Antimicrobial resistance (AMR) is one of the biggest threats to global public health. Selection of resistant bacteria is driven by inappropriate use of antibiotics, amongst other factors. COVID-19 may have exacerbated AMR due to unnecessary antibiotic prescribing. Country-level knowledge is needed to understand options for action.To review AMR in Türkiye and initiatives addressing it. Identifying any areas where more information is required will provide a call to action to minimize any further rise in AMR within Türkiye and to improve patient outcomes.Streptococcus pneumoniae and Haemophilus influenzae, were identified. National and international antibiotic prescribing guidelines commonly used locally for specific CA-RTIs  were also reviewed, plus local antibiotic availability. Insights from both a local clinician and local clinical microbiologist were sought to contextualize this information.National AMR initiatives, antibiotic use and prescribing, and availability of susceptibility data, particularly for the key community-acquired respiratory tract infection (CA-RTI) pathogens Türkiye developed an antibiotic stewardship programme, The Rational Drug Use National Action Plan 2014–2017, prioritizing appropriate antibiotic prescription in the community. Public campaigns discouraging inappropriate antibiotic use were also initiated. Türkiye has a high level of antibiotic resistance and a high level of consumption, however, in 2015 over-the-counter antibiotic sales were prohibited, resulting in a declining trend in overall consumption. There is still a need for physician education on current developments in antibiotic use. Several ongoing global surveillance studies provide antibiotic susceptibility data in Türkiye. Clinicians in Türkiye use several country-specific guidelines for common CA-RTIs plus a range of international guidelines. A more standardized inclusive approach in developing local guidelines, using up-to-date surveillance data on isolates from community-acquired infections in Türkiye, could make guideline use more relevant for clinicians. This would pave the way for a higher level of appropriate antibiotic prescribing and improved adherence. This would, in turn, potentially limit AMR development and improve patient outcome. Even if new medicines are developed, without behaviour change, antibiotic resistance will remain a major threat’.3 The first paper in this Supplement included details about the multiple factors which can drive a rise in AMR along with the global initiatives that are in place to address this threat.2 Each country and/or region must also play their part through local initiatives.Antimicrobial resistance (AMR) is one of the biggest threats to public health throughout the worldStreptococcus pneumoniae and Haemophilus influenzae. National and international antibiotic prescribing guidelines for CA-RTIs, specifically community-acquired pneumonia (CAP), acute otitis media (AOM) and acute bacterial rhinosinusitis (ABRS), commonly used by healthcare professionals in Türkiye were also reviewed, along with how these link to local antibiotic availability. Insights from a clinician and a clinical microbiologist in Türkiye were sought to contextualize this information. In addition, we aimed to identify areas where more information is required and present a call to action to improve clinical outcome for patients and to minimize further rises in AMR within Türkiye.In order to identify how AMR can be addressed in Türkiye in the future, it is necessary to review what is happening now. In this paper, we present the current situation in Türkiye, determined using published information  to ascertain any national initiatives to address AMR in Türkiye, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens 4 many countries began to develop their own National Action Plan (NAP). To address the problem of antibiotic overconsumption and resistance, Turkish health authorities implemented several integrated interventions with WHO guidance and support. Türkiye developed an antibiotic stewardship programme, The Rational Drug Use National Action Plan 2014–2017, which prioritized the proper use of antibiotics in the community. Public campaigns discouraging inappropriate antibiotic use were initiated, and, in 2015, over-the-counter sales of antibiotics were prohibited, resulting in a declining trend in overall consumption with the proportion of antibiotics prescribed by family practitioners dropping by 25% in 2017.5 Current NAP status, as reported by the WHO for 2020–21, shows that Türkiye had yet to finalize an AMR NAP.6Following the formulation by the World Health Assembly in 2015 of a Global Action Plan (GAP) for AMR5 Resistance development is frequently associated with inappropriate antibiotic use.7 According to the WHO, the consumption of antibiotics in in Türkiye was 38.2 DDD per 1000 inhabitants per day in 2015. This was the highest consumption reported in the WHO European region comprising 45 countries.8The Organisation for Economic Co-operation and Development reported in 2015 that, amongst its member countries, Türkiye had one of the highest rates of antibiotic resistance at seven times higher than the lowest rates seen.9 In another study surveying public knowledge about antibiotic use, in 1044 participants in Ankara, about 24% of participants had demanded antibiotics when visiting a physician and 83.5% were successful in their request, indicating that more physician education is needed.10A retrospective year-long descriptive study in 2017 throughout Türkiye of e-prescription data from family physicians showed that around one-quarter of all prescriptions included at least one antibiotic.11 This was further demonstrated by an 8 year evaluation of more than a billion outpatient prescriptions  in all age groups. At the start of the study, in 2011, 34.9% of the prescriptions contained at least one antibiotic but this fell to 24.6% in 2018. In 2011, 14% of all items prescribed were antibiotics, but this reduced to 10.5% in 2018 and during the same period, the percentage of total drug costs accounted for by antibiotics fell from 14.1% to 4.1%. The study concluded that the national interventions had been effective in reducing antibiotic use .12In 2018, the WHO Regional Office for Europe Antimicrobial Medicines Consumption (AMC) Network, revealed that for Türkiye there was an ongoing reduction in antibiotic consumption estimates showing a continued national effort to improve antimicrobial use.13A study investigating knowledge and awareness of physicians concerning rational antibiotic use in a Turkish province revealed that differences in knowledge, attitudes, and behaviours of primary care physicians on the decision to start treatment and its necessity may account, at least in part, for increased rates; and that pressure from patients and their relatives may encourage prescribing. Awareness of multidrug resistance is low. The fear of unproven infection seems to be the most important factor that encourages doctors to start antibiotics. There is still a need for ongoing education for physicians on current developments in antibiotic use. 000 people in Türkiye have pledged their support for the campaign.14A new Turkish version of Antibiotic Guardian, a web-based campaign initiated by Public Health England in 2014, has been made available. Antibiotic Guardian urges everyone to make a pledge to make better use of antibiotics and help save antibiotics from becoming obsolete. To date more than 50S. pneumoniae and H. influenzae, the most commonly isolated respiratory pathogens in the community.15 Key features of SOAR are that it focusses on these pathogens only, and that identification and susceptibility testing are performed in an independent centralized laboratory using standardized methodology (CLSI). SOAR data is analysed based on three different breakpoints: CLSI, EUCAST dose-specific and PK/PD breakpoints. This allows for comparisons to be made between countries or regions and for the identification of trends over time.Several ongoing global surveillance studies provide antibiotic susceptibility data in Türkiye. The Survey of Antibiotic Resistance (SOAR) is a multinational antibiotic surveillance study, ongoing in an expanding range of countries since 2002. The study aims to collect and make available in published, peer-reviewed papers, antibiotic susceptibility data, specifically for 16 Two international organizations define breakpoint values: CLSI and EUCAST. Due to variation in criteria for their definition, there are some differences between CLSI and EUCAST in the clinical breakpoint values for certain bacteria for some antibiotics and this can impact susceptibility interpretation of clinical isolates.17 EUCAST breakpoints are dose-specific and use the EMA-approved doses that are included in the Summary of Product Characteristics of an antibiotic. This means that by application of breakpoints for higher doses, the effect of using a raised dose on the clinical efficacy of a particular antibiotic can be predicted. Türkiye is in a transition period from CLSI to EUCAST guidelines and breakpoints.18 It is therefore possible that dose-specific breakpoints could be used commonly in Türkiye in future. The EUCAST dose-specific breakpoints can also be used retrospectively to calculate the susceptibility of previously collected isolates to show the susceptibility levels that would have been achieved at higher doses.Clinical breakpoints are cut-off MIC values used to classify microorganisms into the clinical categories susceptible (S), intermediate (I) and resistant (R) in order to assist in predicting the clinical success or failure of a specific antibiotic.S. pneumoniae  has over time become less susceptible to amoxicillin/clavulanic acid in some countries21 since the MICs for some isolates have increased. When treating infections, it is important to be able to eradicate bacterial pathogens with raised MICs to optimize clinical outcome while at the same time minimizing the risk of selecting variants with even higher MICs. This is possible because β-lactams, unlike many other antibiotics, have time-dependent killing properties. Their efficacy depends on the amount of time the drug concentration is present at the site of action. Although increasing the concentration at the infection site over a particular concentration will not have any effect on the efficacy, the use of higher doses and/or more frequent dosing allows for successful eradication of pathogens with higher MICs because the time above the MIC is increased.Use of the EUCAST dose-specific breakpoints shows the effect of increasing the antibiotic dose on the susceptibility of a pathogen, providing additional information so the prescriber can decide if a higher dose would be of benefit. For example, S. pneumoniae isolates for all the antibiotics tested.22Türkiye has participated in the SOAR studies over five time periods, starting in 2002 and ending in 2017, and comparative results, as shown in Figure S. pneumoniae isolates in Türkiye for the two most recent time periods studied.22 In 2015 to 2017, 179 S. pneumoniae isolates from outpatients with CA-RTIs were collected from three centres. When applying CLSI breakpoints 78.8% of isolates were susceptible to amoxicillin and amoxicillin/clavulanic acid in 2015–17, with susceptibility of 52% to erythromycin, azithromycin, clarithromycin and between 26.3%–49.2% susceptibility to cefaclor, cefdinir, cefpodoxime, and cefuroxime. Prevalence of susceptibility to ceftriaxone was 83.2%.15Figure H. influenzae isolates in Türkiye for the two most recent time periods studied.22 For H. influenzae isolates (n = 239), in the 2015–17 study, a high susceptibility to amoxicillin/clavulanic acid was seen (99.2%) along with all other antibiotics tested (except trimethoprim/sulfamethoxazole for which susceptibility was 67.4%) when applying CLSI breakpoints. Most isolates of H. influenzae from Türkiye were β-lactamase negative in 2015–17, but the presence of some β-lactamase-positive isolates meant that the susceptibility of H. influenzae to ampicillin was lower.15Figure 23 ATLAS data is analysed based on CLSI and EUCAST breakpoints.The Antimicrobial Testing Leadership and Surveillance (ATLAS) database is a global AMR surveillance programme that is fully accessible and covers susceptibilities of a range of bacterial and fungal pathogens to a bank of antimicrobials with reference to the different breakpoints.S. pneumoniae isolates collected between 2014 and 2017  and for H. influenzae isolates between 2015 and 2017 . Using CLSI criteria, susceptibility of S. pneumoniae isolates to amoxicillin/clavulanic acid was between 75.7% and 95.4% over this period. This contrasts with 41.9%–53.5% for erythromycin, where erythromycin susceptibility also represents that for other macrolides such as azithromycin and clarithromycin based on CLSI guidelines  to amoxicillin/clavulanic acid, intravenous penicillin and ceftriaxone were reasonably consistent and high over the last 4 years tested, whilst the macrolides (erythromycin and azithromycin) and penicillin  showed a much lower level of susceptibility over the same time period. Susceptibility to the fluoroquinolone antibiotics remained high throughout the period under investigation. The results for the susceptibility of H. influenzae isolates confirmed that the respiratory isolates collected were highly susceptible to almost all the antibiotics tested, including amoxicillin/clavulanic acid.The SENTRY Antimicrobial Surveillance Program was established in 1997 and is another long-running antimicrobial surveillance programme. SENTRY monitors worldwide pathogens and changes in resistance patterns over time through centralized testing and utilizing reference susceptibility methods.25 to provide the supporting data required to ensure that countries can design cost-effective, evidence-based AMR response strategies. During the first four years, 91 countries or territories enrolled in GLASS, and data for over two million patients from 66 countries are included.26 Pathogens currently included in GLASS-AMR are: Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Staphylococcus aureus, and S. pneumoniae. GLASS data is analysed based on CLSI and EUCAST breakpoints. A new and important component is the inclusion of antimicrobial consumption (AMC) surveillance at the national level.27 Türkiye is currently not participating in GLASS.In 2015, the WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS). GLASS is the first global system to collect national AMR data for selected bacterial pathogens that cause common infections. The aim is to monitor the prevalence of AMR among major pathogens in clinical settingsS. pneumoniae isolates included, for example, are from blood and cerebrospinal fluid.28Türkiye is a member of the Central Asian and Eastern European Surveillance on Antimicrobial Resistance (CAESAR) network, a joint initiative of the WHO/Europe and partners to survey, contain and prevent AMR emergence and spread. Türkiye has submitted some data, but isolates collected are mainly from hospitals and invasive pathogens. The For management of the common RTIs, CAP, AOM and ABRS in Türkiye, clinicians make use of several country-specific local antibiotic prescribing guidelines plus a range of international guidelines, some examples of which are included in Table 40 and for children the Paediatric Society of America/IDSA 2011 guidelines.32 The British Thoracic Society (BTS) guidelines for adults 2009,31 and for children the IDSA 2011 guidelines are used33 as well as the WHO guidelines, as provided in the List of Essential Medicines.41 For example, a first-line antibiotic treatment recommendation for CAP management from the IDSA 2019 guideline for treating adults with no comorbidities or risk factors for MRSA or Pseudomonas aeruginosa, is amoxicillin or doxycycline or a macrolide  but if the patient has comorbidities, the recommendation is combination therapy with amoxicillin/clavulanic acid or a cephalosporin plus a macrolide or doxycycline, or monotherapy with a respiratory fluoroquinolone. The recommended dosage for adults with comorbidities is amoxicillin/clavulanic acid, 500 mg/125 mg given three times daily, 875 mg/125 mg or 2000 mg/125 mg both given twice daily in combination with a macrolide or doxycycline.40 The BTS also suggests amoxicillin/clavulanic acid, cefaclor, and macrolides as an alternative to first-line amoxicillin in children with low-severity pneumonia.33For the management of CAP, the guidelines principally followed by physicians in Türkiye are the IDSA 2019 guidelines for adults35In AOM, the American Academy of Paediatrics (AAP) guidelines are followed which specifically recommend high dose amoxicillin (80–90 mg/kg/day) given in two divided doses or amoxicillin/clavulanic acid 14:1 formulation, 90 mg/kg/day amoxicillin with 6.4 mg clavulanic acid, given in two divided doses for first-line treatment in children with initial or delayed antibiotic treatment and also recommends the high dose amoxicillin/clavulanic acid 14:1 formulation, 90 mg/kg/day amoxicillin with 6.4 mg clavulanic acid, given in two divided doses or ceftriaxone for patients who have failed initial antibiotic treatment.34 According to the IDSA guideline, amoxicillin/clavulanic acid 90 mg/kg/day given twice daily is recommended as an empirical therapy for children in certain circumstances such as severe disease or in areas where penicillin non-susceptible S. pneumoniae are endemic.For the management of ABRS in adults and children, guidelines used include those from the IDSA.National guidelines in line with more recent international guidelines were published by the Turkish Thoracic Society in 2009 for CAP in children and in 2021 for CAP in adults as contributed by many local associations.41–43Access to antibiotics may be an issue for patients in low- and middle-income countries (LMICs) due to cost and insufficient government expenditure or support in this area. Unreliable drug supply may also contribute to the problem. Limited access to the most appropriate antibiotic to treat a specific infection may result in raised mortality from treatable bacterial infections. The use of suboptimal amounts of antibiotic facilitates resistance development and allows resistant strains to persist.40 recommend for adults, amoxicillin/clavulanic acid 875 mg/125 mg twice daily (or 2000 mg/125 mg twice daily) as an alternative to the three times daily 500 mg/125 mg regimen. In children who are outpatients with bacterial pneumonia and comorbidities, a dose of 90 mg/kg/day in two divided doses (as amoxicillin component) is suggested by the IDSA.32In Türkiye, using amoxicillin/clavulanic acid as an example, several formulations of this antibiotic are currently available, however, the dosage and indication for all forms of amoxicillin/clavulanic acid are currently under review by the Turkish Ministry of Health (MoH). The formulations of amoxicillin/clavulanic acid available in Türkiye are all given twice daily. In outpatients with CAP and comorbidities the IDSA 2019 guidelines34 recommend as standard first-line therapy amoxicillin/clavulanic acid 500 mg/125 mg three times daily or 875 mg/125 mg twice daily in adults and 45 mg/kg/day twice daily in children. ‘High dose’ regimens (2 g given twice daily in adults and in children 90 mg/kg/day given twice daily) are recommended as a second-line empirical therapy or in situations such as severe disease or where there is endemic penicillin non-susceptible S. pneumoniae. In AOM, the AAP35 recommend amoxicillin/clavulanic acid 90 mg/kg/day (as amoxicillin component) in two divided doses (formulation 14:1) as first-line for children for initial or delayed antibiotic treatment or after failure of initial antibiotic treatment.In ABRS, the IDSA guidelines44 The quality of medicines, specifically antibiotics, is an important consideration for countries worldwide. WHO has launched a Global Surveillance and Monitoring System (GSMS) for substandard and falsified products.44 The GSMS aims to work with WHO member states to improve the quality of reporting of substandard and falsified medical products, and, importantly, to ensure the data collected are analysed and used to influence policy, procedure, and processes to protect public health, at the national, regional and the global level. Use of substandard or falsified antibiotics not only compromises clinical outcomes but also risks increased AMR. The most recent summary (2013–17) reported substandard and falsified medicines in 46 member states (including Türkiye). Antibiotics represent 16.9% of all products reported, second only to malaria drugs (19.6%).Substandard poor-quality or falsified antibiotics promote AMR and the spread of drug-resistant infections. Since poor-quality antibiotics are unlikely to contain the full dose needed to eliminate all of the infecting pathogens, use of these would encourage resistance to develop and allow resistant strains to survive and be transmitted.5 There is a need for a robust national AMR surveillance system in Türkiye, which could be included in the already present global systems for providing accurate and reliable surveillance data. Türkiye has two main antimicrobial stewardship programmes established by the MoH, targeting hospitals and the community.Surveillance studies provide important information allowing the identification of trends in pathogen incidence and AMR, including identification of emerging pathogens at local hospital, regional, national and global levels. Local surveillance studies indicate that the incidence of AMR pathogens is increasing and can guide clinician decisions regarding appropriate treatment and to modify treatment guidelines and aid in empirical prescribing. Microbiological methods used for AMR detection in hospitals in Türkiye generally conform to EUCAST and CLSI antibiotic susceptibility testing. Local surveillance studies are also a large part of efforts to assess AMR patterns and provide important information to guide prescribing at individual facilities. At the same time, surveillance studies need to maintain a mix of the small and the large, the local and the global. The Turkish MoH is the main regulator and provider of healthcare in Türkiye. In the absence of standardized national surveillance, some universities generate annual reports of their AST results.23 Many hospitals in Türkiye have electronic medical record systems and most have integrated Laboratory Information Systems. Rarely, vital test results may not be received by the treating clinicians, posing a patient safety threat and reflecting a need for common protocols on who is to receive test results and how to better integrate laboratory IT systems and clinical units. In addition to traditional disease notification systems, it is important to switch to real-time event management, which would significantly reduce turnaround time. Close monitoring of hospital antibiotic use for restricted and unrestricted antibiotics is essential. Resistance rates for key community pathogens should be monitored and antibiotic surveillance needs to be extended to outpatient clinics.Rapid delivery of microbiology test results influences mortality, length of hospital stay and costs, as well as appropriateness of antibiotic prescribing and consumption. In Türkiye, Electronic Health Records include any information recorded, stored, transmitted, accessed, correlated, and processed by using electronic systems relating to physical/mental health conditions or diseases of individuals.New rapid microbiological techniques should replace traditional culturing and susceptibility testing. In addition, revision of testing processes may improve workload, and workflow. Another solution to enhance quality control could be artificial intelligence-based systems. The microbiologists could also supervise clinicians via tele-microbiology service when interpreting Gram stains and results of culture. Providing the right treatment within the first 24–48 h of symptoms is essential.Educational programmes combined with mutual internships for laboratory and clinical staff could contribute to a better understanding of complementary work processes. In performing antibiotic stewardship outreach visits in clinical units, microbiologists can enhance their role as interpreters of clinical processes and patient information to the laboratory technicians. Such visits also provide an opportunity to convey microbiology insights to clinical staff and teach them how to better interpret test results. In future, microbiological methods will evolve and become increasingly sophisticated and the need for professional guidance will increase. This may require a change in how some microbiologists execute their profession, from being predominantly laboratory based to working more closely with clinical staff. It is desirable to provide laboratory evidence of diseases not just at the level of clinical diagnosis, so it is necessary to develop both human and diagnostic capacities of laboratories.Türkiye has very high antibiotic consumption and RTIs are among the diseases for which antibiotics are most frequently used. Administration of treatment in accordance with guideline recommendations not only ensures correct, appropriate, and necessary treatment, but may also reduce the development of AMR. Guideline recommendations, empirical treatment principles and local surveillance data should be considered before prescribing antibiotics. Considering the evaluation and publication times of local surveillance data, shorter intervals and faster and easier access to up-to-date data may be more beneficial for the application of ‘patient-specific treatments’. This ‘patient-focused approach’ should be evaluated together with the guideline recommendations as well as the clinical features of patients, disease characteristics, risks of complication and pharmacokinetic/pharmacodynamic (PK/PD) characteristics of the preferred antibiotic(s).22The results of local surveillance studies conducted in different cities or clinics in Türkiye have been published but they do not show continuity. As described here, SOAR Türkiye studies have been published for five different time periods and continuous participation of three centres is valuable for monitoring national and regional results and changes.45 Recommendations of international guidelines should be considered along with local guidelines when prescribing appropriate antibiotics specific to the disease and the patient. In addition, bacteria-specific local antibiotic resistance data together with endemic resistance rates  should be considered.In Türkiye, there are no local guidelines on the treatment management of diseases such as sinusitis, otitis, tonsillopharyngitis in children and adults; but the booklet entitled ‘Rational Approach to Antibiotic Use in Adult Patients’ published by the MoH, Turkish Medicines and Medical Devices Agency is defined as a ‘local guideline’ that indicates the appropriate and rational use of antibiotics in the treatment of many diseases, including respiratory tract infections.When prescribing antibiotics in Türkiye, the ‘Electronic Prescription System’ program is used in which all antibiotics licensed in Türkiye are listed. Physicians can choose the appropriate antibiotic for their patient with recommended dose and treatment duration. The licensed antibiotics in Türkiye provide sufficient variety for the treatment of many diseases so problems of treatment choice are minimal, especially for adults. Most antibiotic consumption is from primary care and acute RTIs are the most common reason for prescribing antibiotics, particularly in primary care, where antibiotics are often prescribed inappropriately. Ensuring the rational use of antibiotics is a priority within the scope of the National Action Plan for Rational Use of Medicines 2014–2017, which was put into practice in 2014 in order to contribute to slowing AMR.In an era of rising AMR throughout the world, this paper aims to define areas where action is required to address AMR by analysing and understanding the current situation within a country or region. Information is presented for Türkiye concerning antibiotic use and prescribing, approach to AMR, availability of local susceptibility data, use of international and/or local management guidelines and how these link to antibiotic availability. To our knowledge this is the first time this information has been reviewed and presented in detail by country.Antibiotic use in Türkiye is extremely high. This and the lack of knowledge about antibiotic susceptibilities has led to a high degree of antibiotic resistance. Türkiye has the highest consumption of antibiotics per capita in the WHO European region. There is still a need for ongoing education for physicians on current developments in antibiotic use.28 Several global surveillance studies have collected and published data from Türkiye for the principal pathogens in CA-RTIs including SOAR, ATLAS and SENTRY. Surveillance study results consistently show a decrease in susceptibility of respiratory pathogens for several classes of antibiotics, in particular the macrolides. Susceptibility to the fluoroquinolone antibiotics remains high in Türkiye amongst respiratory pathogens but regulatory bodies advise caution for their use, recommending restriction to specific cases, due to safety concerns.In terms of surveillance, Türkiye is a member of the CAESAR network, a joint initiative of WHO/Europe.Initiatives to address the overconsumption of antibiotics and antibiotic resistance in Türkiye have had success, for example, in reducing the antibiotic prescriptions by family practitioners by 25%. However, further improvements, monitoring and investment are required to sustain Türkiye’s antibiotic stewardship programmes.Whilst a range of guidelines are utilized by clinicians in Türkiye, a more standardized inclusive approach is needed to develop local country-specific guidelines These guidelines should be based on up-to-date surveillance data of isolates from community-acquired infections which would make them more locally relevant for clinicians, reiterating the Consensus Principles as described in the introductory paper to this Supplement. This would pave the way for improved adherence and a higher level of appropriate antibiotic prescribing in CA-RTIs which could, in turn, potentially limit AMR development and improve clinical outcomes for patients.


Objective: Employ the hollow fiber infection model (HFIM) to study sequential antibiotic administration  using human pharmacokinetic profiles to measure changes in the rate of antibiotic resistance development and compare this to simultaneous combination therapy with the same antibiotic combinations. Methods: Escherichia coli CFT073, a clinical uropathogenic strain, was exposed individually to clinically relevant pharmacokinetic concentrations of ampicillin on day 1, ciprofloxacin on day 2 and fosfomycin on day 3. This sequence was continued for 10 days in the HFIM. Bacterial samples were collected at different time points to enumerate total and resistant bacterial populations. The results were compared with the simultaneous combination therapy previously studied. Results: Sequential antibiotic treatment (ampicillin-ciprofloxacin-fosfomycin sequence) resulted in the early emergence of single and multi-antibiotic-resistant subpopulations, while the simultaneous treatment regimen significantly delayed or prevented the emergence of resistant subpopulations. Conclusion: Sequential administration of these antibiotic monotherapies did not significantly delay the emergence of resistant subpopulations compared to simultaneous treatment with combinations of the same antibiotics. Further studies are warranted to evaluate different sequences of the same antibiotics in delaying emergent resistance. Antibiotics are used to treat bacterial infections, and their efficacy is threatened by the rapid emergence of resistant bacteria worldwide . BacteriWith the growing need to combat the antibiotic resistance crisis, it has become necessary to reconsider the monotherapy standard and explore combination therapies of existing antibiotics or antibiotics with non-antibiotic enhancing compounds to diminish the emergence of spontaneous resistance . To testPseudomonas spp., Acinetobacter spp., and carbapenemase-producing Enterobacteriaceae have demonstrated synergistic effects [With bacterial mutations, new mechanisms evolve to survive specific antibiotic treatment and thereby diminish the antibiotic efficacy in treating bacterial infection. Different treatment strategies using combination and sequential therapy have been investigated to reduce the emergence of antibiotic resistance. Antibiotic combinations are also increasingly employed to enhance the antibacterial effects of available drugs against multidrug strains. Compared to monotherapy, the advantage of using combinations includes a broader spectrum, synergistic effect, and reduced risk of emerging resistance during therapy ,12,13. S effects ,16,17,18 effects . This un effects . However effects ,22.In our previous study, we utilized the HFIM to study the effect of single, double, or triple simultaneous combination treatments of ampicillin, ciprofloxacin and fosfomycin antibiotics in reducing the total viable population of bacteria, as well as the effect of antibiotic combinations on the emergence of resistant subpopulations using the Escherichia coli CFT073 strain . The stuE. coli CFT073 strain, we simulated human pharmacokinetic exposure to ampicillin, ciprofloxacin and fosfomycin (ACF) in a ten-day HFIM. On day 1 of ampicillin administration, a reduction of about 3 to 4 log10 cfu/mL in the total bacterial population was observed after 4 h. At 24 h post-ampicillin treatment, the total bacterial population increased from the initial inoculum of approximately 108 cfu/mL to 1010 cfu/mL. Further treatment with ciprofloxacin on day 2, fosfomycin on day 3 and subsequent sequential therapy until 10 days did not affect the total bacterial population . Subpopulations resistant to 3× MIC of fosfomycin were present at about 6 to 7 Log10 cfu/mL. The 10× MIC ciprofloxacin-resistant subpopulations (9 to 10 Log10 cfu/mL) constituted most of the total population, followed by ampicillin (5 to 8 Log10 cfu/mL) and fosfomycin (2 to 3 Log10 cfu/mL) subpopulations. The 30× MIC resistant subpopulations were observed at the following concentrations: ciprofloxacin (7 to 9 Log10 cfu/mL), ampicillin (2 to 4 Log10 cfu/mL) and fosfomycin (2 to 3 Log10 cfu/mL)  A–C.The emergence of subpopulations simultaneously resistant to 3×, 10× and 30× MIC for combinations of ampicillin–ciprofloxacin, ampicillin–fosfomycin and ciprofloxacin–fosfomycin are shown in Twenty-four hours after the first cycle of sequential antibiotic therapy, colonies were found that were simultaneously resistant to 3× MIC of ampicillin, ciprofloxacin, and fosfomycin A. No colTreatment with individual antibiotics  resulted in a reduction of the total bacterial population for the first 6 h but had no effect on days 2 and 3 . ColonieE. coli CFT073 using a hollow fiber infection model (HFIM). In this paper, we describe the effects of sequential monotherapy using the same model and compare the results to simultaneous administration.Antibiotic resistance in Gram-negative bacterial pathogens represent a serious threat to public health and results in significant morbidity and mortality worldwide. The World Health Organization (WHO) has published a list of antibiotic-resistant pathogens, and among them, the most common are Gram-negative bacteria ,24. To a10 cfu/mL on day 10. Also, simultaneous combination antibiotic therapy significantly delayed the emergence of any resistant E. coli CFT073 subpopulations compared to the monotherapies. The resistant subpopulations that eventually emerged after combination administration generally showed low-grade resistance (3× MIC) compared to monotherapy, and there was no appreciable outgrowth of multidrug-resistant colonies resistant to all three antibiotics. The emergence of single drug-resistant colonies for all three antibiotics at 3× and 10× MIC were delayed until 96–120 h and 144–168 h, respectively [10 cfu/mL after 24 h. Surprisingly, introducing a second and third antimicrobial in sequence did not appreciably reduce the total bacterial load any further, possibly as the result of the initial induction of efflux pumps or other non-specific resistance mechanisms.Our earlier research showed that simultaneous antibiotic combinations resulted in the sustained killing of the total bacterial population. Only a few viable colonies were observed between 24–96 h, which gradually increased to 10ectively . In contDuring sequential therapy, subpopulations with high-grade monoresistance (30× MIC) to each added antibiotic emerged rapidly. In contrast, following simultaneous combination therapy, high-grade monoresistance was observed for fosfomycin at 168 h post-treatment, but not for ampicillin or ciprofloxacin. When we studied the emergence of multidrug resistance, we found that the sequential antibiotic regimen resulted in the cumulative appearance of single, double, and triple antibiotic-resistant subpopulations following the introduction of each new antibiotic, frequently at high grade (10× MIC). Resistance to more than one drug was rare following simultaneous combination therapy and was low grade (3× MIC) when it did appear. Resistant populations to more than one antibiotic appeared for each of the two antibiotic combinations after 192 to 216 h and for the three antibiotic combinations after 192 h.Our findings on sequential monotherapy showed an accumulation of cumulative and often high-grade multidrug resistance. An in vitro study with quinolones, β-lactams, and aminoglycosides with isolates of commonly encountered nosocomial pathogens showed the development of multiple drug resistance to related and unrelated classes of antibiotics. This unusual pattern of multiple drug resistance was shown to be associated with changes in the outer membrane proteins of bacterial pathogens . SequentSimultaneous treatment with three antibiotics aimed at different bacterial targets constitutes a strong selection pressure and appears to disallow the emergence of significantly resistant mutants. Combination treatments with three antibiotics are gaining acceptance to treat highly resistant pathogens based on their ability to target multiple targets in the bacteria. Several studies, including our earlier study, showed that the administration of single antibiotics resulted in the appearance of resistant bacteria, while combinations reduced their emergence ,23,25. TThe differences observed between the sequential and simultaneous treatment regimens may depend on several factors, such as different mechanisms of action of each drug administered, the sequence of drugs administered, the time interval between each antibiotic administration and the type of resistance that occurs. In the sequential study, ampicillin was administered while the bacteria were in the logarithmic growth phase on day 1. Further treatments with ciprofloxacin on day 2 and fosfomycin on day 3 were administered to the static total bacterial population, along with resistant subpopulations. In the case of simultaneous treatment, all three antibiotics were administered to an actively growing susceptible bacterial population. The antibiotic action on the different growth phases of the bacterial population, along with the generation of resistant subpopulations, might have resulted in the differences observed in the accumulation of resistant mutants between these two treatment regimens. Further hollow fiber experiments with individual antibiotics given for 3 days showed that the effects during the initial 6 h after administration were similar to those observed with sequential therapy. Each individual antibiotic reduced the total bacterial population on day 1, but continued administration of the same antibiotics on days 2 and 3 had no effect on the total bacterial population. The resistance pattern observed with sequential therapy (ampicillin-ciprofloxacin-fosfomycin) might have been different if the same antibiotics had been used in different sequences. This has been observed in several studies conducted with different regimens of sequential treatments involving either a single switch between antibiotics or multiple switches at short intervals depending on both the included antibiotic classes and the particular sequence of antibiotics in treatment. The main findings of these studies were that the efficacy of the sequential treatments depended on both the antibiotic classes and the treatment sequence ,30,31.E. coli cells [The emergence of new genetic resistance is a major public health concern and has resulted in many fatal outbreaks ,33,34,35li cells ,37, as wEscherichia coli strain CFT073-WAM4505 (ATCC BAA-2503) (strain isolated from the blood of a patient with pyelonephritis) purchased from American Type Culture Collection, ATCC,  was used for experimental infection in the hollow fiber infection model for antibiotic resistance development studies. This strain was grown in Luria-Bertani (LB) broth to a logarithmic growth phase; 15% glycerol stocks were made and stored at −70 °C. Prior to each experiment, this strain was streaked for isolation onto LB agar plates incubated overnight at 37 °C. A single colony was isolated, inoculated into 10 mL of LB broth and incubated at 37 °C overnight. The overnight culture was then subcultured (1:100) the next day into fresh LB broth, incubated at 37 °C and was grown until it reached the mid-exponential growth phase. Bacterial growth was determined by measuring the optical density of the culture at 600 nm (OD600). The mid-exponential phase culture  was used as a starting inoculum for the hollow-fiber experiment.The clinical Ampicillin sodium salt, ciprofloxacin hydrochloride and fosfomycin disodium salt were obtained from Sigma-Aldrich, St. Louis, MO, USA. Stock solutions of the antibiotics were prepared in sterile water and filter sterilized. LB broth and agar were obtained from bioWORLD . Sterile LB broths and agar were prepared by autoclaving, and filter-sterilized antibiotic solutions were used for the HF experiments.8 CFU/mL mid-log phase Escherichia coli culture, prepared as described in max of 10 µg/mL, 0.16 µg/mL and 100 µg/mL for amp, cip and fos respectively) in the hollow fiber cartridge by simulating human free drug pharmacokinetic profiles. The antibiotics were administered individually each day in the following order for 10 days: amp (day 1), cip (day 2), fos (day 3), amp (day 4), cip (day 5), fos (day 6), amp (day 7), cip (day 8), fos (day 9) and amp (day 10). The intended PK profiles achieved in the HFIM were confirmed by sampling at different time points  and quantified as previously described [In vitro HFIM experiments were conducted for 10 days as previously described by Garimella, Zere et al., 2020  with sliescribed . In a seBacterial samples (1–2 mL) from the sequential antibiotic therapy HF experiments were collected aseptically from the cartridges for the determination of total viable and antibiotic-resistant subpopulations at baseline, and 1, 2, 3 and 4 h on the first day of antibiotic treatment, then once daily at 24 h intervals for ten days. For the three-day single antibiotic HF experiments, bacterial samples (0.5 mL) were collected 1, 2, 3, 4, 5 and 6 h post-antibiotic treatments each day for 3 days. Serially diluted samples in LB broth were quantitatively cultured onto drug-free LB agar plates to enumerate the total viable bacterial population. A portion of the bacterial suspension was also quantitatively cultured onto LB agar, supplemented with either ampicillin or ciprofloxacin or fosfomycin or their double or triple combinations at 3×, 10× and 30× the baseline minimum inhibitory concentrations (MIC). This was performed to assess the impact of the treatment regimen on the development of resistant subpopulations. LB agar plates were incubated at 37 °C, and viable colonies were counted after 24 h of incubation. For some antibiotic combinations at 10× and 30× MIC levels, the plates were incubated for 48 to 72 h to enumerate the less susceptible resistant subpopulations.The total population and resistant subpopulation bacterial counts geometric mean and geometric standard deviation were determined from three independent sequential therapy HFIM experiments by using GraphPad Prism 8 software.This study compared the efficacy of a sequential antibiotic treatment regimen (ampicillin-ciprofloxacin-fosfomycin) to simultaneous combination therapy in reducing the emergence of resistant subpopulations using a hollow fiber infection model. Sequential antibiotic treatment resulted in the appearance of single and multi-antibiotic-resistant subpopulations, but simultaneous treatment significantly delayed or prevented the emergence of resistant subpopulations. Further studies that evaluate different sequences of the same antibiotics in delaying emergent resistance are warranted.


The greatest weakness of concrete as a construction material is its brittleness and low fracture energy absorption capacity until failure occurs. In order to improve concrete strength and durability, silica fume SF is introduced into the mixture, which at the same time leads to an increase in the brittleness of concrete. To improve the ductility and toughness of concrete, short steel fibers have been incorporated into concrete. Steel fibers and silica fume are jointly preferred for concrete design in order to obtain concrete with high strength and ductility. It is well-known that silica fume content and fiber properties, such as aspect ratio and volume ratio, directly affect the properties of SFRCs. The mixture design of steel-fiber-reinforced concrete (SFRC) with SF addition is a very important issue in terms of economy and performance. In this study, an experimental design was used to study the toughness and splitting tensile strength of SFRC with the response surface method (RSM). The models established by the RSM were used to optimize the design of SFRC in terms of the usage of optimal silica fume content, and optimal steel fiber volume and aspect ratio. Optimum silica fume content and fiber volume ratio values were determined using the D-optimal design method so that the steel fiber volume ratio was at the minimum and the bending toughness and splitting tensile strength were at the maximum. The amount of silica fume used as a cement replacement, aspect ratio, and volume fraction of steel fiber were chosen as independent variables in the experiment. Experimentally obtained mechanical properties of SFRC such as compression, bending, splitting, modulus of elasticity, toughness, and the toughness index were the dependent variables. A good correlation was observed between the dependent and independent variables included in the model. As a result of the optimization, optimum steel fiber volume was determined as 0.70% and silica fume content was determined as 15% for both aspect ratios. Concrete is accepted as brittle, and it has a low fracture energy absorption capacity until failure occurs. In order to improve fracture energy or toughness of concrete, short steel fibers have been incorporated into concrete. Incorporation of steel fibers into concrete increases the ductility and toughness of the concrete ,4,5,6,7.w/c), air content, admixture dosage, and both fine and coarse aggregate content to achieve a target compressive strength, slump (for workability), and air content (for freeze–thaw durability). Within these approaches, the designer can proportion an acceptable concrete mixture. Although standard proportioning methods started as arbitrary 1-2-3 cement–sand–aggregate volumetric ratio methods established in the early 1900s, today they have evolved into the absolute volume approach defined by the American Concrete Institute (ACI) [The mixture proportioning of concrete is the process of selecting the type and quantity of individual constituents to design concrete. This process is very important to design concrete with appropriate properties for a specific type of application. The prescriptive and performance-based approaches are well-known traditional methods for proportioning concrete mixtures. Prescriptive approaches are step-by-step design methodologies. These methods are based on selecting an appropriate water-to-cementitious material ratio (te (ACI) ,16,17.Performance-based techniques are more flexible when compared to the prescriptive approaches. The design of concrete in these techniques is based on many laboratory trial experiments  .It is known that statistics are used in every field in a beneficial way. From the point of view of civil engineering materials research, the studies carried out in recent years have shown that the use of statistical experimental design in cement-based materials has gained attention in the concrete industry. One of the experimental design methods is the response surface method (RSM).The RSM has gained increasing attention for its use in the optimization of engineering problems and/or industrial processes. This methodology is a combination of mathematical and statistical techniques that are widely used in the area of concrete preparation optimization ,20.Kockal and Ozturan investigated the optimization of properties of lightweight fly ash aggregates for suitability in high-strength lightweight fly ash concrete production using RSM via Design-Expert software. In the optimization shown in this research, the relationships between the sintering parameters  and the three experimentally obtained responses  were established .2 values ranging from 78.7% to 98% and the optimization produced NaOH and crumb rubber levels of 10% and 2%, respectively, with a high desirability of 71.4% [Mermerdaş et al., investigated the effect of binder content, curing temperature, and curing time on the compressive strength of lightweight geopolymer mortar. The study was conducted in two stages. In the first stage, the researchers applied experiments with different parameters and obtained 336 data samples. In the second stage, they used the experimental data for optimization through the response surface method. Results show that the experimental verification indicates a good agreement with optimized results . Kaya etof 71.4% . Xia et of 71.4% . Ma et aof 71.4% .Awolusi et al.  demonstrfT) and splitting tensile strength (stf). This method was used to optimize both silica fume and steel fiber content to obtain the highest possible flexural toughness (fT) and splitting tensile strength (stf) with the lowest possible steel fiber content.In this work, an experimental design was used to study the toughness and splitting tensile strength of SFRC with a silica fume addition. The novelty of this research was to perform a multiobjective simultaneous optimization using the response surface methodology (RSM) in relation to the establishment of mathematical models for flexural toughness :The RSM is a combination of mathematics and statistics to build the experimental model. It includes the fitting of regression surface in order to obtain a close-range response. It is also used to design experiments, variations in responses, and designs by the usage of responses approximated. The investigation, which initially generated interest in the package of techniques, was presented by Box and Wilson in 1951. In the earlier works, the RSM was only used to build a model for the experimental responses. Later, the RSM was used for modeling numerical experiments ,37. The With replacements of n. Response surface is defined in Equation (4) and Equation (5):The total experiment number is s is the error of s comes from Equation (8):Variance–covariance matrix of ESS is the sum of the squares of the residuals, and it is given in Equation (9):R2adj in Equation (10) can be utilized.Syy given in Equation (11) stands for sum of squares in total.Understanding the prediction fineness of the response surface, the adjusted coefficient of multiple determination R2adj can have 0 or 1, which is the minimum or maximum. Getting closer to 1 means that the response surface is perfect. Results obtained from the response surface are subjected to the t-statistic of the coefficient, bj, in Equation (12):Cjj is the element of number jj from Equation (7). If the absolute value of the jth term is judged to be significant for regression. In order to find the best regression, methods such as stepwise and decreasing methods may be used [The  be used .fV) were 0.5% and 1%. SF contents (SFV) were 0, 5, 10 and 15% as the percentages of cement weight. The mixture compositions are presented in Water–cement was 0.38 in the fabrication of concretes. CEM I 42.5R as cement was utilized. Silica fume came from a ferrochrome factory in Antalya, Turkey. Limestone-based aggregates were used. Fine aggregates up to 0–4 mm in size and coarse aggregates up to 4–12 mm and 12–19 mm in size were used. In control concrete, which was a reference concrete, steel fibers and silica fume were not used. The slump value aimed for was 120 ± 20 mm. Water-reducing admixture was used at the rate of 1% by cement amount to see changes in workability. The aspect ratios of steel fibers were 65 and 80. The fiber volumes  and steel fiber content (fV) of SFRC with a special emphasis on toughness (fT) and splitting tensile strength (stf). The collection of experimental data is essential in order to build a model or a response surface and to obtain a good agreement between experimental data and the fitted model. After building a model, the optimization is carried out by using the RSM in order to obtain the optimum solution. Without a model, the optimization cannot be carried out. A common method using the response surface to obtain optimum settings is to use two variables (fV and SFV) and to use three fractions of fV  with 65 and 80 aspect ratios and to use four ratios of SFV . The designs for two independent variables contain 3 × 4 = 12 mixtures for each aspect ratio, as shown in The multiobjective simultaneous optimization method is used to optimize both SF  of concretes were fitted to the mathematical model by variance analysis (ANOVA). Regression models of mechanical properties of concretes fitted are given below:Equations (15) and (16) for the mixtures with an aspect ratio of 65:Equations (17) and (18) for the mixtures with an aspect ratio of 80:stf and T as a function of volume fractions of SF and steel fibers with 65 and 80 aspect ratios are given in Response surfaces of 2) for the model of splitting tensile strength (stf) and flexural toughness (fT) were found to be 0.90 and 0.94, respectively. Similarly, the correlation coefficients for the aspect ratio of 80 were 0.94 and 0.98, respectively. Correlation coefficients for each model obtained were quite high. The equations presented here can contribute to pre-experimental mix design by predicting the split tensile strengths and flexural toughness of SFRCs in the range of 0–1% steel fiber volume fraction and 0–15% silica fume content in similar studies.For an aspect ratio of 65, the correlation coefficients , could be applied to optimize simultaneous responses. The desirability function (jd) changed between 0 ≤ jd ≤ 1. The multiobjective design was obtained via a sole composite response (D) presented in Equation (19), which is a geometric average of singular desirability functions. D was maximized upon a possible region of optimization factors presented in Equation (18), in which n is the number of responses used for the design [For optimization of the objective functions, independent variables were changed, meanwhile and separately, a regression model was built. Later, the connections among mixed design variations and responses were calculated. They are expressed in Equations (15)–(18). A numerical design approach, which utilizes the desirability functions fT) and splitting tensile strength (stf) are needed in order to obtain a more ductile concrete. The cost of steel fibers is a crucial factor that should be considered in applications. So, the number of fibers should be kept at a minimum quantity for the economical mixture. Numerical optimization can be carried out by applying each integration of either factors or responses. For this, maximizing the T and the stf is very important while minimizing the fV. Three responses  were thought to have equal significance and to be optimized. For n = 3, Equation (19) has the form:The highest flexural toughness  and cost optimization were involved, ideal results for the following optimization factors were found: fiber amount of 0.71% and a silica fume content of 15% for an aspect ratio of 65 and, similarly, 0.70% and 15% for an aspect ratio of 80.Observations from this study exhibit that predictions of quadratic polynomial regressions are able to aid to finding optimal mixtures of concrete. When both mechanical properties (


Moreover, compressive strength test results show that SF and AW replacements of 0.08% and 0.1%, respectively, resulted in peak outcome of 24.97N/mm2, while replacements of 0.5% and 0.45% resulted in a minimum response of 17.02N/mm2. The ANFIS-model was developed using 91 datasets obtained from the experimental findings on varying replacements of cement and fine-aggregates with AW and SF respectively ranging from 0 to 50%. The ANFIS computation toolbox in MATLAB software was adopted for the model simulation, testing, training and validation of the response function using hybrid method of optimization and grid partition method of FIS at 100 Epochs. The compressive strength behavior is the target response, and the mixture variations of cement-AW and fine aggregates-SF combinations were used as the independent variables. The ANFIS-model performance assessment results obtained using loss function criteria demonstrates MAE of 0.1318, RMSE of 0.412, and coefficient of determination value of 99.57% which indicates a good relationship between the predicted and actual results while multiple linear regression (MLR) model presents a coefficient of determination of 82.46%.This research study presents evaluation of aluminum waste-sisal fiber concrete’s mechanical properties using adaptive neuro-fuzzy inference system (ANFIS) to achieve sustainable and eco-efficient engineering works. The deployment of artificial intelligence (AI) tools enables the optimization of building materials combined with admixtures to create durable engineering designs and eliminate the drawbacks encountered in trial-and-error or empirical method. The features of the cement-AW blend's setting time were evaluated in the laboratory and the results revealed that 0–50% of aluminum-waste (AW) inclusion increased both the initial and final setting time from 51–165 min and 585–795 min respectively. The blended concrete mix's flexural strength tests also show that 10% sisal-fiber (SF) substitution results in a maximum flexural strength of 11.6N/mm The notion of sustainable development offers one potential remedy for this issue which refers to a course of development that protects natural resources and builds assets in a way that ensures meeting current demands of the people without compromising those of the future generations4. One of the few always ever-present concerns is the issue of sustainable and ecofriendly infrastructural development because it is crucial to contemporary society. Given that the current notion is relevant to all types of human activity, the application field for sustainability is almost limitless6. The need to determine viable alternatives to the well-known concrete ingredients to achieve sustainability and eco-efficient environment has led to exploitation of solid-waste and the derivatives7. These solid waste materials handling and poor management around the world and more especially in the developing countries have contributed to severe environmental degradation9. So, introduction of supplementary-cementitious-materials (SCM) which possesses alumina-silicates contents is helpful towards achieving sustainability and ecofriendly concrete materials through reduction of greenhouse gases emissions11.Increase in population, urban growth, and commercialization have been observed to pose a direct impact on rising levels of energy demand and material utilization and, therefore bring about generation of solid wastes12. By investigating and evaluating the efficacy of additional locally accessible materials that are classified as either industrial or agricultural wastes, some researchers have attempted to reduce the total cost of building materials14. Concrete's compressive strength and durability properties are improved by the addition of solid-waste derivatives with alumina-silicate content15. Ayat et al.16 researched on the appropriate ratio of supplementary cementing materials (SCMs), including limestone filler (LF), which was mixed with Portland cement, resulting to environmental, economic and technological advantages. ANN was deployed in the study which were trained using the Tan-sigmoid activation function and the feed-forward back-propagation optimization technique and the results showed strong correlations that exceeded 97%.Utilization or recycling of solid-wastes in production of concrete is also essential towards waste management and involves complex analysis of the factor levels to achieve desired response as regards mechanical and resilience behavior15. Fiber reinforced-concrete is a unique and an important area of research given its significance whereby concrete’s properties such as tensile strength are enriched with fibers which is also devoid of compromise on the compressive strength properties. Concrete under impact force fails extensively as a consequence of its brittle nature16. Therefore, addition of fibers intensifies its many engineering properties such as toughness, flexural strength, impact, resistance to fatigue loads, and thermal shocks. The use of natural fiber reinforced composites is now considered as a new perspective in the construction industry17. Concrete materials possess tough compression characteristics but weak and fragile to resist series of tension forces and hence, there is need to provide support or reinforcement to it. In many instances, steel materials reinforcement was utilized; but a significantly large number of researches are in headway to discover a substitute to steel through the utilization of natural fibers18.Utilization of fibers in concrete helps to improve its post-cracking and ductility characteristics to achieve fiber reinforced concrete. This improved ductility behavior is due to the insertion of fibers that can efficiently transmit tensile stresses throughout a damaged region, hence narrowing the cracks19. Sisal-fiber has the potential to be employed as admixture and offer good properties especially the compressive, tensile as-well-as flexural-strength, but they are influenced by the fiber-length20. Dhumal et al.21, conducted research to compare conventional concrete and sisal-fiber reinforced-concrete with varying length to assess the influence of the fiber-length in concrete. Volume fraction and aspect ratio are crucial batching criteria used to describe fiber. The aspect ratio (l/d) is the proportion of a fiber's length to its diameter which ranges from 30–150 and plays a crucial part in improving the mechanical behavior of the concrete22. Conversely, the volume of fiber in a concrete matrix is known as volume fraction. However, current research suggests that fibers are introduced through weight ratio of cement. According to some studies, the ideal fiber content by cement mass is 1%; any higher additions cause a loss in the concrete's compressive and split tensile strengths, respectively, only for tiny modulus fibers23.According to earlier studies, sisal-fiber reinforced concrete has improved the concrete's performance, including its workability, strength and durability24. Neural networks are used as appropriate tools of pattern matching and can have their weights automatically adjusted to have the response function optimized, despite their inability to infer how to arrive at certain decisions25. On the other hand, fuzzy logic overcomes this limitation by inferring how to arrive at a certain judgment but is unable to educate itself and learn autonomously. As a consequence, the concurrent use of both techniques might serve as complements with the aim of resolving the many problems that non-hybrid soft computing techniques have27. Among these, the Adaptive Neuro-Fuzzy Inference System (ANFIS) is one of the best possible modeling tools, having many applications in several fields of inquiry and study. Jang introduced ANFIS, which provides connections between neural networks and fuzzy logic for modeling complex and dynamic systems28. Its functional set of rules is derived from a combination of the least-squares and back-propagation algorithm that serves as an indicator to adjust the parameters and output of the fuzzy inference system (FIS). The If–then rules are also implemented in the ANFIS data processing mechanism through generating a mapping between the variables of input and output29.Artificial-intelligence (AI) or machine-learning refers to any system that presents features concomitant with a human mind, such as learning and problem-solving. AI copycats’ human intelligence and is used in expert, knowledge-based, and robot systems31. In recent times, smart intelligent models are now developed for the optimization of the response parameter to obtain the mixture ratio with maximum response at minimum number of trials33. Alaneme et al.34 researched on ANFIS optimization of compressive-strength performance of periwinkle shell (PWS) and rice-husk-ash (RHA) concrete. Sixty-two (62) datasets derived in the laboratory outcomes were employed to develop the model. The ANFIS-model performance evaluated through loss-function-parameters presented MSE of 0.3906, RMSE of 0.625, MAE of 0.2244, and coefficient of determination (r2) of 98.67%. Also, Nataraja et al.35, proposed a fuzzy-neuro model for concrete component design. In order to measure out the necessary amounts of fine and coarse aggregates, cement and water for a concrete mix's required mechanical properties, 5-layered FIS and three-layered-ANN were hybridized. It was determined that the model performs adequately when compared to traditional approaches, but provides slightly lower contents than the latter within a tolerable range.In order to obtain a specified design strength and other desired features, concrete-mix-design comprises the evolution of establishing the quantities of the different elements of concrete. Although the assessment of the mixture-response is often an empirical process based on specific design codes, statistical methodologies and response surface methods have been appliedThe research study overall idea is to integrate industrial residue such as aluminum waste (AW) with appreciable pozzolanic comportment and agro waste like sisal fiber to partially replace the core concrete components of cement and fine aggregates respectively, from 0–50%. The essence of this exercise is to recycle solid industrial and agro wastes so as to achieve sustainable, eco-efficient and green concrete with enhanced mechanical and durability performance. This experimental investigation is aimed at the deployment of Soft-computing technique (ANFIS) to appraise the mechanical response to facilitate the determination of the optimal combination of the green fiber-reinforced concrete ingredients.37. It is a network of directed linkages and nodes that can be learned from and adjusted, and it produces learning rules related to membership function requirements that can be obtained from datasets, ensuring proper statistical inference of data38. ANFIS imitates the neural network's capacity to learn in order to generate appropriate MF and similar if–then rules of fuzzy sets from a given input–output model variables connection. Because it joins in together neural-networks and fuzzy-logic codes, it is capable of capturing the advantages of the two in a one outline. Its interpretation structure matches to a group of fuzzy-IF–THEN rules with adaptability and potential to round-up non-linear tasks39.Jang's proposed Adaptive Neuro-fuzzy inference system (ANFIS) provides a realistic procedural environment that combines Neural-Network and Fuzzy-logic philosophies to aid the fuzzy-model research and understanding of details from provided clusters of datai and Bi are the membership function parameters, while Pi, qi and ri are the learnable parameters of the output variables41.Figure The five isolated strata of the ANFIS-framework are explained thus;Layer 1: The first Fuzzy layer possesses adaptive nodes with node-functions that convert inputs into linguistic labels of fuzzy set in order to determine data membership values. This stratum is in charge of the input data-fuzzification, in which the crisp data are plotted into attributes ranging from 0 to 1, allowing the degree of relatedness of the constraints to be calculated. Similarly, the membership function values required for each ith node are anticipated as indicated in Eq.  coming as stated in Eq.  with a 30-percent normal consistency that complied with CEM II standards as stated in Nigerian Industrial Standard (NIS) 444–1 requirements, proportions, and compliance indicators was employed51.Water is a crucial component that has an impact on the rheological and mechanical characteristics of concrete. For this investigation, clean, drinkable water was used, and it complies with ASTM C1602-12 specifications for water to be used in concrete mixturesLocally available sisal fiber from Akwa Ibom state were processed manually and used for the experimental investigation. Sisal fiber was chopped into 20-25 mm pieces and used for the investigation. Using a Hounsfield tensometer, the sisal fiber's tensile strength was measured. To guarantee durability, the fibers were given a silica fume treatment by being submerged in a slurry before being added to the concrete and given 13 min to dry.52.The Aluminum waste was obtained from Aluminum Extrusion Industry (ALEX), Inyishi in Ikeduru L.G.A., Imo State, Nigeria. It is produced by heating aluminum scraps at a temperature of 1980 °C in a furnace. The waste is then sieved through a 150 μm sieve size so as to obtain the particles of waste in a finely divided state in accordance with ASTM C618–78 specification53. The experimental investigation also employed clean, well-graded crushed granite with a maximum particle size of 20 mm that was procured from Abia State for the coarse aggregates, and it likewise adhered to ASTM C125-1654.The studies utilized sand taken from a clean river bed in Cross River State, Nigeria as the fine aggregates. It was made in accordance with BS EN 12,620 requirements and sieved via a 2.36 mm standard size56.A conventional batching concrete mix of 1:1.5:3 and a w/c of 0.55 were the mixture design used, which included the replacement of fine aggregate and cement in the green fiber concrete mixture with sisal fiber and aluminum waste from 0 to 50%. The concrete mixture materials were then completely mixed with water to achieve a homogeneous mixture before placement and compaction in the 150 mm × 150 mm × 150 mm cubic mould dimensions. After 24 h, the concrete samples were placed in a curing tank at room temperature for 28 days. Before crushing, it is weighed to assess its compressive strength, which is computed using Eq. 55,56.9\d57.Concrete beam moulds measuring 100 mm by 100 mm by 400 mm were used to test flexural strength. The concrete beams were cast and hydrated at 28 days with mixing proportion of 1:1.5:3 and water-cement fraction of 0.55 for the mixture design comprising replacement of cement and fine aggregate in the green fiber concrete mixture with sisal fiber and Aluminium waste correspondingly from 0–50%. Three specimens were cast and cured after 28 days. After 28 days of curing, the three specimens for each combination were crushed and the average flexural strength was measured using Eq. 60.Following the extraction of experimental findings from the laboratory, the mixed design data and the accompanying answers created were logically sorted to provide the needed variables for model construction. The datasets are separated into two pieces of 30% and 70% for testing and training the model, accordingly. Following training, the smart model generated learns to generalize the experimental data that it has been given in order to make correct predictions. Figure 61.The effectiveness of the generated model was evaluated in order to confirm that it has a shown capacity for forecasting the target variables with a respectable level of accuracy. Based on relevant research, a number of performance requirements are derived from statistical measurements like R-values and less important function characteristics like MAE and RMSE using the equations in Eqs.  and 12)12).11\do2 (54.79%), Al2O3 (15.89%) and Fe2O3 (0.26%) producing a sum of 70.94% which revealed decent pozzolanic characteristics in agreement with ASTM C618, 98 requirements52. The abundance of CaO, Al2O3 and Fe2O3 at 11.3%, 20.6% and 6.405% respectively in the Portland limestone cement (PLC) component makes sure that the cement is fully hydrated, which enhances the concrete's mechanical strength behavior. The hydration compounds that are created when and aluminates silicon oxide from the admixture react with calcium to produce hard material over time. The results on the physicochemical behavior of test sisal fiber indicates aspect ratio, tensile strength, diameter and density of 250, 352Mpa, 1.1 mm and 1.6 g/cm3 respectively62. The chemical composition of the fiber showed 70% cellulose, 11% hemi-cellulose, 8% Lignin and 9% Pectin. The results of these tests on the compositions of admixtures are consistent with those of Bharath and Srikanth20.Tables 64.A semi-log plot that displays the gradation and distribution of the aggregate components employed in the experimental inquiry is created from the sieve analysis of the test materials. From the data provided in Fig. 65. The high requirement of water is as a result of the presence of silica and increased surface area for the ash sample. This is because of silica-lime reaction require more water in addition to the water presented for the cement hydration reaction process. The obtained result is presented in Fig. 66.Workability test result (Slump) which assesses the workability property of the concrete matrix indicates that value of the slump test reduces with increase in the AW content in the concrete mixture by requiring more water in order to make the mixture more workable in consonance with BS EN 12,350–2 specifications67.From the obtained results, it was perceived that the incorporation of AW effected the increase of the initial setting time from 51 to 165 min, while that of final setting time also rose from 585 to 795 min from 0–50% respectively. This is due to the fact that the inclusion of AW decreases the cement paste's surface area, which slows the hydration process and lengthens setting time. The low rate of heat generation results from the delayed hydration. This is crucial for mass concrete production since it calls for less hydration heat. The graph of cement/AW paste setting time versus %-replacement is presented in Fig. 68.The laboratory tests carried out to derive the mechanical behaviour of the blended concrete mixes were done on the hardened samples after 28 days hydration period. The compressive and flexural strength responses were calculated to determine the mixture combinations with maximum and minimum strength characteristics57. the cured blended green concrete beams of 100 mm × 100 mm × 400 mm with varying combinations of AW-sisal fiber mixtures were tested and the test results is presented in graphical chart in Fig. 2 for 10% sisal fiber replacement while the minimum flexural strength of 4.11 N/mm2 was derived for 50% replacement. The calculated results showed that the addition of sisal fiber is effective at 0–10% replacement, however, from 20–50% replacement the strength properties tend to decrease linearly. This strength increment is due to the bonding and fiber integration in the concrete matrix which is also consistent with the findings of Alaneme and Mbadike10.The experimental methodology for the flexural strength tests is four point loading configuration flexural test according to ASTM C1161-02c(2008)e1 specification as shown in Fig. 669 and BS 8110: Part 170 standards.The graphical interaction of the test admixture of sisal fiber and aluminium waste in respect to the concrete’s compressive strength response computed using Eq.  were pre71. Table 72.Information derived from experimental findings, professional judgment, and pertinent literature that evaluates the mechanical-strength characteristics of created green fiber reinforced concrete. The model's input variables are blended mixtures of cement-AW and fine aggregates-sisal fiber combinations ranging from 0 to 50%, and its output variables are the concrete's mechanical strength qualities after curing for 28 days as seen in Figs. 1073. Furthermore, the learning algorithm used uses a hybrid optimization technique that was put to use during the 100-epoch training of the fuzzy inference system (FIS). The processing settings are shown in Table 29.To properly develop the ANFIS model output-input variables, data from related published articles, experimental findings, and expert judgment were employed. Figure 74.Using the recommended hybrid optimization training techniques and FIS variables, ninety-one (91) datasets that are employed for the modelling process are split and sorted into 2 parts to accomplish the network's training (60) and testing (31), respectively. Figure Following the completion of training, the 31 datasets reserved for testing the trained model are further imported from the MATLAB program workspace to guarantee improved predictive accuracy. As illustrated in Fig. In order to assure a better outcome in terms of predictive performance, the network testing was conducted using the processing parameters originally set out for the ANFIS model training. Using the set-out datasets, a testing error of 2.663 was computed after the testing, as shown in Fig. 75.Figure 76.The membership functions (MF) plots for the model input variables for the developed ANFIS network is generated automatically after the training and testing processes as shown in Fig. 77.The ANFIS model, which was used to evaluate the compressive strength qualities of concrete made with AW-sisal fiber, uses a hybrid learning strategy to learn how to generalize the data sets that are provided to it. A given input space may be correctly mapped by the created smart model to the appropriate output response. The connections between the model variables are assessed to determine their individual significant relevance or impacts, as illustrated in Figs. 78. Additionally, using Minitab 18 statistical software, the generated ANFIS model's performance in terms of prediction accuracy was evaluated by statistically comparing the real or experimental findings with those of the ANFIS model. MLR statistics, RMSE, MAE, and coefficient of determination were used in this comparison. In Table 79.The ANFIS approach was used in the modeling process to optimize the experimental findings acquired to assess the compressive strength characteristics of AW-sisal fiber concrete, and the model result is shown in a graphical chart shown in Fig. 21.34 and Wang et al.80.According to the derived statistical findings, which are represented in Table Figure 2 at 0.08% and 0.1%, respectively, and to a minimum compressive value of 17.02 N/mm2 at 0.5% and 0.45%, respectively.The concrete ingredients passed testing for the essential chemical and physical characteristics for excellent pozzolanic performance. The findings of the particle size distribution highlighted the necessity for strong gradation features to create concrete with enhanced durability properties. Following a 28-day hydration interval, concrete's compressive strength findings were also obtained. The computed experimental findings show that sisal fiber and AW were replaced to a maximum compressive value of 24.97 N/mm2 for 10% sisal fiber replacement while the strength reduces with increase content of sisal fiber in the concrete matrix with minimum flexural strength of 4.11 N/mm2 derived for 50% replacement.The flexural strength test response of the fiber concrete indicated maximum strength of 11.6 N/mmThe slump results showed that value of the slump test reduces with increase in the AW content in the concrete mixture by requiring more water in order to make the mixture more workable. The setting time test results on the cement-AW blend showed that the addition of AW effected the increase of the initial setting time from 51 to 165 min, while that of final setting time also rose from 585 to 795 min from 0–50% respectively.For the creation of the model, the system database was comprised of the sorted information obtained through the experimental approach. The 91 datasets in total were separated into two groups, with the latter group receiving 60 datasets and the former group receiving 31 datasets, respectively, in order to test and train the adaptive network.2 were used to evaluate the generated ANFIS model's ability to predict outcomes. The findings of the statistical analysis show an MAE of 0.1318, RMSE of 0.412, and R2 of 99.57%, while the MLR model shows a coefficient-of-determination of 88.69%. Incorporating the industrial waste residues under investigation in this study offers a viable alternative to the traditional concrete ingredients for the production of green concrete that reduces the amount of carbon dioxide released during the manufacture and use of cement, the cost of infrastructural construction materials, the disposal of industrial waste without regard for its intended use, and other factors that contribute to sustainability and the environment.Additionally, MLR, MAE, RMSE, and rFurthermore, smart-intelligent model development was used to assess the created mechanical strength results of concrete reinforced with AW-sisal fiber. This was accomplished via the use of ANFIS soft computing. The findings of this research demonstrate the versatility of the ANFIS approach with relation to its use in modeling the engineering behavior of cement-additive concrete blends for sustainability.For the modeling of the mechanical strength properties of concrete with fine particles and cementitious fraction partially replaced by aluminum waste and sisal fiber, correspondingly, from 0 to 50%, adaptive neuro-fuzzy inference system (ANFIS) was used. The research study's findings allow for the following deductions:


COL3A1 and ACTA2 are responsible for familial TAAD. In addition, we highlight FBLN5, FBN1, SLC2A10, FBN2, and NOTCH1 as candidate genes. Future studies of crosstalk among the pathways may provide us a step toward understanding the pathogenic mechanism. This finding indicates the necessity of obtaining family medical history and screening of extended relatives of patients with TAAD for the early identification and treatment of TAAD.Thoracic aortic aneurysms and dissections are precarious conditions that often cannot be diagnosed with fatal outcomes. Over the last few years, pathogenic variants in numerous genes have been identified that predispose to heritable presentations of TAAD. An evidence-based strategy for the selection of genes to test in familial TAAD helps inform family screening and intervention to prevent life-threatening events. Using whole-exome sequencing, four members of three unrelated families clinically diagnosed with TAAD were used to identify the genetic origin of the disorder. Variant evaluation was carried out to detect the pathogenic mutation. Our studies suggest that mutations of  Thoracic aortic aneurysms and dissections (TAAD) are one of the major causes of death in humans. TAADs usually grow slowly and quietly to the point of either dissection or rupture, with the most common presenting symptom being death. Non-syndromic TAAD is confined to the aorta and does not have visible clinical signs for early detection . Non-synFBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, COL3A1, ACTA2, MYH11, MYLK, LOX, and PRKG1) for HTAAD as category genes in 2018  was an effective and precise method for the identifying causative variants in TAAD families. The analysis of the cost-effective strategy may provide molecular diagnostics, genetic counseling, and individualized health maintenance measures for patients with TAAD . Sufficivia CT angiography (CTA) when necessary. Peripheral blood samples of all TAAD individuals and their family members were collected. According to standard procedures, genomic DNA was extracted for sequencing and molecular analysis. These three families were named TAAD-1, TAAD-2, and TAAD-3. Thirty-seven TAAD-1 patients, 10 TAAD-2 patients, and eight TAAD-3 patients underwent the test of peripheral blood, respectively.We recruited three unrelated families with the diagnosis of TAAD from the Department of Vascular Surgery, Changhai Hospital, Naval Military Medical University. All experiments were approved by the ethics committee of Changhai Hospital, Naval Military Medical University. Written informed consent was provided by all participants before participation in this study. Detailed clinical evaluation of each TAAD individual was performed by a cardiologist and a geneticist, including somatoscopy, medical history investigation, and image examination Genomic DNA (>1.5 μg for each sample) extracted from peripheral blood was sheared to 200-bp using the Agilent SureSelect Human All Exon v6 (60 Mb) Kit  and whole exomes were sequenced on the Illumina HiSeq platform .http://code.google.com/p/cutadapt/). Cleaned reads were aligned to the human genome release hg19 using Burrows-Wheeler Aligner  software. PCR duplicates were marked and removed with Picard (http://picard.sourceforge.net/). After the realignment to the genome, GATK software (https://www.broadinstitute.org/gatk/) was used for base quality recalibration. Somatic SNP/Indel was called by Samtools (bam file process) and GATK . Variants obtained from previous steps were annotated with ANNOVAR (http://www.openbioinformatics.org/annovar).The workflow of this study is shown in  ANNOVAR  (http://http://browser.1000genomes.org), gnomAD (http://gnomad.broadinstitute.org/), ESP6500, and Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/). In addition, by searching the clinical utility gene card for hereditary TAAD, a TAAD-associated gene list was produced  including the coverage and average read depth of all coding exons and intron–exon adjacent regions, genotype quality scores, and QualByDepth (Q/D) scores was performed to obtain the variant of high quality. The low-quality variants  were excluded from downstream analysis. Next, the remaining variants were filtered according to minor allele frequency (MAF) < 1% in multiple databases, including the 1000 Genome Project (produced  (Supplemproduced . The filhttp://www.ncbi.nlm.nih.gov/clinvar), cosmic70, and HGMD (http://sift.jcvi.org), Polyphen2 (http://genetics.bwh.harvard.edu/pph2), MutationTaster (http://mutationassessor.org), and CADD (https://cadd.gs.washington.edu/). The variants were considered pathogenic variants when pathogenic prediction scores were generated by two or more of the tools mentioned above. Finally, mutations that occur in multiple family members are of particular concern. Enrichment analysis on the candidate genes was performed by clusterProfiler to directly investigate in which KEGG pathway or GO term the mutation genes were significantly enriched  was a 45-year-old male patient who was admitted to the hospital due to chest and back pain for 22 h. In the emergency chest and abdomen CTA, thoracic aortic dissection was shown. The condition improved after receiving thoracic endovascular aortic repair. Postoperative check CTA showed the descending aortic stent arrow in , residuaIn TAAD-3, the index case (II-3) was diagnosed with aortic dissection by CTA due to sudden chest and back pain for 8 h. The Bentall operation was conducted in 2017. The CTA image revealed the reticular stent-like shadow in the aortic arch arrow in  in the 2SLC2A10, which is stop gain.By filtering the variants for 160 known disease genes  for TAADCOL3A1 (NM_000090): c.2753G > A, p.G918E was found in five individuals . The mutation cannot be found in dbSNP. However, mutations in this gene are associated with vascular Ehlers–Danlos syndrome, previously referred to as EDS Type IV. Due to vascular fragility, hemorrhage often occurs in vascular EDS patients that may result in mortality (COL4A5 (NM_000495) was found in six individuals . The mutation is rs78972735 in dbSNP and relates to Alport syndrome 1. Alport syndrome (AS) is always accompanied by a spectrum of phenotypes ranging from progressive renal disease with extra renal abnormalities to isolated hematuria. Since the predominant inheritance pattern of TAAD is autosomal-dominant, the mutation of COL4A5 is not responsible for TAAD-1.In TAAD-1, ortality . In addiCOL4A5 (NM_000495): c.3940C > T, p.P1314S were found in three individuals . The mutation was not recorded in dbSNP. FBLN5 (NM_006329): c.1229T > C, p.I410T were found in two TAAD individuals. Mutations in FBLN5 are associated with age-related macular degeneration 3, cutis laxa. FBN1 (NM_000138): c.5678A > G, p.N1893S were found in four individuals . Mutations in NM_000138 are associated with geleophysic dysplasia, familial TAAD, and Marfan syndrome. Moreover, SLC2A10 (NM_030777): c.136G > T, p.E46X was found in two individuals . Mutations in NM_030777 are associated with cardiovascular phenotype, familial TAAD, and arterial tortuosity syndrome.In TAAD-2, three mutations were present in both index cases II-7 and II-3. ACTA2 (NM_001141945/NM_001613): c.460G > A, p.V154M were found in three individuals . Mutations in NM_001141945 are associated with aortic aneurysm, familial thoracic six, and multisystemic smooth muscle dysfunction syndrome. In FBN2 (NM_001999): c.8254G > A, p.D2752N were found in the TAAD individuals. Mutations in NM_001999 are associated with congenital contractual arachnodactyly and cardiovascular phenotype. NOTCH1 (NM_017617): c.4417G > A, p.G1473S, were found in three individuals . Mutations in NM_017617 are associated with cardiovascular phenotype, Adams-Oliver syndrome 5, and familial TAAD.In TAAD-3, in silico. We performed GO and KEGG enrichment analyses to uncover specific functional categories of the eight genes. Since COL3A1 is related to aortic and arterial aneurysms, it was also included in the enrichment analysis. As a result, the eight genes clustered most significantly in 285 GO functional categories and four KEGG pathways . This analysis revealed that most of the identified genes encode proteins related to the extracellular matrix, kidney development, or relaxin signaling pathways. These results suggest that the variation in these genes might induce extracellular matrix degradation and force a decrease in fibrosis in TAAD biology.In order to understand the functional roles behind the mutation genes in TAAD biology, functional enrichment analysis was performed for gene function annotation COL3A1, ACTA2, FBLN5, FBN1, SLC2A10, FBN2, and NOTCH1 are related to the familial TAAD. Furthermore, the bioinformatics analysis uncovered that these genes are involved in extracellular matrix organization. However, it was difficult to determine whether the mutation is a hereditary mutation in the pedigree since we just observed the disease’s natural course of partial TAAD individuals. Considering the inheritance pattern and damaging score, we focus on the de novo COL3A1 mutations in TAAD-1 and de novo ACTA2 mutations in TAAD-3. The data provide direct evidence that mutations of COL3A1 and ACTA2 are probably the major cause of familial TAADs in TAAD-1 and TAAD-3, respectively.In the current study, we examined three Chinese families with TAAD by screening for mutations in the 160 TAAD-causing genes list. We concluded that compound heterozygous mutations of COL3A1 gene comprises 51 exons and encodes the pro-alpha1 chains of type III collagen. Type III collagen constitutes about 5–20% of the entire collagen content in the human body that is found in extensible connective tissues such as the lung, skin, and the vascular system (COL3A1 was described in an inherited Ehlers-Danlos syndrome type IV patient in 1988 (COL3A1 is involved in molecular function extracellular matrix structural constituent and extracellular matrix structural constituent, conferring tensile strength. The molecular function indicated that the mutation affects the aorta smooth muscle tissue morphogenesis.The human r system . The fir in 1988 . Many mu in 1988 . MutatioACTA2 gene comprises nine exons and encodes one of six different actin proteins. Actins are highly conserved proteins that are involved in cell motility, structure, and integrity. It encodes the smooth muscle actin involved in vascular contractility and blood pressure homeostasis. Mutations in ACTA2 cause a variety of vascular diseases, including thoracic aortic disease, coronary artery disease, stroke, and multisystemic smooth muscle dysfunction syndrome. Mutations in ACTA2 were first reported in 2007 (ACTA2 exon 6 mutations in the TAAD Caucasian populations (ACTA2 in TAAD families. Moreover, the corresponding protein sequence encoded by exon 6 is likely to be the key functional region of ACTA.The human  in 2007 . Since t in 2007 . We reviulations . This reCOL3A1, ACTA2, FBLN5, FBN1, SLC2A10, FBN2, and NOTCH1 mutations with TAAD. Considering familial TAAD, the family history and the disease course in the family members may impact the future planning of TAAD individuals. Thus, screening of family members who have the same mutations but have not developed the disease is essential. From a genetic counseling point of view, we would inform the family with similar mutations of the TAAD possibility in the subsequent child. Judging from the current sequencing results, we speculate that the occurrence of the familial TAAD clinical phenotype requires some incentives, such as the weak with age or unmanageable blood pressure. From the genetic counseling perspective, family members who did not carry the same mutation and developed the disease will be informed of the risk of TAAD in offspring with similar mutations. This work represents significant steps for the selection of genes in hereditary TAAD. Subsequent studies are needed to provide new insights into this inherited disease.In summary, this study adds novel mutation loci to the existing spectrum of


Pulmonary involvement of IgG4‐associated disease is a rare condition with no codified treatment apart from steroid administration. We report here the case of a patient with pulmonary involvement of IgG4‐RD successfully managed with Rituximab, in induction and maintenance therapy. This original case could support the use of Rituximab in rare situations of steroid‐resistant or steroid‐dependent pulmonary IgG4‐RD. We report here the case of a patient with pulmonary involvement of IgG4‐RD successfully managed with Rituximab, in induction and maintenance therapy. This original case could support the use of Rituximab in rare situations of steroid‐resistant or steroid‐dependent pulmonary IgG4‐RD. IgG4‐related disease (IgG4‐RD) is a systemic orphan disease that can affect almost all organs. It is characterized by unifying histopathological fibroinflammatory lesions.The disease is usually treated by steroids, with a remarkable efficacy but relapses are frequent. Second line treatments as monoclonal anti‐CD20 antibodies are usually proposed in patients with relapses as steroid sparing agents. We report here a case of IgG4‐RD with pulmonary involvement and his response to treatment.18FDG uptake of salivary glands, mediastinum, pulmonary nodules and pancreas , moderate renal failure (creatinine 148 μg/ml), increased lipase  and IgG4 serum levels . Flow cytometry showed increased circulating plasmablast counts . Body CT scan revealed numerous lymphadenopathies, diffuse enlargement of the pancreas and homogenous hypertrophy of the submaxillary glands. Renal biopsy showed a dense lymphocytic infiltrate with a ratio of IgG4+ plasma cells to total plasma cells of 50% and 40 IgG4+ plasma cells per HPF confirming the diagnosis of IgG4‐RD Figure . The pats Figure . BiologiWe report here the original case of a patient with multiorgan IgG4‐RD involvement, including lung.The diagnosis of IgG4‐RD requires ideally pathological confirmation because elevation of serum IgG4 is not specific and can be absent. The diagnosis is retained after a workup with the aim to exclude disease mimickers. Classification criteria for IgG4‐RD have been reported recently. Although there are not necessary for the diagnosis they listed exclusion criteria's and when these are absent a scoring by organ that include thoracic radiological abnormalities.There are very sparse reports on efficacy of Rituximab in IgG4‐RD pulmonary involvement. Two main studies have reported the efficacy of anti‐CD20 in IgG4‐RD: Carruthers et al.In conclusion, we report here a case of a patient with ‘highly probable’ lung involvement according to Corcoran et al., illustrating the heterogeneous radiological pattern that can be observed in IgG4‐RD and the response to treatment by steroid and rituximab.Romain Muller, Mikael Ebbo and Nicolas Schleinitz concepted and draft the manuscript. Paul Habert, Julia Torrents and Jean Yves Gaubert revised it.None declared.The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images.