Objective: Surveying public awareness of antibiotic use and antibiotics can identify factors relevant to the design of effective educational campaigns. The aim of this study was to evaluate the knowledge, attitudes, and practices related to antibiotic use and multidrug-resistant pathogens in the general population in Pakistan.Research Design and Methods: Cross-sectional survey was conducted, using a 60 itemed structured questionnaire and recruited individuals by convenient sampling from the general population in the four provinces of the country. Descriptive statistics were used to evaluate the responses and the chi squared statistic was used to assess differences between groups.Results: The response rate was 87.6% . Half of the respondents had received at least one prescription of antibiotics in the 6 months preceding the survey. Knowledge about antibiotic use, (39.8%) individuals scored above the mean (≥3) showed good knowledge about antibiotic use. Urban residents and male showed significant higher knowledge (p < 0.001) about antibiotic use. Approximately 50% of the respondents correctly answered the question about antibiotic resistance. Of the 3,611 received antibiotics, 855 (23.7%) were indicated for cough, 497 (13.8%) for a sore throat, 335 (9.3%) for ear ache, 665 (18.4%) for a burning sensation during urination, 667 (18.4%) for wounds or soft tissue inflammation. MDR pathogen was perceived as an important topic by  60.1% of respondents.Conclusion: Participants were aware of the problem of multidrug-resistant pathogens and understood the responsibility of each individual to avoid the spread of these infectious agents. The development of antimicrobial resistance (AMR) is a major public health concern in Pakistan  and worlIn some developing nations, Gram-negative bacteria are a major cause of neonatal sepsis , and theA considerable number of studies have shown that the general population plays a key role in spreading the AMR due to lack of awareness about antibiotics among population in Pakistan . The WorWe sampled all four provinces of Pakistan . The provinces consist of divisions, districts, tehsils (administrative areas containing towns), and villages. Three districts were randomly selected from Punjab and Sindh and two each from Khyber Pakhtunkhwa and Baluchistan. From each target district, we selected one city and one village. City was conveniently selected by the availability of data collectors. We performed a cross-sectional survey in the four provinces of Pakistan. We used convenient sampling and try to collect data from maximum participants during May to November, 2020. Total 7,631 participants approached in the study during this time frame. Participants who were Immigrants, individuals over 80 or under 18 years, and persons with cognitive impairment (N = 479), participants initially participated in the study and during study refused to continue the study due to personal issues  (N = 219), and missing data from survey (N = 379) were excluded from the study. Finally, sample of 6,684 participants was included in the study. . IndividSixteen data collectors were trained to conduct the research in the provinces. Most data collectors were pharmacy students in their final year of study, who were supervised by teachers from the local university. Training covered the following aspects: 1) presenting a concise introduction of the study rationale to respondents; 2) conducting face-to-face interviews; 3) coping with difficulties in data collection, such as lack of cooperation. The training lasted 3 days and included a demonstration by the primary researcher.The knowledge, attitudes, and practices regarding antibiotic use covered four topics: 1) exposure to antibiotics, evaluated by two questions about symptoms and prescription in the preceding 6 months. 2) Knowledge about antibiotics, evaluated by responses to seven statements related to correct identification of antibiotics and to opinions regarding antibiotic use in general. 3) Attitudes and practices related to antibiotic use, assessed by seven items concerning how participants generally obtained antibiotics and whether they were concerned about antibiotic resistance. 4) Whether participants requested antibiotics for personal use, how they used antibiotics, and whether they developed side effects during use (six items). The questionnaire used in this study differed in its contents from those of previous studies . The secCase 1: Let us suppose that your neighbor, an elderly person residing alone, requires some help and you have been shopping for him for a couple of months. After a hospital stay, he tells you that he has become infected with a hospital-acquired pathogen. How would you behave with him/her?Case 2: Let us suppose that your coworker, with whom you share an office and some office items, tells you after a hospital stay that she has become infected with a hospital-acquired pathogen. How would you behave with him/her?Each case scenario was associated with eight statements, and response options were “strongly agree,” “agree,” “somewhat disagree,” and “disagree.”In addition, demographic information  was also collected and included in the analysis. The questionnaire was modified slightly from a previous study  accordinp < 0.05 was considered significant.Descriptive analysis was performed for the demographic variables. To analyze the knowledge about antibiotics, each correct response was awarded one point. For a positive question, the responses “strongly agree” or “agree” were calculated as one point and “do not know,” “disagree,” or “strongly disagree” were calculated as zero. For a negative question, “disagree” or “strongly disagree” were calculated as one point and “strongly agree” or “agree” and don’t know were calculated as zero. A cumulative score of the seven knowledge items on antibiotics and the four items on MDR was derived in the same manner. The cut point of ≥3 is considered to be good knowledge for the knowledge about antibiotic as it was also chosen in the previous studies that those who scored more than mean were considered in good range. A complete questionnaire is subjected to the analysis those with the missing data were excluded in screening phase. Percentages for each item were calculated and the chi squared statistic was used to check significant differences between demographic groups. The research was carried out in accordance with the tenets of the Declaration of Helsinki and was approved by the Medical Research Ethics Committee of Xi’an Jiaotong University, Shaanxi, China, approved reference number (XJTMD11-2020). Respondents who were literate provided a signed consent form and for those who were not literate, the data collector signed the form on their behalf after explaining the content of the form.Out of 7,631 individuals who were approached for the study, 6,684 (87.6%) completed questionnaire items on antibiotic use and MDR pathogens. The mean age (± standard deviation) of the respondents was 32.97 (10.8) years, 4,746 (71.0%) were male, 772 (11.5%) had education above post-graduation and 2,833 (42.4%) resided in rural areas .p < 0.001). Approximately half of the respondents correctly answered the question about antibiotic resistance. The mean (± standard deviation) of the knowledge score of antibiotic use was 2.51 (1.80) and 2,260 (39.8%) individuals scored above the mean (≥3). Few participants provided correct answers to the questions about antibiotics, and those tended to be urban residents and male showed significant higher knowledge (p < 0.001). Of the 3,611 who received antibiotics, 855 (23.7%) were indicated for cough, 497 (13.8%) for a sore throat, 335 (9.3%) for ear ache, 665 (18.4%) for a burning sensation during urination, 667 (18.4%) for wounds or soft tissue inflammation, and 677 (18.5%) for other reasons. Almost two thirds  of those who were prescribed antibiotics stopped the therapy before completion, because 433 (18.6%) felt better, 544 (23.3%) were worried about side effects, 78 (3.3%) experienced a side effect, 455 (19.5%) forgot to take the medication, 211 (9.0%) experienced “too much stress,” and 611 (26.2%) for other reasons. Almost two thirds 4,211 (63%) of all participants had heard of MDR pathogens, 7.3% 488) reported that they knew an individual who had tested positive for an MDR pathogen, and 12% (802) reported that they themselves had tested positive for an MDR pathogen once in their lifetime. Fifty-four percent of respondents  reported that they were prescribed antibiotics within the preceding 6 months, with a significantly higher proportion of participants from Sindh  than Khyber Pakhtunkhwa  reported sharing medications with family members and 35.8% 2,393) reported that they had antibiotics at home to use when necessary. MDR pathogen was perceived as an important topic by  60.1% of respondents . In termThe first scenario involved an aged neighbor hospitalized with an MDR pathogen infection and the second scenario involved a coworker testing positive for an MDR pathogen. For the first scenario, half of the respondents demonstrated good hygiene practices: 50.6% would wash their hands, 51.8% would disinfect their hands, 49.3% would change clothes after interacting with the neighbor, and 41.8% would avoid the neighbor completely . For theIn this study, we assessed the knowledge and opinions of the general public in Pakistan about antibiotic use and MDR pathogens by interviewing the participants. Individuals showed poor knowledge of antibiotic use and poor medication adherence. Similarly, knowledge about MDR pathogens was scant, but fear of the effects of MDR pathogens was more pronounced.More than half had been prescribed antibiotics in the past 6 months, a lower percentage reported for the United Kingdom (38%) and Germany . The maiStaphylococcus aureus in Scotland  and in Scotland (86%) . Very feScotland .We did not find a significant difference in antibiotic use scores between urban and rural residents, in contrast with the previous study in Pakistan  or with More than half of the participants reported that they were personally concerned about infection with a MDR pathogen, but the answers to the case scenarios illustrated that the majority of individuals exhibited reasonable and non-stigmatizing behavior toward carriers of MDR pathogens in their vicinity. This finding is remarkable, considering that more than half of the participants had stated that they were fearful of contracting MDR pathogens. The percentage of respondents that reported they would avoid the infected person was higher than that reported by Raupach-Rosin and colleagues . A largeThe strength of our study is the comparably large sample and the fact that the study population was sampled from the general public. However, because the knowledge of antibiotics and MDR pathogens was assessed by closed-ended questions, respondents may have selected the most favorable answers rather than accurate ones, and therefore, a qualitative approach might be more suitable to reveal misconceptions. Our study faced some limitations as well such as we selected the districts randomly and targeted the cities, village and participants from the city conveniently. Also, that participants may have given socially desirable answers, rather than actual practices.General knowledge about antibiotic use and MDR pathogens was poor in the population we sampled. However, there was a high awareness about antibiotic resistance as a public health problem, although only a few respondents reported to have been personally affected by MDR. Therefore, information about the implications of MDR pathogens should be made available for the general public. Information campaigns about correct antibiotic use could increase knowledge, thereby improving appropriate practices in antibiotic use and resistance in both humans and livestock. The data in this study provide new information and may serve as the basis for development of interventional programs and policies.We recommend strict government policies should be implemented at hospital level and community pharmacy level to prescribe and dispense antibiotics in the presence of qualified physician and pharmacist. Standard guidelines and proper trainings should be provided to physicians and other health care providers to promote the safe prescribing of antibiotics to avoid the antimicrobial resistance. Community based interventions and programs should be initiated to increase the awareness about antibiotic resistant among general population. Government should increase the awareness and initiate the campaigns on electronic media for safe use of antibiotic in general population. Collective efforts of government, stakeholders, health care providers, patients, researchers, pharmaceuticals companies, policy makers and drug regulatory bodies will help to combat the life threatening scenario of antimicrobial resistant around the globe.


The leading contributors to the total antibiotic volume in the DDDs were macrolides (30.3%), combinations of penicillins, including β-lactamase inhibitors (18.8%), tetracyclines (14.8%), fluoroquinolones (14.6%), and third-generation cephalosporins (13.7%). ‘Watch’ antibiotics accounted for 72.3%, 52.7%, and 64.0% of encounters before, during, and after the outpatient visit, respectively. Acute viral illness accounted for almost half of the total DDDs at the outpatient visit , for which the macrolide antibiotic azithromycin was the most frequently prescribed antibiotic .Antibiotic resistance is a naturally occurring phenomenon, but the misuse and overuse of antibiotics is accelerating the process. This study aimed to quantify and compare antibiotic use before, during, and after seeking outpatient care for acute febrile illness in Ujjain, India. Data were collected through interviews with patients/patient attendants. The prevalence and choice of antibiotics is described by the WHO AWaRe categories and Anatomical Therapeutic Chemical classes, comparing between age groups. Units of measurement include courses, encounters, and Defined Daily Doses (DDDs). The antibiotic prescription during the outpatient visit was also described in relation to the patients’ presumptive diagnosis. Of 1000 included patients, 31.1% ( Antibiotic resistance is a naturally occurring phenomenon, but the misuse of antibiotics is accelerating the process. India, the world’s second-most populous country, is the world’s largest consumer of antibiotics based on total volume . The levIncreasingly, health services are delivered through mixed systems of public and private providers . In IndiUnderstanding how antibiotics are used across the course of illness is necessary to identify areas in need of improvement and to develop context-appropriate interventions to optimize antibiotic use. To assess and compare antibiotic use throughout the course of acute febrile illness (AFI), a secondary analysis was performed on data collected from an observational study on the cause and management of acute febrile illness in children and adult outpatients in India. The aim of this analysis was to quantify and compare antibiotic use prior to study inclusion, antibiotic prescription during the outpatient visit, and antibiotic use during a seven-day follow-up period, by AWaRe classification  and AnatA secondary analysis of data generated by a cross-sectional study investigating the cause and management of AFI . All patients from the original study were included in the analysis. The study took place at the pediatric and medicine outpatient departments of R.D. Gardi Medical College, a tertiary-teaching hospital in rural Ujjain, India. The climate is tropical, with a rainy season from July to September, characterized by the simultaneous occurrence of many fever-causing diseases . Antibiotics can be obtained at pharmacies and drug shops or may be provided as part of the care received from a visit to an informal healthcare provider .Inclusion criteria were children (2 months–17 years) and adult (18–65 years) outpatients with AFI, defined as an axillary temperature ≥ 37.5 °C, measured electronically at initial evaluation or a history of fever in the previous 48 h, a duration ≤ 14 days, and no signs of severe illness.Patients were recruited from June to August 2019. All patients presenting to the pediatric and medicine outpatient departments were clinically managed as per routine care by a resident physician in training or specialized in pediatrics or general medicine. For the purposes of this study, the resident physician made a presumptive diagnosis. This diagnosis was a free text description based on clinical assessment during the outpatient visit, without the support of any diagnostic tests, following standard clinical practice. After consultation, patients were screened for fever, assessed for inclusion, and enrolled in the study. Patients were requested to return for follow-up one week after enrolment; those not reporting by day seven were called by phone until they could be reached. Study activities did not interfere with the treatment of AFI or antibiotic prescription as part of routine practice.Data were collected on antibiotic use throughout the course of AFI: before, during, and after visit to the hospital outpatient department. “Before” was defined as a patient/patient attendant reporting antibiotic use for the current episode of illness (within the last 14 days) prior to presenting to the outpatient department. “During” included antibiotic prescriptions received from the resident physician as part of routine practice at the outpatient visit prior to study inclusion. “After” was defined as additional patient-/patient attendant-reported antibiotic use within the seven-day follow-up period.Using standardized case report forms, a dedicated study clinician collected the presumptive diagnosis and antibiotic prescription by the resident physician from the patient’s outpatient record. Following this, the study clinician interviewed the patients/patient attendants regarding reported antibiotic use before/after the outpatient visit. Patients/patient attendants were asked to name the antibiotics, show the medicines or their packages or send a picture via text message. They were also asked to describe the route, duration, dose, and frequency of their treatment. Antibiotics before/after the outpatient visit could have been obtained over the counter from a pharmacy or drug shop, or from an informal healthcare provider, or another formal healthcare provider outside the study hospital. Antibiotic use after the outpatient visit could have also been provided by a resident physician at the study hospital during the follow-up visit. Patients/patient attendants who reported antibiotic use at follow-up were asked if it was done by a formal/informal provider, or self-medication. Data were also collected on fever duration in days at enrolment, and the time to resolution of illness in days, as reported by patients at follow-up.p < 0.05 was used.Any antibiotics in the Anatomical Therapeutic Chemical (ATC) classes J01-antibacterial agents for systemic use, A07-intestinal antibiotics, and orally administered metronidazole (P01AB01), were considered . Antibion = 311) received one antibiotic course, 8.1% (n = 81) two, 1.3% (n = 13) three, 0.4% (n = 4) four, 0.1% (n = 1) five, and the remaining 59.0% (n = 590) received no antibiotics throughout the episode of illness. In summary, a total of 533 antibiotic courses were reported across 410 (41.0%) of the 1000 patients. A total of 24 different antibiotics were reported. The leading contributors of the total antibiotic volume in the DDDs were macrolides (30.3%), combinations of penicillins, including β-lactamase inhibitors (18.8%), tetracyclines (14.8%), fluoroquinolones (14.6%), and third-generation cephalosporins (13.7%). The patient demographic and clinical characteristics are presented in A total of 7551 patients presented to the pediatric and medicine outpatient departments throughout the study period (June to August 2019), of which 504 children and 496 adults were enrolled in the study, all patients were reached at follow-up. Of all 1000 patients, 31.1% (p < 0.001).Overall, 8.3% (83/1000) of patients reported the use of antibiotics before their outpatient visit. Among them, 91.6% (76/83), 7.2% (6/83), and 1.2% (1/83) used one, two and three antibiotics, respectively, for a total of 91 antibiotic courses. Of all courses, 6.6% (6/91) were parenteral formulations. Of all 1000 fever patients, fewer children received antibiotics than adults  of antibiotics, with just one quarter  belonging to the ‘Access’ group, and the remaining  to the ‘Not Recommended’ category. ‘Watch’ antibiotics were most frequent in ages 18–34 years  .The reported antibiotic use before the outpatient visit amounted to 234.2 DDDs. Fluoroquinolones accounted for the highest volume of DDDs , dominated by ciprofloxacin and ofloxacin. This was followed by third-generation cephalosporins , with cefixime and cefpodoxime as the leading agents in this class. The remainder of the top 90% of DDDs was composed of macrolides [azithromycin] , tetracyclines [doxycycline] , and combinations of penicillins, including β-lactamase inhibitors [amoxycillin and clavulanic acid] . Fluoroquinolones accounted for 17.7% (7.5/42.4) of DDDs among children of all ages. Irrational FDCs were only seen in young children, but accounted for 22.2% (5.0/22.5) of DDDs in ages < 5 years .p < 0.001). Of the prescriptions, 90.4% (283/313) contained one antibiotic and 9.6% (30/313) two antibiotics. Half (15/30) of the prescriptions with two antibiotics were for both doxycycline and metronidazole, and 23.3% (6/30) were for ciprofloxacin and metronidazole. Of the total 343 antibiotics courses recorded, 7.6% (26/343) were parenteral formulations.Of all 1000 fever patients, 313 patients (31.3%) were prescribed an antibiotic at the outpatient department, 11.2% (35/313) of which had reported already receiving an antibiotic before seeking care. Of these patients, 31.4% (11/35) were prescribed the same antibiotics they had reported already taking, and the remaining 68.6% (24/35) had a different antibiotic prescribed. Fewer children received antibiotics than adults . There was one encounter of ‘Reserve’ antibiotics, four ‘Not Recommended’, and one ‘Unclassified’. The proportion of ‘Watch’ antibiotics was highest in ages < 5 and decreased gradually with ages up to 50–65 years  .p < 0.0001). Fluoroquinolone use made up 5.0% (15.5/309.4) of the DDDs in children of all ages , followed by combinations of penicillins, including β-lactamase inhibitors [amoxycillin and clavulanic acid] , tetracyclines [doxycycline] , third-generation cephalosporins , and fluoroquinolones , together which made up over 90% of the DDDs. The proportion of third-generation cephalosporins was greater among children than adults  than children. Among all antibiotic courses reported at follow-up, 11.1% (11/99) were parenteral formulations.Antibiotic use within the seven-day follow-up period was reported by 8.9% (89/1000) of patients. Of them, 88.8% (79/89) reported using one antibiotic and 11.2% (10/89) two antibiotics. p = 0.891)  of all antibiotic encounters at follow-up belonged to ‘Watch’ and one-third  to ‘Access’. The remaining  were ‘Unclassified’ and all three were from informal healthcare providers. There was no significant difference in the proportion of ‘Access’ to ‘Watch’ encounters between patients who received care from informal healthcare providers, formal healthcare, or self-medication  .Over half of the total 394.5 DDDs at follow-up were contributed by macrolides [azithromycin] , followed by combinations of penicillins, including β-lactamase inhibitors [amoxycillin and clavulanic acid] , fluoroquinolones , and third-generation cephalosporins , which together comprised over 90% of the DDDs at follow-up. Fluoroquinolones accounted for 15.6% (17.2/110.4) of the DDDs among children of all ages .Of all patients with the seven most common presumptive diagnoses, acute viral illness had the lowest proportion of antibiotics prescribed , followed by gastroenteritis , then typhoid , malaria , and a urinary tract infection . Of patients who received antibiotics, the diagnoses with the highest proportion of ‘Watch’ were typhoid  and acute viral illness  .Acute viral illness accounted for almost half of the total DDDs , with azithromycin contributing the largest proportion . For urinary tract infections , the greatest proportion of DDDs came from doxycycline , all of which was for adult females, and all but one case was given in combination with metronidazole. Both upper respiratory tract infections (URTIs)  and lower respiratory tract infections (LRTIs) , were treated with amoxycillin and clavulanic acid , and azithromycin . For gastroenteritis , fluoroquinolones were the most frequently prescribed . Typhoid  was treated mostly by doxycycline  .The analysis of antibiotic use showed that 41% of patients had an antibiotic at some point throughout the episode of AFI in rural India. A total of 8%, 31%, and 9% of patients received antibiotics before, during, and after seeking care for AFI at a hospital outpatient department. Examining antibiotic use before, during, and after the outpatient visit gives a more complete picture of antibiotic use throughout the course of AFI. This study identified an additional 10% of patients reporting antibiotic use before or after the outpatient visit that would not have been seen by pure outpatient prescribing estimates. The combined volume of DDDs used before (234.2) and after (394.5) the visit accounted for almost one-third of the total DDD volume  of the study.Antibiotic use before seeking outpatient care for AFI was similar to reports of 5% from Thailand and Myanmar , and 8% In 2019, the WHO adopted a country-level target indicator that at least 60% of antibiotic consumption should come from the ‘Access’ group . The 25–The use of a limited number of ‘Reserve’, ‘Not Recommended’ or ‘Unclassified’ antibiotics were identified. Most ‘Reserve’ antibiotics are parental formulations which may be more common with inpatients or patients with severe illness. All four ‘Not Recommended’ or ‘Unclassified’ antibiotics were irrational FDCs which should be avoided due to their lack of proven efficacy and potential effect on selecting for resistance . The 2–3V. Cholerae, Shigella, amoebiasis, and giardiasis, as recommended by the STGs [Almost half of the antibiotic prescriptions were for patients with a presumed diagnosis of acute viral illness. This might reflect uncertainty of diagnosis in the absence of diagnostic tests and “just in case” prescribing. A surprising proportion of patients with urinary tract infections and LRTI had no antibiotics prescribed. For UTIs, physicians may have refrained from prescribing until receiving culture results. Nonetheless, the observation of both over and under prescribing reflects the access vs. excess dilemma. While in many cases there is overuse of antibiotics, some patients simultaneously suffer from a lack of antibiotics when needed. The unnecessary use of antibiotics is accompanied with the risk for adverse events, out of pocket expenses, and can cause dangerous diagnostic and treatment delays. Both the quantity and choice of antibiotics is concerning as several alternative antibiotic agents were chosen over the Indian national standard treatment guidelines (STGs) . For urithe STGs . HoweverStreptococcus pneumoniae [Azithromycin attributed approximately one-third of the total volume of DDDs across all three time periods . Its low cost and once daily oral formulation makes it one of the most frequently prescribed antibiotics for outpatients in India . The exteumoniae , and an eumoniae . The extFluoroquinolones are an important class of antibiotics traditionally effective for urinary, respiratory, intra-abdominal, and sexually transmitted infections. As a result of their widespread global use in human health and agriculture, resistance is rising and fluoroquinolones have been detected in surface water and groundwater, posing a serious threat as they are known to be toxic to plants and aquatic organisms ,38. The To our knowledge, this is the first study from a LMIC to examine outpatient antibiotic use throughout the course of AFI which could account for the wide range of sources where antibiotics are available in these settings. The monitoring of drug utilization by an ATC class helps to scrutinize the appropriateness of prescribing at a granular level in relation to treatment guidelines, while the WHO AWaRe classification system makes complex prescribing data easier to understand in relation to antibiotic resistance and, therefore, may be a useful tool for policymakers. R.D. Gardi Medical College Hospital has a long tradition of research on antibiotic use, providing a unique opportunity to compare prescribing patterns between departments, patient groups, and over time.There are several limitations in this study: First, antibiotic use before and after outpatient visits were based on patient reports and may, thus, be subject to bias. At the study hospital, patients often brought with them any medicines they were currently using. At follow-up, patients contacted by phone were asked to send pictures of their medicines via text message. This likely helped to ensure the correct drug utilization data were collected and increase the robustness of the data. However, it is not possible to know if any antibiotics were overlooked, if the patients adhered to antibiotic use as per the recommended schedule, or if their prescribed treatment was discontinued or switched to another antibiotic by follow-up. Second, the short period of data collection likely missed the seasonality of diseases and the related prescribing practices which would have been available had a full year had been covered. Third, an oversight of the study was the failure to ask the source of antibiotics before the outpatient visit, and including such data in future drug utilization studies in LMICs is recommended. Fourth, it would have been interesting to see if patients with a presumptive diagnosis of malaria were also prescribed antimalarials; unfortunately, however, we did not collect this data. Fifth, using adult DDDs in children may underestimate the actual antibiotic use children, therefore, we presented antibiotic use data by courses, encounters and DDDs to provide different estimates of use. Finally, differences in seasonality, types of infections, antibiotic policies and resistance patterns may make it difficult for a direct comparison of the study results with other settings.The overuse and misuse of antibiotics within and outside the hospital outpatient department are of major concern. Interventions to support providers in aligning prescribing practices with the WHO AWaRe recommendations and national standard treatment guidelines are urgently needed.


By incorporating smart materials in the structural health monitoring systems the issue of incompatibility between monitored structure and the sensor is surpassed since the concrete element fulfills both functions. Machine learning is an attractive tool to reduce model complexity, so artificial neural networks have been successfully used for a variety of applications including structural analysis and materials science. The idea of using smart materials can become more attractive by building a neural network able to predict properties of the specific nanomodified concrete, making it more cost-friendly and open for unexperienced engineers. This paper reviews previous research work which is exploring the properties of CNTs and their influence on concrete, and the use of artificial neural networks in concrete technology and structural health monitoring. Mix design of CNT/concrete composite materials combined with the application of precisely trained artificial neural networks represents a new direction in the evolution of structural health monitoring of concrete structures.Carbon nanotube/concrete composite possesses piezoresistivity  i.e. self-sensing capability of concrete structures even in large scale.Carbon nanotube/concrete composite possesses piezoresistivity  Both nanotechnology and neural networks were in the making during the first half of the twentieth century. The first idea of nanotechnology from R. Feynman  and the first stage of development of artificial intelligence by B. Widrow and M. Hoff  marked the beginning of a new era of technology. The conference held in Trieste, Italy in 1984  opened ast century that carbon nanomaterials found their use. Even though concrete is a conventional material, it is the most used material in the world after water as humanity's first and basic need. Suffice to say that abundant funds are invested every year in concrete structures and their wellbeing. Every civil structure is designed to meet the criteria of safety, durability, serviceability, and sustainability during its service life so constant supervision over its condition is necessary. For exactly that purpose structural health monitoring (SHM) was developed as a field which deals exclusively with optimizing the structure's behavior during its exploitation period.As for civil engineering, it wasn't until the brink of 212,3 such as transducers,4 electrochemical and optical5 sensors as well as smart materials. While the development of nanoscale devices and nanoelectromechanical systems utilizing the unique properties of carbon nanotubes (CNTs) is an evolving area of nanotechnology, there is also considerable interest in making macroscopic engineered, smart, materials that can exploit novel material properties.6 The existing problem with monitoring devices has been the compatibility between the sensor and the concrete structure. Smart materials with the addition of carbon nanomaterials are being developed in order to surpass this issue of incompatibility by making the whole structure able to give feedback about its condition. Cement-based materials with the addition of CNTs manifest piezoresistive behavior, meaning that it is possible to monitor strain by following the change in the electrical resistance7 under certain type of physical stimuli affecting the structure. This way, structural elements not only fulfill a structural function8 but also behave as sensors apt for self-monitoring, while the data acquired and collected is regarded as usable.SHM has now grown into a highly developed field in terms of new and emerging technologies. The most recent developments are connected to different types of nanosensors9–26 in order to predict primarily the compressive strength of concrete with different additions. Nonetheless, ANNs may be used also for predicting electrical conductivity of smart concrete, similar to the model used for conductive nanomodified polymer materials.132,144 The idea of using smart materials can become more attractive by building a neural network able to predict properties of the specific nanomodified concrete, making it more cost-friendly and open for unexperienced engineers.Machine learning has been used in the field of concrete technology in the past twenty years. Various neural networks were madeThis paper is organized as follows. Section 2 and 3 give an overview of the properties of carbon nanotubes and further focus on the electrical properties of CNT/concrete composite materials. Section 4 establishes the role of artificial neural networks in engineering and their application. Section 5 gives a review on the structural health monitoring and state-of-the-art in this field and proposes a new method of designing smart materials capable of self-sensing.et al.27 described “single-shell tubules” three decades ago, there has been extensive research on this topic and many discoveries have been made regarding the properties and possible applications of single-walled carbon nanotubes. Carbon nanotubes are allotropes of carbon with a cylindrical structure. As a part of the fullerene family, nanotubes are members which have a long hollow structure made of one or more sheets one carbon atom thick. The sheet rolling angles are chiral and the radius of the tube determines the properties of the carbon nanotube.28–30 Some of the highlighted characteristics of CNTs that are superior to other materials are Young's modulus,5,30–35 tensile strength,31,32,34–36 mass density,4,35,37 as well as electrical38–40 and thermal conductivity  where the properties of each scale derive from those of the next smaller scale i.e. processes occurring at the nanoscale ultimately affect the performance of the bulk material.55,59–67 Viewed from the bottom up, concrete is a composite of molecular assemblages, surfaces, and chemical bonds that interact through chemical reactions, intermolecular forces and diffusion. The combination of exceptional mechanical properties of CNTs along with their low density and one-dimensional structure makes them an exceptional candidate for reinforcement in composite materials.35,55,59Concrete is a nanostructured, multi-phase, composite material that ages over time. It is composed of an amorphous phase, crystals, and bound water. The amorphous phase, calcium–silicate–hydrate (C–S–H) is the ‘glue’ that holds the concrete together and is itself a nanomaterial.40,56,57,68–72 When added to concrete, CNTs work as fillers in the mesoporous environment of concrete, improving the hydration of cement and decreasing porosity, thus improving the compressive strength and decreasing autogenous shrinkage of concrete. Nanoscale reinforcement can also inhibit crack growth in the initial stages of damage and so prevent damage propagation, enhance the quality of the paste–aggregate interface and increase the amount of high stiffness C–S–H.35,36,59,72–74 Li et al.75 investigated the mechanical properties of CNT/cement composites and found that compressive and flexural strengths of cement with 0.5 wt% CNT addition were increased by 19% and 25%, respectively, compared to the plain unreinforced cement.48Incorporating some functional materials  such as 76 have researched the strain sensitivity of carbon fiber-reinforced cement compared to that of normal cement since 1993. Furthermore, Thostenson and Chou49,77 proposed that the change in the size of reinforcement, from micron-sized fiber reinforcement to carbon nanotubes with nanometer-level diameters, enables the opportunity for multi-functional sensing capability. By in situ combining reinforcements of different scales, carbon nanotubes can penetrate the matrix-rich areas between fibers in individual bundles as well as between adjacent plies and can achieve a nerve-like network of sensors throughout the arrays of fibers in a composite. More studies evaluated the influence of hybrid reinforcement, such as a combination of carbon fiber (CF) and carbon black with CNTs on the self-sensing properties of cement-based sensors.25,40,72,78–81 Anthony et al.80 developed computational models of a composite for predicting the benefits of grafting CNTs at the CF surface in order to create a conductive network that can be used for in situ damage detection. Moreover, Han et al.81 showed that the flexural strength and electrical conductivity in the cement mortar matrix with self-assembled CNT/nano carbon black composite fillers are enhanced and stable, and sensitive piezoresistivity is achieved at a low filler content. Since the fillers are mainly in order of magnitude of microscale or nanoscale, the potential filler–matrix and filler–filler interface surface areas of the composite are enormous. Since these interfaces have an effect on the quality of the electrical communication between CNTs and concrete matrix and also on communication between adjacent CNTs, they are affecting the conductive network and the overall electrical conductivity.56Fu and Chung4,48,72,82–84 resulting in an extremely large surface area. The aspect ratio must be sufficiently large to maximize the load transfer between the CNTs and matrix material and thus to achieve enhanced mechanical properties.4,35,85 Proper dispersion of the nanoparticles in concrete or cement mortar matrix is vital for improvements in the mechanical performance of the composite but it is difficult to achieve for nanomaterials with a large aspect ratio such as CNTs due to their high surface energy and strong interparticle  forces. If not properly dispersed, the addition of nanomaterials to cementitious materials may finally result in a decline in the mechanical properties.35,36,43,59,73,81,86 It has been found that CNTs tend to dramatically hamper the mechanical properties of fabricated nanocomposites.4The mechanical properties and electrical conductivity of concrete/CNT composite depend on the dispersion state of the nanofillers. The nature of the dispersion problem for CNTs is somewhat particular because of their small diameter in the nanometer scale with a high aspect ratio (>1000)56,87 Especially for fibrous fillers, high aspect ratios combined with high flexibilities increase the possibility of fiber entanglement and close packing.81 Studies have emphasized repeatedly74,88–90 that CNTs tend to agglomerate, which hinders their uniform distribution within a matrix.91 Poor dispersion of nanoparticles may lead to segregation, weak zones or potential areas for aggregation of concentrated stresses.36,86 As an alternative, Anthony et al.80 showed how CNTs can be directly grafted on carbon fibers in order to form a hierarchical reinforcement, combining nanoscale and microscale reinforcements92,93 and avoid such dispersing problems. Uniform dispersion can be achieved using various types of mechanical and chemical methods including ultrasonication, shear mixing, calendaring, ball milling, stirring and extrusion. Various methods of chemical modification have been proved quite successful in contributing to better nanotube dispersion. In terms of tensile modulus, it has been established by numerous studies94,95 that chemically modified nanotubes exhibit a significant increase as compared to the matrix resin. This is mainly due to the fact that functionalization improves both dispersion and stress transfer.82 Some studies have proved that the carboxyl CNT41 is much easier to disperse in water and concrete matrix than the plain CNT because of the carboxyl groups situated on the surface of the carbon sheet. Moreover, the carboxyl CNT achieves a better bond with the concrete matrix than the plain CNT.41 Surely that the selection of a proper method or a combination of several methods and their processing conditions need to be based on the desired properties of the end product.35Namely, the one-dimensional fillers are more difficult to disperse and easier to damage during the fabrication of concrete. As the surface area of the filler increases, the attraction forces between the fillers also increase.−10 S cm−1  the distribution of functional filler in a binder, (ii) distribution of the binder with fillers among fine aggregates, and (iii) distribution of the fine aggregates with binder and fillers among coarse aggregates.41,118 As previously stated, the potential filler–matrix and filler–filler interfaces affect electrical bonds between fillers as well as between the concrete matrix and the filler material,119 thereby influencing the comprehensive conductive network and electrical conductivity of the composite.41,56Concrete cannot be used for sensor purposes in its basic form, but if a metallic material is dispersed into it a conductive composite can be manufactured. Moreover, a non-conductive or poorly conductive matrix has greater sensitivity to changes in electrical resistance3,48,79,121–124 Han et al.41 manufactured a concrete/CNT composite with a compressive strength of about 40 MPa. It was proven that electrical conductivity strongly depends on the morphology of the CNT network56,92i.e. the number of contact points and that the electrical resistance of self-sensing CNT concrete can be changed when it is deformed under applied loading. Han et al.41 showed that the changes in resistance coming from the alteration of nanotubes' geometry under external loading may be neglected since they are small and reversible. However, the contact resistance must be taken into consideration since the distance between adjacent nanotubes may considerably differ when under load, implying the sensing ability is stable and reversible as long as the deformation is elastic.40,41Carbon nanotubes express interesting electromechanical properties. When subjected to stress, their electrical properties change indicating a linear and reversible piezoresistive response with respect to different stress levels.105,125,126 and simulations of the processes taking place in these materials are taking a lot of interest. Most recently, the authors107 presented a 3D generalization of the micromechanics model of the overall conductivity and uniaxial piezoresistivity of CNT-reinforced composites subjected to arbitrary strain states. Changes in conductance, as the origin of the piezoresistive behavior, expected when the CNT/concrete composite is subjected to external mechanical strains105,107,127–129 are here prescribed to the changes in the volume fraction, to filler reorientation, and to changes in the inter-particle properties. The authors107 proposed an approach shown to be capable of determining the piezoresistivity coefficients by two virtual tests, namely laterally constrained dilation and distortion, which is efficient in terms of computational cost and, more importantly, it reliably presents the complex electromechanical behavior of CNT/concrete composites. Piezoresistive behavior of CNT/concrete composites is continuously investigated because it is the base point on which the majority of sensoring systems used in SHM today were built on.Piezoresistivity of composites with conductive fillers has been the subject of several numerical and analytical works,1 Although the original idea of ANNs was to solve problems in the same way as a human brain would, over time it deviated from biology and advanced towards the direction of solving specific tasks . Recently, machine learning has emerged as an attractive tool to reduce model complexity.132 As a subset of machine learning, ANNs have been successfully used for a variety of applications,133 some including structural analysis134 and material science, ranging from studies of atomic properties135 to the mechanical properties of individual CNTs.136Based on Hebbian learning, as it is conceived on principles of the biological systems' dynamics, artificial neural networks (ANNs) have been developed as an interconnected group of nodes inspired by a simplified representation of neurons in the brain. According to Braitenberg (1977), changing the stimuli conditions may induce clusters to form and later “evaporate” in order to build new configurations, more or less mimicking the way the corresponding neurons are involved in a dynamic succession of different assemblies, “ignited” by the changes in the external world.137 The architecture of the ANN is formed by input and output layer and a series of hidden layers, each of which is formed by a finite number of neurons.138 This kind of ANN architecture is called a multi-layer network and is the most widely used. It should be noted that artificial neurons in some cases may have a threshold such that the signal is only sent if the total signal crosses that threshold. After building the database which is divided into subsets, the neural network is trained for a specific task needed.The neural network approach is a way of modeling data based on computer learning in order to perform complex functions. The ANNs are composed of simple elements operating in parallel, adaptive and distributed systems. In most ANN implementations, the output of each artificial neuron is computed by some non-linear function of the weighted sum of its inputs. Artificial neurons, which are processing elements, are divided into multiple layers , and the139 The training of a network is a process where the set of adjusted weights is optimized to make the best prediction of the target variable on the basis of background variables. Multiple layer networks are trained with the standard back-propagation error algorithm.137,139,140 This algorithm presents training examples to the network, examining the shift between the network output and the desired output while modifying the weights and biases of connections until the network produces an output very close to the desired one.139 The back-propagation algorithm consists of a forward step where the signal propagates through the computational units until it gets to the output layer and a backward step where all the synaptic weights are adjusted accordingly to an error correction rule.136 There are some factors that affect the ability of ANNs to model problems such as network structure, training and testing dates, network algorithms and finally transfer, training, learning, and performance functions.141The training phase makes it possible to calculate the synaptic weights leading to each formal neuron.139 Currently, it's mostly used in the field of medicine for developing motor neurons138 or improving the monitoring of neural activity.142 Some works have been dedicated to predicting the sensitivity of strain sensing of piezoresistive materials, however computational cost can be considerably reduced by using machine learning.143ANNs offer fast solutions for a wide range of problems, especially the ones with real-time constraints.et al.139 developed a finite element model for an armchair CNTs using ANSYS simulator and examined their ability of detection of the applied force. It was found that the sensitivity is proportional to the nanotube length and that the non-linearity of the sensor depends on the applied force range. Moreover, this smart nanoforce sensor was proposed and investigated using ANN-based computation.139 Matos et al.144 made a FEM simulation of a CNT/polymer composite to produce training data for an ANN in the goal of developing a technique of precise tailoring of the electrical properties of composites. The authors conducted simulations of conductivity of different composites by adopting different conductivities of CNTs and the matrix, geometry of CNTs and their volume fractions of the composites. The objective was to reduce the number of problem parameters and it was concluded that their ANN model could facilitate other types of conductive composites.144 A further study conducted by the same authors included a non-uniform multiaxial strain field.132In terms of sensor systems, various problems regarding CNTs and their composites have been covered, processed or solved using an ANN approach. Menacer et al.141 developed an ANN to predict the natural frequencies of CNTs with different types, lengths, and diameters and the effects of the attached masses on the first five natural frequencies of a special CNT. The well-known molecular mechanics and FE models are used to describe the vibrational behavior of this discrete structure before and after attaching a particle on it. The authors141 used MATLAB ANN toolbox to create a structure that consisted of several layers: one input, few hidden and one output layers. Hidden layers relate the input layer to output one by multiplying them to weight factors and adding some biases. First, the input data were inserted in ANN and the errors between real target values and the proposed target values by ANN were calculated. Next, the sensitivities start to propagate from the output layer toward the first layer and then the weights and biases are updated.141Fakhrabadi et al.137 proposed an ANN for modeling and simulation of the semiconductive SWCNT transistor device. The proposed ANN model was optimized based on data obtained by using the MATLAB script.137 The application of machine learning implies very low computational cost and can be immediately used by relatively inexperienced users.132 ANN can be a powerful tool to model different problems, especially in cases that there are not accurate governing mathematical relations for them.141Hayati 9–26 included investigating the compressive strength and workability of plain concrete or concrete with different aggregates and additions. Probably the largest contribution in this area was given by Dr I.-C. Yeh which created the first database containing 1030 sets of data about properties of concrete specimens . The concrete specimens contained the additions of blast furnace slag, fly ash and superplasticizer in different proportions. The database was given by the UCI repository and has been used in research works since then.11,14The topic of using neural networks for predicting optimal concrete mix proportions has been explored many times in the past few decades. The research work9,10 green,16,17,22 self-compacting,18 and lightweight concrete. Khan et al.15 estimated the compressive strength of plain concrete using MATLAB ANN simulation and after comparison of the mathematical results and 55 experimental results, concluded that multilayer feed-forward neural network gives satisfactory results. Although the multilayer feed-forward approach is mostly used,9,10,14,17,21,24 researchers often use other techniques for predicting the compressive strength of concrete, such as fuzzy logic, decision tree or support vector machine.Some investigations have been directed toward specific types of concrete such as high-performance,et al.23 showed how ANNs can be applied in civil engineering practice. The authors designed the optimal ANN architecture and fed it with an extensive database including 741 concrete mix recipe record and a corresponding laboratory destructive test. The final product was a system for obtaining optimal concrete mixtures which can be used by unexperienced users in everyday practice. Some authors showed the efficiency of ANNs by comparing the results from the ANN model with the results from fuzzy logic model,20 multiple linear regression model,25 random forest model,21 decision tree,21 support vector machine,24etc.The study carried out by Ziolkowski etc. These damages manifest in the form of cracks, corrosion, displacement, delamination, rotation, strain and more, potentially resulting in a catastrophe if not regularly and properly monitored.Civil engineering structures are generally the most expensive investments, with concrete (cement) as the second most widely used material in the world. Civil and industrial structures are constantly exposed to damage caused by the weather conditions in combination with poorly designed material properties, damage due to the over-usage of the structure, accidental occurrence, earthquake, 71,145 Having this in mind, the field of structural health monitoring (SHM) was developed in order to follow structural changes taking place under different environmental influences and loads.Because a civil structure should meet the criteria of safety, durability, serviceability, and sustainability during its service life, constant supervision over the condition of the structure is necessary. Structural assessment is done manually by visual inspection and in the intervals prescribed by the designer and/or regulations. These examinations are expensive, time-consuming and labor-intensive, as well as highly subjective and can only consider the damage that is visible on the surface of the structure.124,146 NEMS is currently the object of intense research activity in the field of detection, where the main objective is to minimize the MEMS surface and power consumption using carbon nanotubes.139,147,148 Nevertheless, more advanced and accurate methods for structural health monitoring are available and continue to be developed.36,149The field of SHM has, since its inception, been developed in terms of sensor systems going from cabled to wireless, to MEMS  and ultimately developing NEMS .40 accelerometers and strain gauges,150 which provide real-time information that may be continuously monitored from a central location and give insight into the condition of the structure.36,145 One of the main limitations of sensors is that they are discrete fixed-point directional sensors, not being a part of the material or structure that is being monitored. This on occasion leads to discrepancies in readings, showing the incompatibility between the sensor and the monitored element. The need to develop sensors that can be a part of the material and used for multidirectional and multiple location sensing is, therefore, imposed.79 Self-sensing CNT/concrete composite is considered a good candidate for solving the durability and incompatibility issue between conventional sensors and concrete structures because it itself functions as both the structure and the sensor.41,92,151 With the development of smart, self-sensing materials including CNT/concrete composite which provide an electrical response to stress or strain,36,56,152 self-monitoring structures can become a civil engineering practice.More advanced methods typically involve the analysis of measurements from sensors such as displacement transducers,etc.) in a controlled fashion. These composite materials are usually designed as a compound of the main insulating material and a filler material which gives it multifunctionality. Within the general field of civil engineering, structural design and SHM are the areas which mostly emplore these materials. Multifunctionality is introduced to structures exposed to severe loading, such as skyscrapers, bridges, structures in seismically active areas and aesthetically important structures. Saafi97 presented how the use of CNTs has opened the door for new smart and advanced sensing materials that could effectively be used in structural health monitoring where wireless and real-time sensing72,145 could provide information on damage occurrence and progression. CNTs have exhibited extraordinary mechanical properties, providing structural and functional capabilities simultaneously, including actuation,52,153 sensing, and generating power. These abilities represent the possibility for developing actuators capable of high stress and strain operating at low voltage, and multi-functional electrochemical and mechanical sensors.34Smart materials are engineered intelligent materials with properties adaptable to external stimuli  in order to be used as the sensor element. The authors proved92 that this CNT thread is suitable as a distributed sensor material, allowing the development of a robust and efficient SHM system.Carbon nanotube networks are highly valuable in SHM for strain mapping, damage detection and identification of crack initiation and propagation.et al.37 proposed a new approach for developing smart composite material able to monitor its own health while the actuation capability allows it to actively improve the performance of the structure and extend its life. The high strength and elastic modulus, as well as piezoresistivity, indicate the possibility to produce a long continuous sensor that would be able to measure strain over a large surface area for SHM of concrete structures. The authors37 developed a long continuous strain sensor  as an element in a biomimetic artificial neural system used for SHM, which is capable of measuring large strain and forming a grid over a large area of a structure, and on the other hand, simultaneously has a load-carrying capability as a structural component.Kang 163,164 The artificial neural system is primarily developed within the medical field where the application of CNTs includes electrical interfaces for neuronal stimulation and recording,29 neural prosthetic devices for artificially restoring impaired neural function,165 encouraging neuronal growth,28 retinal implants,165etc. In a functioning nervous tissue, neurons carry electrical signals via switching of the membrane potential caused by electrically and chemically gated ion channels in the cell membrane. Although the signal propagating along the length of the neuron is primarily achieved electrically, neurons communicate with each other at chemical junctions, synapses,142 much so like the communication between CNTs by electron hopping at their junctions. The same principle constitutes the artificial neural system which uses multiple neurons for real-time monitoring of strain of the structure. Since the production of neurons involves a mask to define the pattern, it can take any shape including a grid that resembles that of the peripheral neural system of the human body. Sensors act as nerves and actuators act as muscles where the control center is the brain.Fabrication of CNT neurons as long films has been proposed by some authors.etc. and the ultimate form of intelligent structures are the ones that have the additional ability to learn in contrast to the pre-programmed response. This learning feature is realized by employing artificial intelligence, namely machine learning and vis-à-vis artificial neural networks.145,166 The choice of ANNs to model so many different systems is, in part due to their flexibility, adaptability and generalization capabilities and their easy application in software and hardware devices138 and materials.144The idea of a self-sensing structure is to mimic a biological system through sensing, actuation, adaptability, self-repair, e.g. high permeability, assigned porosity, high flexure, self-cleaning ability, etc.The authors of this paper propose a novel SHM system that would imply concepts of self-sensing concrete containing an artificial neuron system in combination with artificial neural networks. Real-time effective and long-distance monitoring of concrete structures is possible to obtain by developing a cementitious material representing a good host for the intricate branches of long continuous CNT neurons on one side and on the other – trained neural network capable of disregarding negligible data and processing relevant information about the structure's condition, so it may generate a 3D model showing possible damage and current state of structural elements. In the case of the maintenance planning of a new structure, ANN could also be used to obtain the optimal mixture of CNT/concrete composite in terms of desired specific properties of the end product such as 79,150,167 made with nanomaterials as fillers. The goal is not only higher strength of the composite material, but also obtaining multifunctional smart and cost-effective structures. The behavior of CNT-based sensors and actuators involves multiple phenomena at length scale hierarchies ranging from quantum to macroscopic scale. Although there are still some challenges regarding the proper dispersion168 of CNTs in the cement matrix and the exact volume fraction at the percolation threshold,43 the results of the work presented in this paper are very promising and indicative of the beginning of a new direction in the development of structural health monitoring. Fundamental knowledge of the structure/property relations of CNT composites is still somewhat lacking which highlights the need to develop both experimental and analytical techniques to bridge these scales toward the optimization of SHM systems.49 Nevertheless, smart nanomodified cementitious materials provide promising and innovative applications.38This paper reviews previous research work which is exploring the properties of CNTs and their influence on concrete, and the use of artificial neural networks in material science and concrete technology. The paper shows how incorporating carbon nanotubes in concrete mixtures or alternatively grafting short CNTs to produce excessively long carbon fibers, combined with the application of precisely trained ANNs represents a new direction in the evolution of structural health monitoring of concrete structures. The capability to tailor materials at the nanoscale has opened-up a new field of research aimed at both tailoring their properties as well as developing intelligent systems that are capable of sensing and actuation. Several studies were conducted on the topic of different types of strain sensors140 Future investigation of the authors will focus on developing the methodology of production of self-sensing CNT/concrete composites and specifying, modeling and training the ANN by using the obtained results of sensitivity testing.Moreover, despite much work devoted to making a perfect ANN structure, there are no conclusive rules on how to achieve it. The only way to produce a suitable ANN structure includes the ‘trial and error’ process, so the number of hidden layers and the number of neurons at each layer is adjusted for each specific problem.There are no conflicts to declare.


We also measured the ketogenesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) in relation to jejunal mitochondria. Methods: A single-centre, randomized, unblinded crossover study in 15 healthy volunteers ingesting strictly controlled equicaloric diets (either HCD or HFD), with 60% energy from the respective source. An enteroscopy was carried out after 2 weeks of each diet and jejunal mucosal biopsies were acquired. Conventional histology, immunofluorescent staining, transmission electron microscopy and confocal microscopy were used. Results: The villi did not demonstrate any change in the epithelial enlargement factor. Despite an increased mitosis, there were no changes in apoptotic indices. However, the ultrastructural analysis demonstrated a significant increase in the enlargement factor at the bases of the villi. The mitochondria demonstrated increased amounts of cristae after the HFD. The confocal microscopy revealed increased HMGCS2 per mitochondrial marker at the top of the villi after the HFD compared to the HCD. Conclusion: There is a morphometric adaption in the jejunal mucosa following the 2-week diets, not only on a histological level, but rather on the ultrastructural level. This study supports the notion that mitochondrial HMGCS2 is regulated by the fat content of the diet and is involved in the expression of monosaccharide transporters.Background and aims: The conditions for jejunal glucose absorption in healthy subjects have not been thoroughly studied. In this study we investigated differences in  After sonication, each homogenate was centrifuged 6 in 0.05 M sodium cacodylate, for 2 h at 4 °C. A bloc staining followed in 0.5% uranyl acetate in distilled water for 1 h at room temperature in darkness. The tissues were then rinsed in distilled water, dehydrated in rising concentrations of ethanol, followed by 100% acetone, and were infiltrated and embedded in Agar 100 resin. Sixty nm ultramicrotome sections were photographed in an electron microscope  at primary magnifications ×4000, ×8000, ×25,000 or ×31,500. To standardize the measurements, the orientation of the sections must meet certain criteria: straight microvilli bordering the lumen; clear cell nucleus in the middle; and infranuclear cytoplasm sitting on the basement membrane. We counted the number of intersections between a square grid (square size 12 × 12 mm2) and the cytoplasm, which gives an estimate of the size of the cytoplasm [The biopsies were kept in ice-cold Ringer solution for ~30 min before fixation in ice-cold Karnovsky fixative . After fixation overnight, the specimens were post-fixed in 1% OsOImmunofluorescent double stainings were performed with the mitochondrial ketogenesis enzyme HMGCS2 and mitochondrial marker TOMM20. The sections were rehydrated, antigens were retrieved and sections were blocked in 5% normal goat serum  before incubation with primary antibodies HMGCS2  and TOMM20  overnight at 4 °C. Slides were then washed and incubated with the secondary antibodies Alexa Fluor 488 goat anti-mouse  and Alexa Fluor 568 goat anti-rabbit  for 2 h at room temperature and counter-stained with Hoechst staining . Sections with blocking buffer instead of primary antibodies were included as negative controls. Confocal imaging was performed at the Centre for Cellular Imaging at Sahlgrenska Academy using an LSM 700 inverted laser scanning confocal microscope  with Zen 2012 software . All images were captured with the same settings using an apochromatic 20X/0.8 objective. For the longest wavelength, the pinhole was set to 1 airy unit and this pinhole size was applied to both channels. For each sample, 24 optical slices (0.998 μm thickness) were made in z-dimension around the center of the sample. For each sample, scanning images were acquired from the top, middle and base of the villus. Acquired images were quantified for intensity signal of each channel using Fiji version 2.1.0/1.53c [p-value of ≤0.05 was considered significant. All analyses were performed using Prism 9 for Mac OS X .Statistical outcomes of the paired Western blot, immunohistochemistry, etc. were analysed with the Wilcoxon signed-rank test. The 2-way ANOVA with Sidak’s multiple comparison test was used for confocal microscopy. The standard errors of the mean (SEM) are presented in figures. A Fifteen healthy volunteers  with a mean body weight of 72 ± 3 kg ingested two weeks of each iso-caloric diet; beginning either with the HCD or the HFD, in a crossover randomized order .p = 0.027).p = 0.034). Despite this, the ratio between pro- and antiapoptotic proteins (as a measurement of apoptotic probability) was not significantly different between the two diets . This was not the case concerning the length of the microvilli in the top region of the villus. The diameter of the microvilli did not differ depending on the diets (p = 0.020). No such difference was present in the top region of microvilli.he diets E. The ulsupranuclear part at the top of the villi demonstrated a significantly increased number of mitochondria following HCD as compared to HFD (The number of mitochondria were counted in relation to the nucleus of the enterocyte see A. The sud to HFD B. No oth2) occupied in the respective parts of the enterocytes in relation to the number of mitochondria  compared to the HCD, while in bases of the villi the diet had no effect  following HCD compared to HFD .mitos, “thread”, chondrion, “granule”, was coined in 1898. The first high-resolution electron micrographs of mitochondria were published in 1952. In 1957, the term “powerhouse of the cell” was coined by Philip Siekevitz [matrix granules may create a level of “connectivity” between the inner and outer mitochondrial membranes that allows for homeostasis of the mitochondrion [cristae within the mitochondria was significantly lower at the tops (both in the supra- and infranuclear parts), as well as in the supranuclear part in villi subjected to HCD compared to HFD. The infranuclear mitochondria at the bases showed a similar pattern, but this did not attain statistical significance. The present results thus demonstrated an increased number of mitochondrial cristae in the enterocytes upon following the HFD compared to following the HCD. The cristae host the majority of respiratory enzymatic actions and energy production in the form of ATP.Mitochondria play a crucial role in the lifecycle of the cell by performing energizing oxidative reactions involving amino acids, lipids and ketone bodies, and create the vast majority of adenosine triphosphate (ATP) that is the energy storage necessary to support all cellular functions. The term “mitochondria”, from the Greek μίτος, iekevitz , and in iekevitz ,14,15. Ihondrion . We founIn the mitochondria, the energy-rich ketone bodies can be produced. We have recently shown that the jejunum of the small intestine can express the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) and produce ketone bodies during prolonged consumption of a diet high in fat . In anotIn summary, a morphometric study was done on the jejunal mucosa in healthy volunteers who consumed both a HCD and a HFD in a random crossover fashion. The study did not demonstrate any change in the mucosal surface enlargement factor, but indicated an ultrastructural larger surface enhancement of the microvilli following the HCD compared to the HFD. The mucosal epithelium also showed signs of increased mitotic activity after HCD compared to HFD, but no clear evidence of increased apoptosis was found. The ultrastructural investigation of the mitochondria showed an increased number of cristae in the mitochondria (indicating a larger metabolic activity) following the HFD compared to the HCD. Confocal microscopic assessment found the highest HMGCS2 count per mitochondrial marker in the tops of the jejunal villi following HFD compared to HCD. The study supports that there is a morphometric adaption in the jejunal mucosa following the two-week diets, which is not mainly on a histological level, but rather on the ultrastructural level.


MET oncogene benefit from treatment by specific inhibitors. Previously, we reported that discontinuation of MET tyrosine kinase receptor inhibition causes ‘rebound’ activation of the oncogene, with a post-treatment transient hyperphosphorylation phase that culminates into a dramatic increase in cancer cell proliferation. The molecular mechanisms behind the ‘MET burst’ after treatment cessation are unknown but critically important for patients. Here we identify a positive feedback loop mediated by the AKT/mTOR pathway leading to (a) enhanced MET translation by activating p70S6K and 4EBP1 and (b) MET hyper-phosphorylation by inactivation of the tyrosine-phosphatase PTP1B. The latter effect is due to m-TOR-driven PTP1B phosphorylation of the inhibitory residues Ser50 and Ser378. These data provide in vitro evidence for the use of mTOR inhibitors to prevent the ’flare effect’ in MET targeted therapy, with potential applicative ramifications for patient clinical management.Targeted therapy significantly impairs tumour growth but suffers from limitations, among which the ‘flare’ (‘rebound’) effect. Among cancers driven by tyrosine kinase receptors, those relying on alterations of the  MET, the receptor tyrosine kinase for hepatocyte growth factor, stands among the top five proteins for which targeted therapies have been developed with successful applications in the clinic2. Genetic alterations or gene amplification unleash MET-driven invasive growth in a large panel of cancer types3. Accordingly, kinase inhibitors or antibodies induced objective responses in patients with  MET-addicted tumours, including non-small cell lung cancer (NSCLC), lung squamous carcinoma, gastric cancer, colorectal adenocarcinoma, melanoma, gliomas, and renal cancer14. However, even in cases experiencing exceptional responses, therapeutic resistance inevitably ensues17. In current clinical practice, the line of treatment is dismissed when resistance arises. In many patients, discontinuation of kinase inhibitors results in rapid tumour regrowth: this phenomenon is known as disease ‘flare’ or tumour ’rebound’21. Its real incidence is poorly known, and the prognosis is dismal22. The biology and molecular mechanisms behind the ‘flare’ (rebound) phenomenon, either cell-autonomous or mediated by the tumour microenvironment, are largely unexplored. We previously found that abrupt termination of MET kinase inhibition unleashes the ‘flare’ effect23. In this paper, we provide the mechanistic explanation for this effect by describing a dual regulation of the MET burst: sudden discontinuation of the targeted therapy leads to restored kinase activity followed by the activation of the AKT/mTOR pathway, resulting in (1) enhanced translation of MET and (2) inactivation of the PTP1B phosphotyrosine-phosphatase, one of the major negative regulators of MET26. Cancer cells adopt this shrewd dual strategy to increase MET total protein quantity and MET phosphorylation (activation), leading to a rapid resumption of cancer cell proliferation. We demonstrate that the ‘flare effect’ is avoided by specific AKT or mTOR inhibitors and thus provide a proof-of-concept foundation for strategies improving MET targeted therapy.Targeted therapies were a breakthrough in cancer research, proving our ability to translate cutting-edge understanding of cancer cells’ vulnerabilities into the design of mechanism-specific treatment strategies. They significantly improved the disease-free survival time of patients with tumours dependent on the inhibited target (‘oncogene addiction’)30. However, inhibitor withdrawal causes rapid MET re-phosphorylation at levels higher than those at the steady state, with the subsequent reactivation of downstream pathways and increased proliferation of cancer cells23. Yet, the mechanisms of this phenomenon remain obscure. To enlighten what is behind the flare effect, we used two cell lines derived from two cancer cell types (EBC1 isolated from lung squamous carcinoma and HS746T from gastric carcinoma). These cell lines were carefully chosen to recapitulate the most common alterations of MET in cancer leading to MET ‘addiction’: gene amplification (EBC1 cells) and exon14 skipping (HS746T cells)31. Overnight JNJ-605 treatment abolished MET phosphorylation . The activation (phosphorylation) status of known MET transducers, i.e., P-AKT and P-ERK, was analysed. Remarkably, P-AKT levels were significantly enhanced after WO compared with the pre-treatment steady state, whereas no significant change was observed in P-ERK. The concomitant increase of P-MET and P-AKT after WO led us to hypothesise that the MET/AKT axis might be involved in the flare effect. Notably, the specific AKT inhibitor MK-2206 prevented MET hyperphosphorylation after WO  blocks proliferation in vitro and halts tumour growth in vivo34. mTOR phosphorylation levels were gradually increased after MET kinase reactivation following JNJ-605 inhibitor WO in both EBC1 and HS746T cells  in response to different stimuli25. These pieces of evidence prompted us to investigate the possible role of PTP1B in cells released from MET kinase inhibition. EBC1 and HS746T cells were treated overnight with JNJ-605 and released through a rapid washout. Concomitant with the peak of MET tyrosine re-phosphorylation, we detected a substantial increase in PTP1B serine phosphorylation on two residues (S50 and S378) both in EBC1 and in HS746T cells. The S352 was phosphorylated to a lesser extent after MET kinase inhibitor washout . We found that mTOR phosphorylation in patients was positively correlated with PTP1B phosphorylation, especially on S50 and S378 . PTP1B wild type (wt) or mutants were transfected in EBC1 or HS746T cells. Overexpression of PTP1B-wt reduced phosphorylation of MET. S378A further decreased levels of MET phosphorylated  40. It should be noted that the receptor accumulated at the cell surface is not phosphorylated, and therefore inactive, but potentially ready to be re-activated after inhibitor withdrawal40. Here, we show that the phosphatase PTP1B is inhibited by AKT-mTOR-dependent phosphorylation upon inhibitor withdrawal. PTP1B is central to MET regulation because it dephosphorylates the activating residues Y1234/1235 on the kinase domain, inhibiting all receptor functions. PTP1B can be phosphorylated in serine, which inhibits phosphatase activity41. After MET kinase inhibitor withdrawal, PTP1B is inhibited by AKT-mTOR-dependent phosphorylation. PTP1B-dependent MET tyrosine dephosphorylation is therefore impaired, and MET is hyper-activated. In particular, we observed that phosphorylation of serines 50 and 378 of PTP1B play a critical role in the process, as demonstrated by the use of PTP1B non-phosphorylatable mutants. Notably, AKT and mTOR inhibition both affect phosphorylation of PTP1B and blunt reactivation of MET after JNJ-605 washout, showing that the AKT-mTOR axis is part of a MET positive feedback loop.MET phosphorylation drives its oncogenic potential32. In this article, we demonstrate that the AKT/mTOR pathway controls total MET levels. Altogether, we describe the ‘flare’ effect as the consequence of a strategy of cancer cells enhancing both MET phosphorylation (activation) and MET synthesis.The AKT/MTOR pathway triggered by discontinuation of MET blockade is responsible for MET hyperphosphorylation and MET synthesis. Indeed, mTOR is a crucial regulator of translation42. This study shows its pivotal role during the ‘flare’ effect and its emergence as a promising target against tumour ‘rebound’. We demonstrate that AKT/mTOR activation follows the withdrawal of MET inhibitors, initiating a positive feedback activation of the MET pathway, responsible for the ‘flare’ effect. If these conclusions can be generalised, pulsatile treatments alternating tyrosine kinase and mTOR inhibitors could provide a promising strategy to hamper tumour growth, reducing the risk of disease ‘rebound’.The critical role of mTOR in cancer is well-knownThe pJ3H PTP1B plasmid was a kind gift from Ben Neel (Addgene plasmid number 8601). Q5® Site-Directed mutagenesis kit  was used following manufacturer’s instructions to generate Serine to Alanine mutations of PTP1B. Primers used for the mutagenesis reactions are listed in Supplementary Table EBC1  and HS746T  cell lines were purchased from indicated companies and were cultured respectively in RPMI and DMEM media supplemented with 10% FBS and 1% L-Glutamine. The medium was reefed every 3–4 days. All cells were regularly assessed for the absence of mycoplasma contamination. Cells were transiently transfected with Lipofectamine™ 2000 reagent following manufacturer’s instructions .5 cells in 6 cm dishes. Twenty-four hours later, the MET kinase inhibitor JNJ-38877605 (indicated as JNJ-605) was added at a concentration of 500 nM overnight (ON). Cells were washed three times with medium. After adding fresh medium, cells were left in culture for indicated durations. DMSO-treated cells (VEH) were processed as the JNJ-605-treated ones. When indicated, inhibitors were added to the medium immediately after the JNJ-605 washout. The different inhibitors used in this study are listed in Supplementary Table EBC1 and HS746T cells were plated at the concentration of 5 × 1031. Cell lysates were quantified using Pierce BCA Protein Assay Kit , and 15 µg of total proteins were resolved in polyacrylamide gels under denaturing conditions. After transfer, membranes  were blotted against antibodies listed in Supplementary Table Cells were lysed in Laemmli Lysis Buffer as previously describedCells were plated in chamber slides . Immunostainings were performed as previously described using antibodies listed in Supplementary Table Cells were plated in 96-well plates at a density of 5000 cells / well. After the ON JNJ-605 treatment, cells were washed as described above and treated with the indicated inhibitors. Cell viability was assessed using CellTiter 96® Non-Radioactive Cell Proliferation Assay  every day for eight days. The culture medium was renewed every three days.The publicly available TCGA dataset was used to assess PTP1B and mTOR phosphorylation levels in human cancers. On the day of analyses (June 2022), 97.3% of TCGA phosphoproteome data was based obtained from breast invasive carcinoma patients. For some patients, PTP1B and/or mTOR phosphorylation levels were unavailable and therefore not analysed. In the end, a cohort of 42 breast carcinoma patients was obtained. Patient information is available in Supplementary Table Supplementary Information 1.Supplementary Information 2.