.