Antimicrobial resistance has become a global health emergency and is contributed to by inappropriate antibiotic use in community clinical settings. The aim of this study was to evaluate the antimicrobial use pattern in infants from birth until 18 months of age in Indonesia.A post-hoc analysis was conducted in 1621 participants from the RV3BB Phase IIb trial conducted in Indonesia from January 2013 through July 2016. Any health events were documented in the trial as adverse events. Concomitant medication surveillance recorded all medications, including antibiotics during the 18 months of follow-up. Information included the frequency, duration of usage, formulation, classes, and their indications, including prophylactic antibiotic and perinatal use.Of 1621 participants, 551 (33.99%) received at least one antibiotic for treatment of infections during the 18 months observation period. Additionally, during the perinatal period, prophylactic antibiotics were used in 1244 (76.74%) participants and antibiotics consumed in 235 mothers of participants (14.50%). A total of 956 antibiotic consumptions were recorded for 18 months follow up, 67 (7.01%) as part of antimicrobial combinations. The average duration of antibiotic course was 4.92 days. Penicillin and sulfonamides were the most common antibiotic classes consumed .Despite the low community consumption rate, the overuse of antibiotic in URTIs and non-bloody diarrhea in our setting represents a major opportunity for antimicrobial stewardship, particularly in early life. Antibiotics are widely used among adults and children in the community. In children, antibiotics are considered a potential lifesaving treatment for bacterial infections. International guidelines for Integrated Management of Childhood Illness (IMCI) recommended antibiotic use to treat dysentery and acute respiratory tract infection . HoweverInappropriate use of antibiotics can also lead to antimicrobial resistance which is recognized as a serious global health threat . In infaAs the fourth most populated country in the world, Indonesia’s population in 2018 is 266 million people, with 29% under 15 years of age. Among the medications given to children, antibiotics are the most frequently prescribed therapy . Key to Studies of antibiotic use have been performed in diverse health-care settings and in community-based surveys. However, such studies are still very limited in Indonesia. In Indonesia, antibiotics require a prescription to be dispensed, however, previous studies have shown high rates of pharmacy provision of antibiotics without a prescription . The RV3NEJM.org). The complete study design of RV3-BB phase IIb trial has been described previously [The secondary data from a phase IIb randomized, double-blinded, controlled trial for RV3-BB rotavirus vaccine  was used in this study  and 2 hospitals in Sleman district (Yogyakarta province) as urban area and Klaten district  as rural area. After initial provisional maternal written informed consent in pregnancy, healthy neonates were enrolled in this study in less than 6 days of age. The inclusion criteria were children who were healthy, born full-term, weighed between 2500 and 4000 grams. Eligible participants were randomly assigned to one of the three groups with a 1:1:1 ratio. Randomization was completed based on a computer-generated code with a block size of 6, with stratification according to province. All investigators, trial monitors, data managers, statisticians, other trial staffs, and participants’ families remained blind to the trial-group assignments until the completion of the trial. At the central pharmacy in each province, pharmacist who was aware of the trial-group assignments drew the doses of RV3-BB vaccine or placebo for dispensing .The detailed explanation on sample size calculation has been described previously . In briePuskesmas , medications were also documented in medicals records. If the participants were hospitalised, medications given to them were extracted from their medical records. All the follow up data were inputted into an electronic case report form.Antibiotics as a part of concomitant medication were recorded from perinatal exposure from antibiotics given to the mother during the labour up to 18 months of age. Antibiotic use by participants’ mother during perinatal period in this study were recorded from 30 days to several hours prior to labour. After gave birth, during weekly follow up phone calls, research assistants would ask the mothers about the condition of the participants, including when there was any medication given to the participants from previous contact. Study midwives visited participants at their home monthly and monitored medications given to participants during the study by asking the mothers. If the participants were ill and visited Puyer, derived from “powder” in Dutch, contains medicines all ground together and has been commonly used for a long time in Indonesia, including for antibiotics [From the concomitant medications data, the information on antibiotics used by the study participants were extracted, both prescribed and non-prescribed antibiotics. Collected Information on antibiotics included antimicrobial type, class, dose, formulation such as oral , parenteral or topical, mode of administration, duration of course, and their indications. ibiotics . InformaAdverse event (AE) episodes were defined as all illnesses or symptoms occurred during the 18 months observation period following the first dose of RV3 vaccine or placebo was given at day 0–6 of life. Adverse events were classified using Medical Dictionary for Regulatory Activities (MedDRA). The final classification of morbidities was narrowed down to 13 disorders mainly according to their affected organ systems.This study received the ethical approval from Medical and Health Research Ethics Committee of Faculty of Medicine Universitas Gadjah Mada–Dr Sardjito General Hospital. Written informed consent from the parents or guardians of every child were already obtained during the RV3-BB phase IIb trial. The subjects who were included in this study were those who agreed that their data will be used for further studies.All statistical calculations were conducted using SPSS version 23. Results were presented as mean, median, frequency and percentages for descriptive data. Bivariate chi-square test and binary logistic regression of association was carried out to explore the relationship between gender, vaccination group and the number of AE episodes with antibiotic use. The independent t-test was conducted to compare the differences in antibiotic course duration per participant between males and females. One-way ANOVA was also performed to explore the effect of vaccination to the antibiotic course duration per participant.551 of 1621 (33.99%) participants received at least one therapeutic antibiotic during the 18-month observation period. Baseline characteristics of the study participants can be seen at A total of 956 antibiotic courses were recorded in 551 infants during a 18-month observation period, with an average of 1.74 antibiotic use per infant . The meaOral antibiotic formulations were used in 64.33% of all antibiotic consumptions, which consisted of syrups, pulveres/puyer, and unknown oral formulations. All parenteral antibiotics were administered intravenously. Most of antibiotic use was for outpatient therapy.Penicillin was the most common antibiotic class used for treatment , with 68.46% of penicillin utilization aimed to treat respiratory system disorders. Although mostly consumed as single antimicrobial (322/371), penicillin was the most frequent antibiotic class used in combination therapy (49/67), especially with aminoglycosides (22/49). The penicillin-aminoglycosides combination therapy was preferred in the pneumonia (6/22) and neonatal sepsis (5/22). The second largest antibiotic class was sulfonamides , overwhelmingly as cotrimoxazole and mostly indicated for treating gastrointestinal disorders (50.85%). The antibiotics and their usage indications are summarized in Aside of therapeutic indications, prophylactic usages of antimicrobials were also observed during the study. Single topical-prophylactic agents, including chloramphenicol, gentamycin, and tetracycline, were used in 76.74% (n = 1244/1621) participants directly after birth to prevent ophthalmia neonatorum (ON). None of the participants received more than one type of antibiotic prophylaxis during the study.There were 235 participants’ mothers (14.50%) who received antibiotics during the perinatal period with start dates ranged between 30 days to several hours prior to labour. Ceftriaxone, cefotaxime, cefadroxil, amoxicillin, and metronidazole comprised 98.30% of all perinatal antibiotic use in this study . None of the participants’ mother used more than one antibiotic during the observation period. All the prophylactic and perinatal use of antibiotics can be seen at One in three participants 33.99%) had at least one antibiotic used for treatment of infections within the 18 months observation period. This number was surprisingly low and may reflect a strength, especially since the study captured both prescribed and non-prescribed antibiotic use. Some studies in other developing countries showed much higher percentages of antibiotic consumption among children, including: India, Sudan, and Nigeria  –16. This9% had atHaemophilus influenzae type b (Hib), and pneumococcal conjugate vaccine (PCV). This may be due to the directly-diminished primary pathogens causing infections and also through the declining of febrile illnesses which often led to antibiotic prescriptions [Gender was significantly associated with antibiotic use in this study, with more male used antibiotic than female. This result was similar with other previous studies in which the male predominance in antibiotic prescription rate might reflect the difference of infectious disease burden between boys and girls in younger age ,8. Severriptions –20. Reporiptions ,20.The mean duration of antibiotic courses was 4.92 (±1.86) days per participants. This correlates well with a similar study of antibiotic use patterns in eight countries in South America, Sub-Saharan Africa and Asia . HoweverOf all antibiotics used in this study, two thirds contained oral antibiotic formulations. This is reasonable as oral is the preferred route for patients of all ages in terms of convenience . Syrup aAmong the numerous kinds of antibiotic recorded, penicillin was the most frequent class used, followed by sulfonamides. The most common reason (68.46%) for using penicillin was respiratory system disorders, while sulfonamides were mostly (50.85%) aimed for gastrointestinal disorders. Similarly, studies showed that penicillin was the most frequent antibiotic class used in children , especiaHowever, among adverse events that were treated by antibiotic, URTIs comprised 84.78% of the respiratory system disorders, whereas non-bloody diarrhea comprised 74.85% of the gastrointestinal disorders. These results were contrary to international guidelines for the treatment of childhood illnesses which recommended that antibiotic should not be routinely prescribed for non-bloody diarrhea and URTIs , 2, 31. Chloramphenicol utilization comprised 11.30% of all antibiotics and mostly aimed for ocular disorders. Since the dosing is not weight-adjusted, ocular treatment administration in children should be well-observed as they are at greater risk of systemic side effects, and drug metabolism is reduced in the immature blood-brain barrier . MacroliTopical ocular antibiotics were widely used among participants as the national guideline in essential newborn care requires all the newborns to receive these antimicrobials to prevent ophthalmia neonatorum (ON) ,35. AbouE.coli infections at birth [Almost 1 in 6 participants’ mother consumed antibiotic during the perinatal period. Recent study suggested an association between intrapartum ampicillin with the emergence of ampicillin-resistant at birth ,44. Moreat birth –47. TherConsidering a large sample size used in our study, the results might represent the antibiotic utilization in wider population of young children in Yogyakarta and Central Java provinces. However, different result might occur in different area in Indonesia. Many aspects could affect clinician’s decision making for prescribing antibiotics in children. Misinterpretation of parental concerns over their ill children and seeking of additional information, and lacking of time during consultation are the problems most commonly encountered . Thus, bIn Indonesia, although there are policies that only allow antibiotic purchase by prescriptions, antibiotics are still relatively easily obtained without a prescription. These behaviors may contribute to evolving antimicrobial resistance. Antimicrobial resistance is a complex global health issue, and no simple strategy will be enough to conquer it, because of that, the development and implementation of holistic strategies to restrict the emersion and spread of antimicrobial resistance are vital .Despite the low community consumption rate, the overuse of antibiotic in URTIs and non-bloody diarrhea among infants in this study indicated inappropriate antimicrobial use and further opportunities for education. Moreover, different classes of antibiotic were inappropriately used and may risk development of antimicrobial resistance. Training on rational use of antibiotic is needed to improve prescriptions behavior of practitioners in clinical settings.S1 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Extensive antibiotic use makes the intensive care unit (ICU) an important focus for antibiotic stewardship programs. The aim of this study was to develop a set of actionable quality indicators for appropriate antibiotic use at ICUs and an implementation toolbox, which can be used to assess and improve the appropriateness of antibiotic use in the treatment of adult patients at an ICU.A four round modified-RAND Delphi procedure was used. Potential indicators were identified by a multidisciplinary panel of 15 Dutch experts, from international literature and guidelines. Using an online survey, the identified indicators were rated on three criteria: relevance, actionability and feasibility. Experts discussed and rated the indicators for the second time during a face-to-face consensus meeting. During a final consensus meeting the toolbox was developed, containing potential barriers and improvement strategies which were identified using a validated checklist by Flottorp et al., and if available also containing supporting material.The first round resulted in 24 potential indicators. After the final meeting a set of three process indicators, one structure indicator and one quantity metric remained: 1) perform at least two sets of blood cultures before start of empirical systemic therapy; 2) perform therapeutic drug monitoring in patients treated with vancomycin or aminoglycosides; 3) perform surveillance cultures if selective digestive or oropharyngeal decontamination is applied at the ICU; 4) biannual face-to-face meetings between ICU and microbiology staff in which local resistance rates are discussed; and 5) quantitative antibiotic use at the ICU expressed in days of therapy (DOT). The toolbox contains 24 unique barriers and 37 improvement strategies.Our study identified a set of four actionable quality indicators and one quantity metric, together with an implementation toolbox, to improve appropriate antibiotic use at ICUs. Infection is a major cause of morbidity and mortality in critically ill patients, resulting in a high percentage of patients using antibiotics in intensive care units (ICUs), up to 71% . ASPs arA requirement for an effective stewardship program is the ability to measure the appropriateness of antibiotic use. Quality indicators are defined as measurable elements designed to evaluate aspects of quality of care . PreviouIndicator scores can be used to develop tailored interventions. Tailored interventions are designed to achieve improvements in healthcare, based on the assessment of local barriers in clinical practice . SystemaThe aim of this study was to develop a set of actionable quality indicators and an implementation toolbox, which can be used to assess and improve the appropriateness of antibiotic use in the treatment of adult patients at an ICU.We performed a modified-RAND Delphi procedure, consisting of four rounds, to develop quality indicators and a toolbox for appropriate antibiotic use in the treatment of critically ill patients at an adult ICU –21. An oFirst, we performed an inventory of potential indicators among a panel of Dutch experts. The multidisciplinary panel consisted of fifteen experts: three anesthesiologists-intensivists, three internist-intensivists, one intensivist-infectious diseases physician, three internists-ID (infectious diseases) physicians, two clinical microbiologists and three clinical pharmacists. The panelists were invited based on their (inter)national involvement in guideline development, working groups or societies regarding antibiotic use or intensive care  foundation, the Dutch Working Party on Antibiotic Policy (SWAB) or the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)). They represent nine university and non-university hospitals. The experts were instructed to individually propose potential quality indicators representing appropriate antibiotic use, based on their knowledge and expertise in the field. In addition, we performed a systematic literature search in Medline to identify already available indicators on appropriate antibiotic use. The search included all articles available in Medline up to January 2014. The search was updated in April 2017, which showed no additional indicators relating to antibiotic use at the ICU since 2014. The search strategy is listed in In the second round we converted the list with potential quality indicators into an online survey and instructed experts to appraise each indicator. Based on a 9-point Likert scale , the experts independently rated the potential indicators on two criteria: 1) relevance, the impact of the indicator on disease or on healthcare expenditure and 2) actionability, the extent to which an indicator offers direction for improvement in clinical practice. Indicators with a median score between 4–9 on both relevance and actionability were defined as potentially suitable and indicators with a median score between 1–4 were defined as not suitable . We selest 2015, we presented the results of the second round to the expert panel. All 15 experts from round 2 were invited, and 10 of them were available at the selected meeting date. The indicators were discussed, rephrased, merged and rated for the second time based on three criteria: 1) relevance, 2) actionability and 3) feasibility of data collection, the ability to use routinely collected electronic data. In addition, the experts appraised each indicator with a validity score, reflecting whether the indicator is associated with and appears to measure quality of antibiotic use in clinical practice . A blinded survey tool was used to support independent rating. After the second rating all indicators with median scores of 7–9 on relevance, actionability and feasibility without disagreement (i.e. 80% of the assessment rates were within the range of the median scores of 7–9), together with a validity score in the highest quartile of all validity scores, were selected  did not apply to this study, since the study subjects were to receive treatment according to standard care and had no burden of the study (August 2016).The expert panel proposed in the first round 31 unique quality indicators for appropriate antibiotic use, based on their clinical experience. The literature search resulted in 133 scientific articles. Based on predefined exclusion criteria 105 articles were excluded. The remaining 28 articles were selected for full-text screening. Two additional articles were selected from literature references and added to the final list. Thirteen indicators were extracted from these studies. Seventy-eight indicators were extracted from six guidelines regarding antibiotic therapy in critically ill patients . After dThe panel members rated the 24 potential indicators on their relevance and actionability. All indicators were considered potentially suitable and were therefore selected for the next round .During a three-hours consensus meeting all 24 indicators were discussed. As a result, three indicators were rejected, eleven indicators were rephrased and seven interrelated indicators were merged into three indicators. One indicator was considered a quantity metric rather than a quality indicator, as this metric provides relevant context to the other indicators despite the absence of a target value. Sixteen resulting unique indicators and the quantity metric were rated for the second time. Thirteen indicators and the quantity metric received a median score of 7 or higher on relevance, actionability and feasibility, of which four indicators and the quantity metric received a validity score in the highest quartile , Table 2This resulted in a final set of three process indicators, one structure indicator and one quantity metric: 1) perform at least two sets of blood cultures before start of empirical systemic therapy; 2) perform therapeutic drug monitoring in patients treated with vancomycin or aminoglycosides, within 48 hours after start of antibiotic therapy; 3) perform surveillance cultures if selective digestive or oropharyngeal decontamination is applied at the ICU; 4) biannual face-to -face meetings between IC staff and microbiology staff in which local resistance rates and trends in the ICU population are discussed; and 5) quantitative antibiotic use at the ICU expressed in days of therapy  . TargetsThe implementation toolbox shows for each indicator a list of potential barriers in the antibiotic care process, and associated improvement strategies to overcome these barriers. Barriers can be applicable to more than one indicators and improvement strategies can be applicable to more than one barrier. A total of 24 unique barriers and 37 unique improvement strategies were identified and grouped into four categories: A) barriers related to the guidelines, e.g. the local antibiotic guidelines are inadequate or incomplete; B) barriers related to the individual health care professional, e.g. health care professionals are not (sufficiently) familiar with the antibiotic protocol; C) barriers related to professional interactions, e.g. there is insufficient communication within ICU teams; D) barriers related to incentives and resources, e.g. there is a lack of culture media bottles at the ICU ward. Supporting materials were provided for eight of the unique improvement strategies .In this study we used a modified-RAND Delphi procedure to systematically develop a set of four actionable quality indicators and one quantity metric for appropriate antibiotic use in adult ICUs. In addition, we developed an implementation toolbox, containing a list with possible barriers that lead to poor performance on the selected indicators, and a list with improvement strategies to overcome these specific barriers, which can be used to support stewardship actions to increase performance on antibiotic use.A quantity metric for total antibiotic use was selected next to the four quality indicators, as the experts found it important to include this metric that enables benchmarking and provides context to the other indicators despite the absence of a specific target value.Previous studies have described several quality indicators for ICUs –26. De VThe use of quality indicators to measure the appropriateness of antibiotic use requires extensive data collection from patient records, which is labor intensive . Since tNot every clinical setting needs the same level of improvement. Improvement strategies should therefore be tailored, depending on the local barriers in clinical practice. , 28 LiteOur study has several strengths. First, we used a modified-RAND Delphi approach, a systematic rigorous procedure in which scientific evidence and expert opinions are combined. , 19, 20.th 2017, which showed no additional indicators relating to antibiotic use at the ICU since 2014. Third, we did not involve ICU nurses or patients in the expert panel. In the Netherlands antibiotic agents are solely prescribed by doctors. We realize that nurses are increasingly involved in antimicrobial stewardship and should be included in future expert panels. Patients were not involved in our expert team as this was logistically challenging, and also given the heterogeneity of reasons for ICU admission. Fourth, while some of our indicators have been described before and are applicable to general wards as well as to the ICU, they were selected because they were considered particularly relevant to the ICU setting based on literature and expert opinion, because they offered direction for improvement in clinical practice and because data collection was considered feasible. Moreover, indicator 3 and 4 are specific for the ICU setting only and have not been described as quality indicators before. Finally, although our indicators have been developed in a national setting, our method of development was according to current international standards and as the indicators were based on international literature, guidelines and consensus of a multidisciplinary team of experts with international experience in their field, we believe that the resulting indicators are generalizable to other ICU settings. Furthermore, one is able to select indicators tailored to one’s own healthcare setting (e.g. some countries do not apply SDD).Our study also has some limitations. First, for pragmatic reasons the literature search was performed only in Medline. However, we used a wide range of search terms regarding quality measurements and antibiotics and we therefore assume that the terms should identify those studies reporting on quality indicators. Second, the Medline search was performed in 2014 when the project started. The search was therefore updated on April 5The results of our study are a first step towards the use of a new set of actionable quality indicators, together with an action implementation toolbox, to monitor and improve the appropriateness of antibiotic use in adult ICUs. The toolbox will support professionals in selecting improvement strategies that work best in their setting, based on careful assessment of local barriers. Clinimetric properties of the indicators and feasibility in daily practice of electronic data reuse from the EHR or PDMS will be tested during an evaluation study using time series analysis.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


This study aimed to retrospectively assess the clinical outcome with nitrofurantoin and pivmecillinam for lower urinary tract infections (UTI) in men. Patients treated with trimethoprim were also included for comparison.All prescriptions of the study antibiotics to adult men in Uppsala County, Sweden, during 2012 were extracted. Data on patient characteristics, therapy, clinical outcome and microbiological results were obtained from the electronic medical records. The relative impact of antibiotic therapy, patient factors and pathogens on clinical outcome was assessed with univariate logistic regression using a 95% confidence interval (CI).P = 0.022) and prostate cancer was associated with a higher incidence of relapse .832 prescriptions were identified, and 171 patients treated with nitrofurantoin (n = 69), pivmecillinam (n = 57) and trimethoprim (n = 45) met the inclusion criteria. Treatment failure occurred in one patient treated with nitrofurantoin and in four patients treated with pivmecillinam. New prescriptions of UTI antibiotics and relapse within 3 months after completion of therapy were more frequent with nitrofurantoin (34% and 15%) and pivmecillinam (30% and 17%) than trimethoprim (22 and 7%). However, these differences were not statistically significant and substantial heterogeneity was noted between the treatment groups. Urinary tract catheterization was associated with a higher risk for new antibiotic prescriptions (OR 2.34, 95% CI 1.14–4.80; Escherichia coli and limited impact on the intestinal microbiota.The clinical outcome with nitrofurantoin and pivmecillinam was acceptable in comparison with the results of previous studies. These antibiotics are suitable for empirical treatment of lower UTI in men considering their high activity against  Escherichia coli [E. coli are more commonly isolated and resistance levels are generally higher than in other patient groups [Oral treatment options for urinary tract infections (UTI) are increasingly limited due to emerging resistance and the lack of new antibiotics , 2. Resihia coli –5. Lowerhia coli . PathogeE. coli [As a response to the increasing resistance to standard treatment, old antibiotics that are still effective have been revisited in recent years –10. NitrE. coli , 11, alsE. coli , 8, whilE. coli isolates were resistant in 2012 according to national surveillance data) [E. coli (c. 20%) [E. coli isolates in Sweden [The increasing resistance rates are considered in the revised guidelines for UTI provided by the Swedish Society of Infectious Diseases published in 2013 . CiproflIn this study, we describe patient characteristics, medical conditions, causative pathogens, treatment regimens and clinical outcome in male patients prescribed nitrofurantoin and pivmecillinam for lower UTI in Uppsala County, Sweden, during 2012. Data on patient characteristics, treatment, microbiological results and outcome were extracted through retrospective review of the electronic medical records and were obtained also for patients treated with trimethoprim during the same period to allow a comparison between the three drugs.All prescriptions of nitrofurantoin, pivmecillinam and trimethoprim to adult men during 2012 were identified through the electronic medical records database of Uppsala County. Data on patient characteristics, antibiotic treatment, clinical outcome and microbiological results within a 3-month follow-up period were extracted. The study was approved by the Regional Ethical Review Board of Uppsala and was conducted in accordance with the Declaration of Helsinki. All data were analysed anonymously and informed consent was waived due to the retrospective observational design of the study.3 colony forming units, cfu/mL) pure culture growth of bacteria in urine and (4) susceptibility of the isolated bacteria to the prescribed antibiotic. Exclusion criteria were (1) concurrent therapy with other systemic antibiotics, (2) suspected prostatitis, (3) other indication than UTI, (4) antibiotics intended as prophylaxis, (5) death during follow up, (6) incomplete or lacking medical records and (7) fever > 38°C after onset of symptoms. Patients with prostatitis were not included because they usually receive trimethoprim for 3–12 weeks, which is much longer than the normal treatment duration for lower UTI (7 days).Inclusion criteria were (1) age ≥18 years, (2) at least one of the following conditions: dysuria, frequency or urgency of micturition, suprapubic pain, urinary retention or haematuria, (3) significant  a new prescription of antibiotics within 3 months after completion of therapy, (2) recurrent urinary tract symptoms and (3) isolation of the same bacterial species with a similar antibiotic susceptibility profile as in the first episode.Enterobacter sp., Citrobacter sp. and Serratia sp. was tested and interpreted as recommended for E. coli in accordance with local guidelines during the study period.Microbiological results as reported from the Department of Clinical Microbiology at Uppsala University Hospital were extracted from the medical records. Bacterial cultures and species determination were performed using either conventional methods or a Vitek 2 instrument . Antibiotic susceptibilities were determined with disk diffusion or the gradient method  and interpreted according to EUCAST definitions . SusceptP< 0.05 (two-sided). The statistical analysis was performed using R version 3.3.2 .Patient characteristics are described with median (range) for numerical variables and with numbers and percentage for categorical variables. Outcome in relation to antibiotics, duration of therapy, potential risk factors and Gram-negative vs. Gram-positive bacteria was assessed with univariate logistic regression. The results are presented with odds ratios (OR) and 95% confidence intervals (CI). The association of relapse and duration of treatment was explored with antibiotic type as an additional covariate with a log likelihood ratio test comparing the full model with an interaction term (antibiotic type and duration) and the smaller model without the interaction term. Subgroup analyses to evaluate the risk of relapse with different treatment durations (≤ 7 vs. > 7 days) were performed using Fisher´s exact test. Statistical significance was set at During 2012, 832 prescriptions of nitrofurantoin, pivmecillinam and trimethoprim were made to adult male patients in Uppsala County. 171 patients were eligible for inclusion in the analysis. The most common reasons for exclusion were a lack of documented urinary tract symptoms (n = 153) or urinary cultures (n = 164), as shown in E. coli was the most frequent pathogen (49%), followed by Enterococcus faecalis (23%), which was predominant in patients treated with nitrofurantoin. More than 95% of E. coli isolates were susceptible to nitrofurantoin and pivmecillinam, whereas 31% were resistant to ciprofloxacin and trimethoprim  were reported to produce ESBLs.Notable differences existed in causative bacteria between the treatment groups . Most isethoprim . Of the The median duration of therapy was 7 days in the nitrofurantoin and pivmecillinam groups, in accordance with current guidelines, and 10 days for trimethoprim . Five daTreatment failure was observed in one patient with diabetes mellitus treated with nitrofurantoin  who had P = 0.204) and pivmecillinam  than with trimethoprim (22%). However, these differences were not statistically significant. Treatment duration > 7 days was not associated with a reduced risk . A higher incidence of new prescriptions was noted in patients with urinary tract catheters  (New antibiotic prescriptions for suspected recurrent UTI were made in 49 of the patients Tables . Two pat= 0.022) .P = 0.209) or pivmecillinam  than with trimethoprim (7%). These differences were not statistically significant. Overall, duration of therapy > 7 days did not result in a lower risk for relapse. However, in the subgroup of patients treated with pivmecillinam a longer treatment duration was associated with a significantly lower risk; none of the 18 patients treated for more than 7 days relapsed (17 patients were treated for 10 days and one patient for 14 days), as compared to nine of 35 (26%) patients treated for ≤ 7 days (P = 0.045). No association was found between the dosage of pivmecillinam and relapse (16% with 200 mg q8h and 12% with 200 mg q12h). Prostate cancer was associated with a significantly higher risk for relapse   .E. coli; four of 23 (17%) of patients treated with nitrofurantoin, eight of 41 (20%) of patients treated with pivmecillinam and one of 20 (5%) patients treated with trimethoprim. In patients infected by ESBL-producing E. coli one of nine patients treated with nitrofurantoin relapsed and treatment failure or relapse were recorded in two of three patients treated with pivmecillinam. All 40 patients infected by E. faecalis were prescribed nitrofurantoin and relapse was noted in six cases (15%). No subgroup analysis was performed for other bacterial species due to the low number of cases.Relapse occurred in 13 of 84 (15%) infections caused by E. faecalis probably due to the selection of patients prescribed nitrofurantoin. Resistance to ciprofloxacin and trimethoprim in E. coli was 31%, which is higher than reported national data  [E. coli were noted for nitrofurantoin and pivmecillinam.In this study, prescriptions of nitrofurantoin, pivmecillinam and trimethoprim were reviewed retrospectively using strict criteria for inclusion and outcome. As expected, older age, recent antibiotic treatment and presence of urinary tract abnormalities and catheters were frequent in the study group. The causative bacteria were also in line with previous reports , 15 but ctively) . This fiThe overall clinical response was comparable to previous studies considering that risk factors for recurrent infections were present in most patients. Treatment failure was noted in 3%, a new antibiotic prescription in 30% and relapse with repeated bacterial growth within 3 months in 13% of the patients. In a recent study on the efficacy and safety of nitrofurantoin in male patients with lower UTIs, clinical cure was reported in 77% of the cases . Still, A large proportion of patients did not meet the inclusion criteria resulting in a small sample size in each treatment group included in the analysis. Inappropriate antibiotic prescriptions based on isolation of bacteria in urine despite a lack of urinary tract symptoms  were frequent. Urinary cultures, which are always recommended in these patients according to national treatment guidelines because of the high risk for resistant bacteria, were performed in only 71% of the cases. In this study, the presence of a urinary tract catheter and prostate cancer were the only statistically significant risk factors for new antibiotic prescriptions and relapse. Our results further support the recommendation to remove urinary tract catheters when possible to reduce the risk for UTI . ScreeniThis study has several limitations, including the small sample size, multiple testing, the retrospective study design associated with a risk of bias in the selection of antibiotic therapy and differences between the treatment groups. The great variation in bacterial species between the groups is an important limitation, which may have influenced clinical outcome. A shorter duration of therapy was noted for patients treated with nitrofurantoin or pivmecillinam, which may have increased the risks of treatment failure and relapse. Several potential risk factors that were unevenly distributed between treatment arms (less frequent in the trimethoprim group) may also have affected clinical outcome although they were not found statistically significant in this study. Because of the low number of events, possible confounders could not be explored. Symptoms, bacterial growth and relapse rates may have been underestimated and some infections misclassified as lower UTI rather than prostatitis or pyelonephritis because of the lack of standardised data collection and follow up. Furthermore, new prescriptions of UTI antibiotics during follow up, used as an indicator of suspected relapse, may in some cases have reflected recurrent infections, other indications for antibiotic therapy or unnecessary treatment for asymptomatic bacteriuria.Therefore, our results should be interpreted with caution and the results regarding treatment regimens are not conclusive. Still, due to the paucity of other data we believe that our study provides new important information of interest in the management of these patients. The strict criteria for inclusion and relapse and the consecutive inclusion of all patients in the region based on reliable data on individual prescriptions, are strengths of this study in relation to many previous publications on lower UTI in men.In absolute numbers, clinical outcome was worse in the nitrofurantoin and pivmecillinam groups. Use of these antibiotics has previously been discouraged for the treatment of UTIs in men because of their pharmacokinetic properties resulting in low antibiotic concentrations in prostate . In thisE. coli and favourable ecological profiles, the high resistance levels to trimethoprim and the need to preserve quinolones and other key antibiotics for more severe infections. However, individual assessment of patient-specific risk factors for resistant bacteria is always required. Prospective randomized studies with a larger sample size are needed to establish the potential differences in efficacy between antibiotics, dosage regimens and treatment durations for these infections.We conclude that nitrofurantoin and pivmecillinam are suitable for empirical treatment of lower UTI in men considering their high activity against


To curb antibiotic over-prescriptions, improving knowledge itself is not enough. The lack of motivation of physicians to change needs to be addressed through a systematic approach.The aim of this paper is to measure the knowledge and attitudes of primary care physicians toward antibiotic prescriptions and their impacts on antibiotic prescribing. A questionnaire survey was conducted on 625 physicians from 67 primary care facilities in Hubei, China. Structural equation modelling (SEM) was applied to test the theoretical framework derived from the Knowledge, Attitudes, and Practices (KAP) theory. Physicians’ knowledge, five sub-types of attitudes, and three sub-types of behavioral intentions towards antibiotic use were measured. Physicians had limited knowledge about antibiotic prescriptions (average 54.55% correct answers to 11 questions). Although they were generally concerned about antibiotic resistance , and were reluctant to be submissive to pressures from consumer demands for antibiotics  and the requirements of defensive practice , there was a lack of motivation to change prescribing practices  and strong agreement that other stakeholders should take the responsibility . The SEM results showed that poor knowledge, unawareness of antibiotic resistance, and limited motivation to change contributed to physicians’ high antibiotics prescriptions ( Antibiotic resistance (AR) has become one of the most serious global issues of concern for health development today, threatening our ability to treat common infectious diseases . AR can Physicians play a critical role in the global campaign against AR, simply because prescriptions are required for antibiotic usage ,12. It iChina has the largest consumer population for antibiotics in the world. Meanwhile, the overuse of antibiotics is also prevalent in China. Inappropriate and over-prescription of antibiotics are widespread across the entire health industry, whether in hospitals or in primary care facilities. Over half of the vast volume of patient visits (43.67 billion in 2016 ) involveThis study was conducted in Hubei Province of China, with a focus on primary care facilities. In China, about 60% of outpatient visits occur in primary care facilities . Hubei iThis study covered both urban community health centers (CHCs) and rural township health centers (THCs). There are 342 CHCs and 1139 THCs in Hubei. In 2016, they received 20.44 million and 58.00 million patient visits, respectively . A studyComplacency: prescribing antibiotics to satisfy patient demands and expectations;Fear: prescribing antibiotics for fear of losing patients or losing in potential disputes with patients;Ignorance: a lack of concern in relation to antibiotic resistance resulting from over-prescriptions of antibiotics;Indifference: a lack of motivation to change antibiotic prescribing practices; andResponsibility avoidance: a belief that others  are responsible for the problem of antibiotic resistance.We adapted the theoretical model proposed by Teixeira . The modThe model hypothesized that knowledge can directly link to prescribing practices. Meanwhile, the impacts of knowledge on antibiotic prescriptions can also be enhanced or jeopardized by the above five aspects of attitudes. In addition, the personal characteristics of prescribers may also exert some influence on certain knowledge, attitudes, and prescribing behaviors .A 54-item questionnaire  was deveKnowledge was measured using 11 questions, asking the respondents to make a judgment on the circumstances in which antibiotics (or a type of antibiotic) should or should not be prescribed. Eight of these questions were borrowed from previous studies conducted in Peru, DR Congo, and Laos ,32,33. WAttitudes were measured using 27 items along a five-point Likert scale, with each subscale containing a minimum of three items. These items were adapted from two validated instruments ,37, takiIntentions to prescribe antibiotics were measured in this study as a proxy indicator for prescribing practices. Such a strategy has been successfully applied in previous studies ,40,41,42The personal characteristics captured in this study included age, gender, education, income, medical sub-department they worked at, job title, years of practice, and training in relation to antibiotics. n = 625) through confirmatory factor analysis (CFA) and Cronbach’s alpha. The CFA results demonstrated an excellent fitness of data into the hypothetical model [The development and modification of the questionnaire followed the guidelines of the KAP survey  and the al model ,48: rootal model . HoweverA stratified cluster random sampling strategy was adopted. Hubei Province is geographically divided into western, central, and eastern regions. We estimated that at least 17 clusters with a sample size of 167 responses would be required in each region using the sample size calculator developed by Dhand and Khatkar  . In eachIf less than 70 physicians were identified from a city or county, an additional primary care facility (if available) was added to the sample. This resulted in a final sample of 67 primary care facilities, including 19 urban CHCs and 48 rural THCs . Data were collected over the period from 23 April to 6 June in 2018. Each facility was visited by a pair of trained investigators . The recruited investigators have learned theories and methods of social investigation from their postgraduate courses and received a one-day intensive training, covering the background of the current survey, detailed interpretation of survey instrument, and a simulation survey test. Finally, a total of 10 investigators were recruited. They approached all of the physicians on duty, but only those who prescribed antibiotics were invited to participate in the survey. The physicians working in the administrative departments and those whose tasks rarely involved antibiotic prescriptions  were excluded from the survey. The investigators explained the purpose and procedure of the study and obtained written informed consent from each respondent before the respondent was asked to self-complete the questionnaire. On average, the survey took 10–15 min to complete. The completeness of the returned questionnaire was examined by the investigators, with missing items (if existing) being amended through complementary interviews on the spot. A token gift (roughly $1.65) was given to the participant upon completion of the survey. In total, 712 questionnaires were distributed and 664 were returned. Of the returned questionnaires, 625 contained no missing items and were included for further analyses. This represented an effective response rate of 87.78%. Knowledge about antibiotic prescriptions was assessed using 11 questions. The percentage of respondents giving a correct answer to each question and the total number of correct answers per respondent were calculated. Each attitude item was coded along a five-point Likert scale, with a negative score indicating disagreement and a positive score indicating agreement with the relevant evidence-informed good practice. The scores in the same sub-domain were added and averaged (ranging from −2 to 2). Prescribing behavioral intentions were assessed using three indicators: percentage of prescriptions containing antibiotics for URTIs, average score for the efforts to prescribe antibiotics, and average score for the efforts to reduce antibiotic prescriptions. The latter two were coded in a similar way as the attitude measurements , Kruskal–Wallis rank tests, or one-way analysis of variance (ANOVA). p < 0.05). The fitness of data into the SEM model was assessed using several recommended criteria [Structural equation modelling (SEM) was applied to establish the associations between knowledge, attitudes and practices . Means acriteria ,48: RMSEp-value < 0.05 was considered statistically significant.The statistical analyses were performed using STATA (version 12.0)  and Mplus (version 6.0) . A The 625 respondents had a mean age of 43.27 years  and most (69.76%) were male. The majority (78.08%) of respondents came from rural THCs. On average, the respondents had 16 years of clinical experience and over three quarters (76.32%) had received training in relation to antibiotics over the last year prior to the survey . p < 0.001).On average, the respondents answered six questions correctly (SD = 1.46) of a total of 11 . IncorreOn average, the respondents reported a positive attitude toward rational antibiotic prescriptions in response to pressures from patient expectations  and the requirements of defensive practice . There was a relatively high level of concern about antibiotic resistance resulting from over-prescriptions . However, a shortage of motivation in changing antibiotic prescribing practices was evident: a negative score (−0.29) was shown in indifference (SD = 0.70). The respondents were inclined to believe that the solution to antibiotic resistance went beyond their own responsibilities  . The respondents reported that they would prescribe antibiotics to about 40% (SD = 22%) of patients with URTIs. However, a relatively strong intention to reduce antibiotic prescriptions  was reported, compared with the intention to prescribe antibiotics .p = 0.002).The attitudes and behavioral intentions toward antibiotic prescriptions were consistent across different subspecialties, except for the gynecologists and Chinese medical practitioners who reported less pressure of defensive practice (fear) compared with other sub-specialties , less fear , and less ignorance , but not less indifference. Knowledge had indirect effects on intentions to prescribe antibiotics through the attitude of ignorance. However, the attitudes of complacency and fear were not linked to intentions to prescribe antibiotics (p > 0.05). Poor antibiotic knowledge was also directly linked to the intention to prescribe antibiotics for URTIs .Overall, a high level of knowledge was associated with a more positive attitude and behavioral intention for containing antibiotic prescriptions. Higher knowledge scores were found to be linked with less complacency  and high intentions to reduce antibiotic prescriptions . Intentions to reduce antibiotic prescriptions were also associated with the motivation of behavioral changes .A high level of concern about antibiotic resistance (attitude of ignorance) was linked with low intentions to prescribe antibiotics , gynecologists  and Chinese medical doctors  had poorer knowledge about antibiotic prescriptions; but the latter two were less submissive to pressures from patient demands and defensive practice  and lower intentions  to prescribe antibiotics. Higher income was associated with higher intentions to prescribe antibiotics . Increased years of clinical practices was associated with higher intentions to reduce antibiotic prescriptions . Antibiotic training appeared to be associated with better knowledge  antibiotics. Better knowledge may ease the pressure of antibiotic prescriptions resulting from patient demands or defensive practice. However, this may not eventually result in a reduction of antibiotic prescriptions. On the other hand, knowledge bears no connection with the motivations of physicians for behavioral changes, in spite of the importance of motivations for changing prescribing practices. One potential pathway for knowledge to play a role in changing prescribing practices, however, is to promote the acknowledgement of the importance of prescribing practices in curtailing antibiotic resistance. This study revealed low levels of antibiotic knowledge in the study participants. The respondents scored on average 55% of correct answers about antibiotic prescriptions (6.04 out of 11 questions), compared with 60–86% for physicians in hospitals from Lao, DR Congo, and Peru. This knowledge gap is even larger in antibiotic treatments for URTIs: 5% versus 35–76% ,32,33,34Training can contribute to knowledge acquisition, as indicated in the SEM results. However, the current training programs may have contributed little, if any, to the improvement of the antibiotic knowledge of physicians. Over three-quarters of respondents in this study reported experience of training in relation to antibiotics. However, their antibiotic knowledge level remained low. Since 2009, China has made great efforts in training primary care workers . HoweverAttitudes can undermine practices. We found that our study participants reported highly positive attitudes in dealing with pressures from consumer demands for antibiotics and the requirements of defensive practice. However, these attitudes are not connected with intentions to prescribe (or not to prescribe) antibiotics. A survey in Shandong revealed a similar result, in which 88% of physicians would refuse to prescribe antibiotics they considered unnecessary even when patients insisted on it . HoweverThe attitudes of ignorance and indifference were found to be associated with antibiotic prescribing intentions. The study participants had already demonstrated quite a positive attitude toward acknowledging the negative consequences (antibiotic resistance) of over-prescriptions, although it may be further strengthened through better knowledge. Therefore, the greatest challenge may lie in the lack of motivation of physicians to change practices as indicated by the negative average score in the attitude of indifference. Indifference may lead to low intentions to reduce antibiotic prescriptions. The current high level of antibiotic prescriptions is likely to continue if the motivation issue is left unaddressed, especially when high workloads are common in physicians . HoweverIt is a serious issue of concern that primary care physicians may find excuses for not taking responsibility themselves in fighting antibiotic resistance. There was an overwhelming belief by the study participants that other people are responsible for antibiotic resistance. However, empirical evidence shows that antibiotic prescribing in primary care has contributed significantly to the development of antibiotic resistance . China iThe campaign for reducing over-prescriptions of antibiotics should take a systems approach, addressing problems associated with both the knowledge and attitudes of prescribers. Training programs and practice guidelines should target major gaps in the knowledge of prescribers , involving not only general practitioners but also other sub-specialists, such as surgeons and gynecologists ,64. GreaThis study adopted a SEM approach to test the theoretical framework based on KAP, which enabled us to explore multiple factors associated with antibiotic prescriptions . This fiThere are several limitations in this study: (1) The study was conducted in primary care facilities in one province (Hubei) of China. Attempts to generalize findings of this study should be cautious. (2) We measured behavioral intentions instead of practices. This may result in an overestimation of the tested effects since intentions do not always translate into practices. (3) Responsibility avoidance, an important construct of attitudes, was not included in the SEM simply because of its low Cronbach’s α. Further studies are warranted to explore the underlying reasons. Some researchers recommend separate measurements of attitudes toward the responsibilities of different stakeholders  ,37. (4) Physicians in primary care facilities in Hubei have low levels of knowledge about antibiotic prescriptions. This is connected with a high level of antibiotic prescriptions for URTIs in particular. These physicians are concerned about antibiotic resistance resulting from over-prescriptions of antibiotics. Generally speaking, they are reluctant to be submissive to pressures from consumer demands for antibiotics and the requirements of defensive practice. However, there is a lack of motivation to change prescribing practices although high levels of antibiotic prescriptions are evident in China. There is a tendency to shift responsibilities to other stakeholders. Improving knowledge may lead to higher motivation and result in fewer antibiotic prescriptions. However, responsibility avoidance can be a serious barrier for mobilizing health professionals, which should be addressed through a broad systems approach that goes beyond training and practice guidelines.


MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p < 0.001). Furthermore, MET mutations occur at high allele frequency and in the presence of co-occurring TP53 mutations (n = 7), as well as MDM2 (n = 7), CDK4 (n = 6), and HMGA2 (n = 5) co-amplifications. No other potential driver mutation has been detected. Conversely, in the MET-amplified group, we identify co-occurring pathogenic NRAS and KRAS mutations (n = 5) and a significantly higher number of TP53 mutations, compared with the MET-mutant cohort (p = 0.048). Of note, MET amplifications occur more frequently as subclonal events. Interestingly, despite the significantly (p = 0.00103) older age at diagnosis of stage IIIb/IV of MET-mutant patients (median 77 years), compared with MET high-level amplified patients (median 69 years), MET-mutant patients with advanced-stage tumors showed a significantly better prognosis at 12 months (p = 0.04). In conclusion, the two groups of MET genetic alterations differ, both clinically and genetically: our data strongly suggest that MET exon 14 skipping mutations represent an early driver mutation. In opposition, MET amplifications occur usually in the background of other strong genetic events and therefore MET amplifications should be interpreted in the context of each tumor's genetic background, rather than as an isolated driver event, especially when considering MET-specific treatment options.Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a  ALK), epidermal growth factor receptor (EGFR), ROS1 proto-oncogene receptor tyrosine kinase (ROS), and RET proto-oncogene receptor tyrosine kinase (RET) were introduced [Lung cancer is the leading cause of cancer deaths in women and men with more than 230.000 expected new cases and an estimated 150.000 cancer-related deaths in the United States in 2018 [troduced –10, the troduced –14. Overtroduced , 15, 16.troduced , 17, 18.MET alterations are described in about 5% of non-small-cell lung cancer cases [MET alterations alone are sufficient to drive carcinogenesis [MET alterations include copy-number gains and amplifications, as well as single-nucleotide variants, insertions/deletions (indels) being the so-called MET exon 14 skipping mutations [MET high-level amplification, mostly defined as at least ten gene copies per cell [The MET proto-oncogene receptor tyrosine kinase and its ligand, the hepatocyte growth factor were first characterized in the mid-1980s and their involvement in non-small-cell lung cancer tumorigenesis was first described in the 1990s , 19–21. er cases , and it ogenesis . MET altutations –26. At tutations , 28. PatMET exon 14 skipping mutations or MET high-level amplifications [As MET inhibitors were developed to treat both, patients with either ications , we aimeMET amplifications with gene copy ≥ 10, as previously defined [MET exon 14 skipping mutations [MET high-level amplification as a mechanism of resistance to EGFR tyrosine kinase inhibitors were excluded from further analysis.The archive of the Institute of Pathology of the University Hospital of Cologne, Germany was retrospectively searched for non-small-cell lung cancer cases showing high-level  defined  or MET eutations , 30, 31.MET exon 14 skipping mutation and 24 harboring MET high-level amplification presented sufficient clinical follow-up and sufficient material for further analyses. For each of the 50 cases, the material was derived from the biopsy at the time of diagnosis of stage IIIB/IV.According to selection criteria, we were able to identify 86 biopsies of lung tumors originating from 86 patients. Of those, 26 tumors harboring MET high-level amplification using FISH analysis described above.Furthermore, 25 resection samples of primary resectable adenocarcinoma of the lung in early stage were collected. This group was used as an independent validation cohort for Histology was reviewed by two experienced pathologists (AMS and RB) according to the current World Health Organization classification criteria . Prior tAll samples were fixed in 4% neutral-buffered formalin at room temperature and embedded in formalin  by routine processing methods. For cases with sufficient material Table , 3 µm-thFor the assessment of the ALK status, ALK expression was classified as positive if strong granular cytoplasmic staining in tumor cells was present . PositivPD-L1 was graded according to the internal guidelines as described in the literature , 34. ScoMET expression was determined using immunohistochemistry according to the literature , 35, 36 ® 16 FFPE Plus Tissue LEV DNA Purification Kit  on the Maxwell® 16 (Promega), and DNA was eluted in Tris-HCl (pH 7.6) for parallel sequencing approach.For each case, the tumor area was marked on a haematoxylin-eosin stained slide by two senior pathologists . Six 10-µm-thick sections were cut for each case. After deparaffinization, tumor areas were macrodissected from unstained slides. Tissue was lysed with proteinase K overnight. DNA was purified with the Maxwell® analysis, DNA was purified manually using the QIAamp® DNA Mini Kit (Qiagen). Samples were diluted to a working concentration of 150 ng/μl. All extraction procedures were done following the manufacturers’ instructions.For the NanoString nCounterARAF exons 7, 10, 15, BRAF exons 11, 15, CTNNB1 exon 3, DDR2 exons 4–19, EGFR exons 18–21, ERBB2 exons 19, 20, FGFR2 exons 8–10, 12, 17, 20, FGFR3 exons 7, 10, 15, KEAP1 exons 2–6, KRAS 2–4, MAP2K1 exon 2, MET exons 14, 16–19, NFE2L2 exon 2, NRAS exons 2–4; PIK3CA exons 9, 20, PTEN exons 1–8, TP53 exons 5–8.The DNA content was measured using a quantitative real-time PCR. For multiplex PCR-based target enrichment, the isolated DNA (10 ng each) was amplified with a customized GeneRead DNAseq Targeted Panel V2 , targeting 17 cancer genes most frequently mutated in lung cancer, and the GeneRead DNAseq Panel PCR Kit V2 (Qiagen) according to the GeneRead DNASeq Gene Panel Handbook (Qiagen). The panel comprised a subset of cancer relevant genes including: Libraries were constructed using the Gene Read DNA Library I Core Kit and Gene Read DNA I Amp Kit (Qiagen). After end-repair and adenylation, NEXTflex DNA Barcodes were ligated . Barcoded libraries were amplified and then the final library product was quantified with Qubit dsDNA HS Assay Kit  on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific), diluted and pooled in equal amounts. Finally, 12 pM of the constructed libraries were sequenced on the MiSeq  with a MiSeq reagent kit V2 (300 cycles) (Illumina) following the manufacturer’s recommendations.http://www.broadinstitute.org/igv/, Cambridge, USA). A 5% cutoff for variant calls was used, and results were only interpreted if the coverage was > 200x.Data were exported as FASTQ files. Alignment and annotation was done using a modified version of a previously described method . BAM filFISH was performed using commercially available FISH probes provided by Zytovision  according to manufacturer’s instructions. Further information is available in Supplementary Table MET, ROS1, and RET were performed at time of diagnosis. ALK FISH was performed in case of any positive or ambiguous immunohistochemistry result. FISH for MDM2, CDK4, and MYC was performed only in selected cases in order to validate gene amplifications as described in the Results section.FISH for Slides were reviewed at high magnification power ( ×63) and scored according to appropriate respective guidelines , 38.® platform . For detection of copy-number alterations, nCounter Copy Number Variation CodeSets were used with 600 ng of genomic DNA extracted as described above. DNA purity was measured by NanoDropTM 2000c Spectrophotometer , and only DNA samples with an OD A260/280 ratio between 1.7 and 1.9  were used for further studies.All samples were analyzed for copy-number alteration analysis using the NanoString nCounterDNA was fragmented into small pieces (~500 bp) via AluI digestion (37 °C for 1 h) and subsequently denatured to produce single strands (95 °C for 5 min). Fragmented DNA was hybridized with the CodeSet of 87 genes in the nCounter v2 Cancer CN Assay Kit  for 16–18 h at 65 °C and processed according to the manufacturer’s instructions.The nCounter Digital Analyzer were counted and tabulated the signals of reporter probes. The data were normalized to the invariant control probes and to positive and negative controls in each hybridization reaction. Positive and negative controls as well as the probes were derived from formalin-fixed paraffin-embebbed material, in order to detect any fixation artefacts. Finally, data analysis was performed using nSolver™ Analysis Software 3.0. Copy number was determined by averaging over three probes per region.Each assay contained 6 positive dsDNA control probes, 8 negative control probes, and 54 invariant genomic control probes designed for autosomal genomic regions predicted not to contain common copy-number alterations.Based on the manufacturer’s protocol, the gene was considered to be a single copy if the average copy number was below 1.4, two copies if between 1.5 and 2.4, three copies if between 2.5 and 3.4 and continued.t test was performed. To investigate if the two groups differed in their chromosomal stability, a Brown–Forsythe test was applied as previously described [Graphpad Prism software  was used for statistical analyses. To test if the two groups differed in terms of age at diagnosis, smoking status and gender a Student's escribed . This inescribed . For oveMET-mutant cases were classified as adenocarcinomas and 2 as squamous cell carcinomas of the lung. Twenty-one of 24 MET-amplified cases were considered as adenocarcinomas, two as squamous cell carcinomas, and one as large cell carcinoma of the lung  than in the high-level amplified cohort (median age 69 years). The MET high-level amplified cohort comprised mostly men , the MET-mutant cohort predominantly women , in line with the current literature [p = 0.0002) in the MET-mutant cohort. Conversely, the MET-amplified group was dominated by smokers  or 3 . For two cases (8%) immunohistochemical analysis was not possible, due to the lack of available material. In contrast, in the MET-mutant cohort MET-immunohistochemistry showed heterogeneous results. In six of 26 cases (23%) MET was scored as 0, in four (15%) as score 1, in three (12%) as score 2, in nine (35%) as score 3. In four cases (15%), immunohistochemistry was not performed, due to lack of sufficient material.MET status was evaluated immunohistochemically by two board-certified pathologists, blinded to genomic or clinical data Fig. . The entMET aberration and PDL-1 expression. In the MET-mutant cohort, six of 26 cases (23%) were scored for PD-L1 as 0, 2 (8%) as score 1, one (4%) as score 2, four (15%) as score 3, none as 4 and ten cases (39%) as score 5. In the MET-amplified cohort, five of 24 cases (21%) were scored as 0, one (8%) as score 1, three (13%) as score 2, two (8%) as score 3, one (4%) as score 4, and four (17%) as score 5 (Table p = 0.510).PD-L1 status showed highly variable results in both cohorts, indicating no detectable relationship between  5 Table . No signMET exon 14 skipping mutations were localized between nucleotide 2842 and 3082. As described in Table MET mutation was detected in the MET-amplified cohort. The MET-mutant cohort contained seven (26.9%) cases with concurrent TP53 mutation leading to a non-functional protein. No concurrent driver mutations were detected in this cohort.All TP53 mutations occurred with higher frequency in the MET-amplified , compared with the MET-mutant cohort . Only the former cohort was characterized by a high frequency of other co-occurring pathogenic driver mutations, such as KRAS  and NRAS   showed a subclonal MET amplification with high heterogeneity among the tumor cells  were detected with an average gene copy number per cell of five, with peaks up to 20 gene copies per cell detected in case M5. CDK4 , HMGA2 , and DYRK2  were also found to be amplified in this cohort. Amplifications of CDK4 and MDM2 were confirmed using FISH analysis. Copy number gains were detected for CCND1 , MYC , and TERT .As shown in Table MET high-level amplification detected by FISH showed high concordance with the copy-number alteration analysis results in 16 of 24 cases, in 8 of 24 cases we noted a high discrepancy in the amplification level of MET. For all eight discordant cases, we were able to identify either the presence of high intratumoral heterogeneity using FISH analyses or a high number of intratumoral lymphocytes. High-level amplified tumor cells with cluster amplification were identified adjacent to tumor cells with normal signal pattern or lymphocytes with normal signal pattern  were detected in three cases (12%) and MYC copy number gains in eight further cases (33%). CCND1 and CCND2 showed a recurrent low copy number gain in 14 cases (58%).MET-mutant and MET-amplified cohort revealed diffuse genetic instability, pointed out on the chart as deviation from average gene copy number of two, but the global amount of copy number alterations was significantly lower in the mutant cohort, when compared with the amplified group (p < 0.001), suggesting a reduced genetic instability in the former group.As shown in Fig. MET-amplified and MET-mutant cases were evaluated using immunohistochemistry. The entire cohort of MET-amplified cases was evaluated as positive (scores 2–3), confirming the FISH results: six of 24 cases were scored as 2 + and 16 cases as 3 + . In two cases, immunohistochemical evaluation was not possible, due to lack of material.Both MET-mutant cohort  compared with MET high-level amplified group .Stratification of patients by genetic alterations was statistically significant: the subset of MET exon 14 skipping mutations or MET high-level amplifications, defined by a gene copy number of at least 10.Non-small-cell lung cancer driven by MET aberrations is currently considered as a heterogeneous group of tumors . At the TP53 mutations are present in both groups, however at higher frequency in the cohort of MET-amplified cases . As described in the literature, the higher frequency of TP53 mutation may be explained by the high prevalence of smokers in this group [MET-mutant cohort does not have any co-occurring driver mutations, confirming that MET exon 14 skipping mutations may represent a strong carcinogenic driver. Conversely, MET-amplified cases frequently show co-occurring driver mutations, such as KRAS and NRAS, which has also been described previously [KRAS mutations in MET-amplified cells lead to resistance against MET inhibitors, in a similar way to EGFR-inhibitors. They suggest that the impact of KRAS mutations should not be undervalued during treatment planning and could be responsible for reduced/no response after treatment with MET inhibitors. Similar to KRAS alterations, we detect the presence of co-occurring high-level MYC amplifications in 12 MET-amplified cases. MYC high-level amplification is also detectable in three MET-mutant cases. According to Shen et al. [MYC amplifications can be responsible for acquired resistance to MET inhibition in MET-addicted cancers. In mouse models, the resistance could be overcome by double-MYC-MET inhibition. Further studies are necessary to clarify the role of KRAS mutations and MYC amplifications in MET-altered non-small-cell lung cancer.The analysis of the genomic alterations of lung cancer detected by targeted next-generation sequencing reveals that recurrent is group . Of inteeviously . Leiser eviously  have demn et al. , MYC ampMET signal distribution in the MET-amplified cases, using FISH. Interestingly, we show that intratumoral heterogeneity is present at high frequency and is represented by different tumor clones either intermingled or located closely to each other. Of note, it results in a lower level of total gene copy numbers, when using extraction-based copy number variation analysis, compared with single-cell methods, such as FISH  in all amplified cases. However, MET-immunohistochemistry results are extremely heterogeneous in the mutant cohort, with scores between 0 and 3+. In conclusion, immunohistochemistry seems to be a highly sensitive method for the detection of suspected high-level amplifications. In contrast, it does not seem to be a reliable screening tool for the detection of MET high-level amplifications are predominantly younger  when compared with the amplified patients and by a longer average survival time (p = 0.003) as depicted in Fig. Finally, the analysis of the clinical features of the cohorts highlights that the two groups differ not only genetically but also clinically. Patients harboring MET-altered lung cancer in an advanced stage are limited to clinical trials, using crizotinib or other more recently developed MET inhibitors. Our results confirm that MET-altered tumors are a biologically heterogeneous group of tumors and we suggest that an accurate characterization of the genetic background may be useful for a better understanding of patients’ outcome with the ultimate goal to improve tailored therapeutic options.Currently, the therapeutic options for patients affected by The current study is limited as it is based on the analysis of a restricted number of genes frequently altered in lung cancer and a relatively small number of patients.MET-mutant and -amplified lung cancers, but only few studies directly compare both groups. In spite of sampling number limitations, the study underlines interesting issues that are crucial in both predicting and understanding response to MET inhibitors. This new side-by-side genetic characterization shows that MET exon 14 skipping mutations can be interpreted as early strong driver to carcinogenesis in lung cancer cases, whereas MET amplifications seem to occur as subclonal genetic event usually in the context of other strong driver mutations and therefore must be interpreted in the context of each tumorʼs genetic background, rather than as isolated event.Many studies have already described the composition of patient cohorts in The present study provides the scientific basis for the performance of furthermore comprehensive studies analyzing a larger series of cases.Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Figure S1Supplementary Figure Legend


Once abstract concepts are acquired, they can work as social tools: the social metacognition mechanism (awareness of our concepts and of our need of the help of others) can evoke the presence of a “ready to help actor” in an implicit way, as a predisposition to ask information to fill the knowledge gaps.Sociality influences both concrete and abstract concepts acquisition and representation, but in different ways. Here we propose that sociality is crucial during the acquisition of abstract concepts but less for concrete concepts, that have a bounded perceptual referent and can be learned more autonomously. For the acquisition of abstract concepts, instead, the human relation would be pivotal in order to master complex meanings. Once acquired, concrete words can act as tools, able to modify our sensorimotor representation of the surrounding environment. Indeed, pronouncing a word the referent of which is distant from us we implicitly assume that, thanks to the contribution of others, the object becomes reachable; this would expand our perception of the near bodily space. Abstract concepts would modify our sensorimotor representation of the space only in the earlier phases of their acquisition, specifically when the child represents an interlocutor as a real, physical  Sociality is pivotal for survival and for well-being of our species. It would be difficult to deny that sociality permeates a cognitive process like language, since when talking we need to have an interlocutor, i.e., a person that takes part to the conversation/dialogue with us. In contrast, the role of sociality for processes such as perceiving, categorizing and thinking, is not always sufficiently emphasized. Concepts are “bricks” to build an internal world; they serve to filter the surrounding world, to understand the incoming stimuli for acting and to create new systems of meanings. Here we will argue that sociality is relevant for the formation and representation of concrete and abstract concepts , but in different ways. We will first clarify what we intend with concrete and abstract concepts, then we will formulate our theoretical proposal, illustrate evidence supporting it and discuss some open issues.Concepts have been defined as the “glue” that links our current with our past experience , 2002. WWe will here focus on the distinction between abstract and concrete concepts . We do not intend such a distinction as a dichotomy but we rather conceive it as blurred and not stable, for a number of reasons . First oOn the negative side, abstract concepts are typically less associated than concrete ones to sensorial and perceptual modalities , they arIn sum: in our view both concrete and abstract concepts are grounded in perception-action, in sociality and in linguistic experience, even though the weight of the sensorimotor experience is higher for concrete concepts, that for the social and linguistic experience higher for abstract concepts. In this paper we will focus on the role social experience plays in acquisition and representation of both kinds of concepts.The main thesis of this paper, that we will articulate and defend, is that acquisition, learning, and representation of both concrete and abstract concepts rely and are influenced by social experience. However, we will qualify this social experience and contend that different kinds of social relationships are involved during processing of concrete and abstract concepts.Let us consider concrete concepts first. In our view the social dimension is less important for the acquisition of concrete concepts compared to that of abstract ones, since the referents of concrete concepts are perceptually similar and are clearly bounded objects. There is a clear and unique relation between the concept and the referent, that can be autonomously learned. For example, children can form the category of “entities that move on their own,” even if learning the correspondent word, e.g., “animals,” can contribute to refine and render more compact their category . Hence, Taking into account abstract concepts , we argue, instead, that sociality is crucial for their acquisition. Since the referents of abstract concepts are not perceptually similar and are not clearly bounded objects, we need the others’ linguistic and social input in order to form categories. Consistently, we will advance the new hypothesis that abstract concepts might include a sensorimotor representation that affects the perception of the environment. Because in the early phases of abstract concepts acquisition we might need the another person sufficiently close to us to explain the word meaning, this might impact our space representation. Indeed, we would implicitly assume the “real” presence of a social referent, at least in the earlier phases of conceptual acquisition. The presence of another person, his/her explanations, would be fundamental in order to allow us to form concepts composed by a variety of heterogeneous events and situations, as the members of abstract categories are. Once abstract concepts are acquired, we contend that they always refer to the social dimension, but in a more implicit way. Differently than for concrete words, we might not be able to use abstract words as physical instruments, e.g., to ask others for an object. However, at a metacognitive level we might be less satisfied of our knowledge related to abstract than to concrete concepts, and we might want the help of authoritative others to fill these gaps. Hence, we may continue to need others to complement the gaps of our knowledge .One important note: in distinguishing between concrete and abstract concepts we mentioned “their referents.” Because we intend words as tools that modify our relationship with the surrounding world, it is worth of note that we do not intend referents as something static, that is simply out there in the world. Words are not only pointers to referents, they are rather tools that modify the environment and the space. In this perspective, in keeping with In the human being, the social self emerges quite early compared with the other mammals. At 3–6 months infants are already involved in complex interactions with the mother/caregiver . Imitatibodily tool or a sort of instrumental referent to reach the object when it is outside the infant’s reaching area. She rather starts to be considered also as an intellectual referent, somebody to draw knowledge from, who can help to build meanings, to acquire the vocabulary and to construe the concepts useful to interpret the daily life. This shift of the infant’s interest opens to the connection between two minds with the beginning of the cultural development.Negotiation of activity is the less complex level among the pragmatic abilities, indeed it belongs also to non-human primates since it is based on the instrumental use of language . At thisThis is the phase in which language acquisition occurs. When the child acquires new concepts and new words, both concrete and abstract, he/she needs the presence of others. Which are, then, the differences in the acquisition of concrete and abstract concepts and words?We contend that the presence of others is more crucial during the acquisition of abstract concepts, because their members are quite diverse and heterogeneous. Consider the difference between the concepts “table” and “freedom.” Different tables share many similarities, and often they can be reconducted to a prototypical image; so the child can quite easily learn on her own to abstract from the more idiosyncratic features and to form the category of “table.” This does not mean that children learn concepts on their own, solely on the basis of the perceptual inputs. The linguistic and social input is clearly determinant to refine and render more compact the categories they have formed , as wellOne further difference is that learning of concrete concepts and words typically occurs in presence of an object/entity, the conceptual referent. Abstract words like “freedom,” instead, do not refer to an object with which the child can interact and that the adults/others can indicate. In order to learn concrete concepts a single label might be a sufficient input, while to learn abstract concepts more extended explanations of the word meaning are generally required in order to gather the multiple experiences abstract concepts assemble together. The guidance of the other/adult and of a rich linguistic input is therefore of paramount importance  space . This exIf the body is our bridge with the world, the word is our bridge with the others. In general we communicate with other people through verbal language or gestures if we are too much distant to be heard, still we can scream. Either the words and the body serve to communicate meanings, but the words are able to convey more complex meanings and allow us to be more precise, for example to refer to a specific object among many. When an adult talks usually there is at least interlocutor to promote a dialogue.instrumental referent who can give us the pen, making the pen closer to us and expanding our near space. In the second case, the sentence invokes the help of an intellectual referent, who help us to learn the meaning of new words. These two functions can be summarized as follows: (1) The word, like a physical tool or like the body of another agent, serves to reach distant objects that are outside our action domain; (2) The word is useful to understand the meaning of words, to create and build new conceptual networks. We propose that the first function of language concerns primarily concrete concepts, while the second function of words concerns both concrete and abstract concepts, but is particularly prominent for abstract ones.Let us consider two different roles among the many that language can play. Words can help us to find a solution to reach concrete aims, e.g., “Can you pass me the pen?,” or to create new knowledge e.g., “Do you think that this object/entity/event belongs to this category? What does this word mean?” In the first case, the sentence invokes the help of an Empirical evidence supports the idea that concrete words work as physical tools. Experimental results obtained by So far we have claimed that sociality is more crucial for abstract than for concrete concepts acquisition, and that, once concepts are acquired, we might use concrete words as tools. Concrete words namely implicitly evoke the presence of others who may help us in reaching objects, and this impacts our representation of the reachable space. While the representation of abstract words likely does not impact and modulate the borders of our bodily space, in our view sociality continues to influence abstract concept representation in different ways.Once abstract concepts have been acquired, to what extent does their processing involve the presence of others? Can this presence be evoked only in an implicit way? Let us consider separately the three mechanisms that we briefly illustrated in section. We propose that these mechanisms, that are not mutually exclusive and can co-exist, underlie abstract concepts processing, and explain why the mouth motor system is activated , concrete concepts like “glass” or “table” have a sensorial well-defined referent and their acquisition stems from the sensorimotor experience of the physical object/entity to which concepts refer. Abstract concepts are more detached from the sensorial experience, and evoke more social and linguistic experience than concrete ones ,c. Both instrumental referent to reach an object, she becomes an intellectual referent to catch meanings. The role of the other as intellectual referent is particularly crucial for the acquisition of abstract concepts: due to the heterogeneity of their members and to their detachment from sensory modalities they are more difficult to learn relying exclusively on the perceptual inputs.The main thesis of this paper is that sociality influences both concrete and abstract concepts acquisition and representation, but in different ways. We revised developmental literature showing how the point of contact between the infant and the surrounding reality/environment is the mother/caregiver. Far from being considered just an Once concrete and abstract concepts have been acquired, sociality continues to be determinant for their representation. We briefly illustrated theoretical proposals and evidence showing how concrete words can act as tools : similar“ready to help actor,” with a consequent interpersonal bodily representation of the physical reality  until the moment in which the dialogue between the infant and the real interlocutor becomes internalized. When the infant masters a solipsistic inner language, three possible mechanisms underlie and explain the activation of the mouth motor system during abstract concepts processing. For the re-enactment and re-explanation mechanisms the reference to a possible companion/other would be implicitly evoked. The social metacognition mechanism can evoke the presence of a “ready to help actor” in an implicit way, but it can lead to a stronger activation of the mouth motor system in presence of real others, to whom to prepare to ask information and help (Here, we propose that also abstract concepts, like concrete ones, might influence the perception of the environment but following two different modalities. In the earlier phases of abstract concepts acquisition, the child might represent an interlocutor as a real, physical and help .New research is necessary to investigate how the social component of abstract concept evolves, from being external and more embodied, to be internal and more semantic and how this is reflected in a different perception of the world.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LW declared a past collaboration with one of the authors AB.