This systematic review aimed to assess antibiotic use in Brazilian hospitals in the 21st century, as well as to understand the different drug utilization metrics adopted to assess the consumption of these drugs. Literatura Latino-Americana e do Caribe em Ciências da Saúde]) for observational or experimental studies that assessed antibiotic utilization in Brazilian hospitals. The main outcomes were the drug utilization metrics and the consumption of antibiotics.  We systematically reviewed five databases  and the percentage of antibiotic prescriptions. The most commonly used antibiotic classes were third-generation cephalosporins, carbapenems, fluoroquinolones, and combinations of penicillins when the DDD was the adopted drug utilization metric.  Although few studies have been conducted, existing data indicate a high use of broad-spectrum antibiotics. We found that the lack of standardized antibiotic utilization metrics impaired the mapping of drug consumption at the national level. We aimed to synthesize the existing studies by conducting a systematic review to assess antibiotic use and consumption in Brazilian hospitals in the 21st century and to understand the different drug utilization metrics adopted to assess the consumption of these drugs.A systematic literature review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelinePublications that answered the guiding question: “What is the pattern of antibiotic consumption in Brazilian hospitals?” were selected. Observational and experimental studies performed in Brazilian hospitals that analyzed the general pattern of antibiotic consumption, consumption of specific classes, or use according to a specific purpose  in the entire hospital or in specific care units were included. Articles were excluded for the following factors:data was collected before the beginning of the 21st century;conceptually described/discussed consumption of antibiotics in Brazil, comments or expert opinions, study protocols, reviews , dissertations or theses, editorials, news reports, and abstracts published in conference proceedings;published in languages other than English, Portuguese, or Spanish;did not have full text available.The search strategy construction incorporated terms that characterized the guiding question, structured by the PICOS acronym This strategy was developed using a combination of terms from the Medical Subject Headings (MeSH) thesaurus of the PubMed database . The MeSH terms selected are descriptors of each PICO component: anti-infective agents, antibacterial agents, hospitals, and Brazil. For each MeSH term, relevant keywords were selected to expand the search and were combined with the Boolean operator “OR” as follows:anti-infective agents OR antibacterial agents OR antimicrobial* OR antibiotic* OR antimicrobial OR antibacterial agents [Pharmacological Action];hospital OR hospital*;Brazil.The terms used in 1, 2, and 3 described above were combined using the Boolean operator “AND.” Literatura Latino-Americana e do Caribe em Ciências da Saúde). The search was conducted in April 2019.This database has been adapted for other databases, namely CENTRAL , EMBASE® (Excerpta Medica Database), Scopus (Elsevier’s abstract and citation database), and LILACS , a critical appraisal designed to assess systematic reviews of mixed studies. It appraises the methodological quality of five categories: qualitative research, randomized controlled trials, nonrandomized studies, quantitative descriptive studies, and mixed methods studies. There is no overall score because it does not provide information on what aspects of the studies are problematic. When the study receives a “yes” to any item of this tool, it means that the category assessed adequately meets the criteria. Conversely, “no” means that the category does not adequately meet the criteria and “can’t tell” means that the category does not report sufficient information to answer “yes” or “no” according to the criteriaThe study hospitals were classified according to their sizesThe included articles presented two antibiotic drug utilization metrics: the relative frequency of antibiotic prescriptions  and the defined daily dose . The DDD/beds-days, patients-day, or admissions unit was used in some studies divided by 100 and in others by 1,000. Thus, to facilitate the comparison, the studies that divided by 1,000 were divided by 10 to present them as DDD/100 bed-days, patient-days, or admissions. Furthermore, this study, included antibiotics in the consumption analysis classified as J01 in the 2019 Anatomical Therapeutic Chemical (ATC)/DDD index.If one study presented results of antibiotic consumption from more than one place or for more than one type of treatment , all results were included in this study as multiple presentations of antibiotic use.After applying the search strategy in the databases, 3,566 publications were selected for title and abstract evaluation. Publications for which there was disagreement were excluded or included after discussion and consensus. Finally, 29 studies were selected for full reading, and 17 were considered suitable for inclusion in the systematic review. By manually searching the reference lists, 6 more articles were included, totaling 23 articles. Supplementary Material (Table 1)-The 23 included articles were published between 2003 and 2018, of which 69.5% were observational studies .One study presented results of the two previously reported drug utilization metrics (DDD and frequency). Furthermore, 14 (60.9%) studies described antibiotic use in the treatment and prophylaxis of infections, while 9 (39.1%) described only use for treating infections  shows the study characteristics according to the antibiotic assessed, inclusion criteria of patients, and drug utilization metrics applied. Of the 13 studies that used the DDD, only 6 described the calculations and ATC/DDD index version . Of the 13 studies not conducted in ICUs or pediatric units, only 5 described the units where the research took place.The utilization metrics found in this review were DDD/bed-days in 4 studies, DDD/patient-days in 8, and DDD/100 admissions in 1. Among these, two presented antibiotic consumption data arising from multiple locations .Nevertheless, only one study described how the relative frequency of antibiotic prescriptions was calculated . In three studies, we determined how the relative frequency of antibiotic prescriptions was measured by calculating the percentage ourselves from the absolute prescription frequency presented in the manuscript . Furthermore, seven studies described the source of data collection . Only two studies presented the ATC index version Regarding the geographic distribution of works published in Brazil, the state of São Paulo was the most represented (five publications); followed by Minas Gerais (four); Paraná, Santa Catarina, Rio Grande do Sul, Distrito Federal, Mato Grosso do Sul (two each); and Pernambuco, Paraíba, Bahia, and Rio de Janeiro (one each). Thus, the South and Southeast regions were the most represented.Staphylococcus aureusPseudomonas aeruginosaAmong the studies that analyzed antibiotic consumption using the DDD, we found that according to 5 of the 16 results for antibiotic use , which comprises a public set of actions and services (from the most basic to the most complex) aimed at providing healthcare for the entire Brazilian populationS. aureus or P. aeruginosa (P. aeruginosa),,,Third-generation cephalosporins were the most commonly used antibiotic class in the included studies, but were not among the three most used classes in articles that evaluated the treatment of infections caused by a particular bacteria, such as ruginosa . This is,In a Chilean study covering 15 hospitals with medical clinics and intensive care and surgical units, similar results were observed, since third-generation cephalosporins were the most commonly usedThe excessive use of cephalosporins, especially third generation, is associated with the selection of enterobacteria that produce extended-spectrum beta-lactamase (ESBL); this complicates treatment, since the only effective class of beta-lactams is carbapenemsS. aureus  study and the Chilean studies (ICU)Enterobacteriaceae, has been more prevalent over the past decade, with few therapeutic options remainingCarbapenem antibiotics have a broad spectrum of action, including gram-positive cocci, fermenting and nonfermenting gram-negative bacilli, and gram-positive and -negative anaerobes. In addition, the carbapenem class is one of the few options for treating infections caused by ESBL-producing bacteria, and as a consequence of these increased infections, its use has become increasingly more frequent. As a result, the occurrence of carbapenem-resistant bacteria, such as carbapenem-resistant Combined with the use of the DDD , some stIn a systematic review undertaken by Zanichelli et al. (2018)-The DDD is a drug utilization unit developed by the WHO and is defined as the average daily dose of a drug for its main indication in adults. Thus, use of the DDD is important, as it is a globally standardized drug utilization metric that allows comparison of multiple studies. Although the DDD is a globally standardized indicator, it alone does not provide complete information about how the drugs are used in a given location. Although the DDD considers the length of treatment, number of people who use them, and prescribed dose; however, it does not clarify which of these most significantly influences total consumption. Therefore, it is suggested that studies on antibiotic use choose more than one type of drug utilization metric, such as the average daily dose prescribed, days of treatment, and costConsidering the 23 studies included in this review, only two drug utilization metrics were used to assess antibiotic use: the DDD and the percentage of antibiotic prescriptions. Moreover, only one study analyzed antibiotic consumption using two metricsEven among studies that adopted the DDD as a consumption metric, the comparison of results is questionable, because some antibiotic classes were revised in the DDD over the years. Few studies have clarified which version was used; however, using the year of publication, it is possible to infer which different version may have been applied. This reinforces the importance of using more than one drug utilization metric. Other factors that hinder comparisons include the lack of standardization in the adoption of the DDD/bed-day, patient-day, or admissions; moreover, some manuscripts even describe how the calculation was performed. Additionally, one study used the DDD for pediatric patients-The application of the DDD as an antibiotic utilization metric is common in countries around the world. Europe, the United States, Japan, and Chile frequently report antibiotic consumption based on well-designed studies presenting complete information for the whole country that allows for comparisons and identification of problems that need interventions to improve antibiotic useIn the present review, in addition to evaluating the prevalence of articles that adopted insufficient drug utilization metrics to determine the pattern of antibiotic use, we attempted to compare them with more reliable international studies, but their quality was unsatisfactory according to the MMAT criteria. The main reasons for this low quality were the data collected and measurements used, which did not allow us to address the research question or the confounders unaccounted for in the study analysis. The absence of studies of excellent quality may indicate a lack of training or an incentive to register, analyze, and publish data on antibiotic consumption in hospitals, as well as the difficulty Brazilian researchers have in obtaining financial resources to conduct high-quality, well-designed, multicenter studiesThis systematic review obtained data on the use of antimicrobials in municipalities in 11 Brazilian states, covering four national regions , with a prevalence of studies carried out in the Southeast and South (69.6%). These differences probably reflect the greater investment in science and research in regions where most of the country’s research centers are concentrated, but they may also be a sign of structural differences in organization, coverage, and provision of health services in Brazil.Inappropriate antibiotic use by means of unnecessary prescriptions, prolonged use, and antibiotics with a broad spectrum of action for conditions that could be otherwise treated with restricted-spectrum drugs increases the risk of resistant bacteria selection. Microbial resistance to antibiotics is one of the fastest growing public health concerns. It is estimated that by 2050, it will be among the most lethal health problems, in addition to generating an additional billion dollars in healthcare costsOne of the main limitations of this systematic review is the absence of studies published in gray literature. Nonscientific documents on antibiotic use in hospitals may have been published but were not included in this study. These data could make the scenario of antibiotic use in Brazil more realistic. Another limitation of this review is the lack of standardized reliable metrics and methods for assessing consumption, which would provide a clearer dimension of antibiotic consumption in Brazil and facilitate comparisons with the global scenario. Nevertheless, this study computed the available information and presented a scenario still unknown in Brazil: the national pattern of antibiotic consumption.,Studies on the use of antibiotics have become essential to support health managers with consumption data for a region, country, or even internationally. Consumption data obtained from well-designed and -conducted studies may support campaigns, strategies, and interventions at the local or global level to rationally use antibioticsThe few studies found in this review show antibiotic use with high consumption of broad-spectrum antibiotics, mainly when compared to international drug utilization studies. However, the low quality of the studies, the absence of good antibiotic utilization metrics, and the lack of studies with results covering national patterns hinder the understanding of the actual antibiotic consumption in Brazil and confirm the need for high-quality drug utilization research about antibiotic consumption on a national level.


Given the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs.B0AT1, ATB0,+, rBAT, ASCT2, IMINO, LAT4, y+LAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of B0AT1, ASCT2, LAT2, and LAT4.A total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of B0AT1, rBAT, y+LAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB0,+ showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (y+LAT1) intestine. Protein expression of B0AT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met  and L-Met .Expression of all investigated transcripts differed significantly along the GIT. A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues. As stated earlier, however, other scientists considered a functional relevance of transporters like ATB0,+ for large intestinal absorption of amino acid derived from microbial metabolism . A quite similar finding was reported earlier for the glucose transporter GLUT2. Despite lower mRNA levels in the distal ileum compared with the middle jejunum, GLUT2 protein abundance was higher in the distal ileum than the middle jejunum [An interesting finding of the present study was that mRNA expression of two transporters, namely B jejunum . ConsideThe present study was performed in pigs; however, Met supplements gain increasing popularity also in human nutrition . As huma0AT1) of apical Na+-dependent transporters in the present study cannot explain the de novo induction of a Na+-dependent uptake system in the mid jejunum identified in our previous study upon feeding a DL-Met–containing diet [The present study showed that known Met transporters have a distinct longitudinal pattern of expression along the different sections of the GIT in pigs. A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in AA absorption. However, some transporters showed rather high expression in segments that do not have a proven role in AA absorption . Dietary Met source changed the expression of some transport systems. From these changes, it may be extrapolated that a diet containing DL-Met has potential to increase apical Met transport in the small intestine, which is congruent with recent functional findings of our group. On the other hand, a diet containing DL-HMTBA has potential to increase basolateral Met transporters in the small intestine and, partly, other gastrointestinal tissues. However, this was not previously found to improve functional methionine absorption and may be a result of a system compensation for lower free Met to be used for epithelial cell metabolism or as an exchange molecule for transport of other AA. Overall, the degree of regulation appeared small to moderate and, likely attributable to this fact, changes in mRNA expression did not clearly correlate with changes in protein expression. Importantly, the small changes in mRNA (ASCT2) and protein expression (Bing diet . Therefo


The c-mesenchymal–epithelial transition factor (C-MET) is an oncogene encoding a tyrosine kinase receptor that plays an important role in tumor growth and metastasis. The National Comprehensive Cancer Network (NCCN) guidelines have approved carbatinib/crizotinib for advanced non-small cell lung cancer (NSCLC) patients with MET exon 14 skipping.In June 2020, the Department of Respiratory and Critical Care Medicine of Peking University People’s Hospital admitted a 72-year-old male patient with lung adenocarcinoma (LADC) with a history of interstitial lung disease secondary to antineutrophil cytoplasmic antibody-associated vasculitis. Genetic examination by next-generation sequencing showed an intergenic fusion of MET, and crizotinib was administered on August 14, 2020. Follow-up showed that tumor volume was significantly reduced. However, crizotinib was discontinued in November 2020 because of the patient’s worsening interstitial lung disease, and CT scans showed continued partial response (PR) for 5 months. In April 2021, right lower lobe mass progressed, and disease progression was considered.This was the first case of a patient with LADC with MET intergenic fusion who significantly benefited from crizotinib. Even after crizotinib was discontinued for 5 months, the patient continued exhibiting PR, suggesting that MET intergenic fusion may have carcinogenic activity in LADC and was sensitive to crizotinib. Lung cancer is the main cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of the mortality. NSCLC diagnosis mainly depends on pathology, and most patients are often diagnosed at late stage with poor prognosis. The emergence of targeted therapy has greatly improved the progression-free survival (PFS) and overall survival of patients with advanced NSCLC. Therefore, routine genetic testing is required for patients with NSCLC. The c-mesenchymal–epithelial transition factor (C-MET) gene is an oncogene locating on chromosome 7q21-31 and encoding a tyrosine kinase receptor . PreviouCrizotinib is a small-molecule tyrosinase inhibitor that effectively inhibits MET (high-level MET amplification or MET exon 14 skipping mutation), anaplastic lymphoma kinase (ALK), and reactive oxygen species (ROS) proto-oncogene 1 (ROS1) –10. The The patient was a 72-year-old male smoker with advanced NSCLC. He had a history of interstitial lung disease (ILD) secondary to antineutrophil cytoplasmic antibody-associated vasculitis and no other major medical problems. The patient presented in June 2020 with a 4-month history of dyspnea. Chest CT scans revealed a right lower lobe mass (long-axis diameter was 3.7 cm) and bilateral enlarged mediastinal/hilar lymph nodes. In July 2020, he underwent CT-guided lung biopsy and was pathologically diagnosed with poorly differentiated LADC Figure 1To our knowledge, this was the first case of a patient with LADC with MET intergenic fusion who significantly benefited from crizotinib.MET fusion genes are composed of MET lacking the regulatory para-membrane domain and have different N-terminal partners. These fusion proteins lead to the activation of the kinase domain and are involved in tumorigenesis and progression , 11. PreThe present case described a novel MET fusion pattern, MET intergenic fusion, in LADC. According to the genome annotation of the regions containing the breakpoints, fusions are classified into three types . Among these three types, the intergenic–intergenic fusion does not generate chimeric transcripts and upregulates a gene by a different promoter or enhancer on the upstream of the gene . HoweverIn conclusion, we found a case of LADC with novel MET intergenic fusion response to crizotinib, suggesting that this fusion may have carcinogenic activity in LADC. Further, gene sequencing should be routinely performed in patients with NSCLC.The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Ethics Committee of Peking University People’s Hospital. The patients/participants provided their written informed consent to participate in this study.HL: resources, formal analysis, validation, and writing—original draft. DZ: resources. LD: resources and visualization. MZ: resources. ZG: resources. XM: conceptualization, formal analysis, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.This study was supported by the CAPTRA-LUNG (Grant No. CAPTRALung2021001).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Treatment of Blood Stream Infections (BSIs) with a combination of a β-lactam and an aminoglycoside antibiotic is widely used in intensive care units (ICUs) around the world. However, no studies have systematically examined how these drugs interact and potentially influence the antimicrobial efficacy of the overall treatment.coli isolates from the Uppsala University hospital that were isolated from blood of patients with suspicion of infection. Of those we tested the efficacy of combinations of 2 common β-lactam antibiotics (Ampicillin and Cefotaxime) combined with 2 common aminoglycosides (Gentamicin and Tobramycin) on 254 isolates. The efficacy of all 4 pairwise combinations in inhibiting bacterial growth was then examined on all susceptible strains. That was done by quantifying the Fractional Inhibitory index (FICi), a robust metric for antibiotic combinatorial behaviour, of all possible treatments on every strain. When non additive interactions were identified, results of the original screen were verified with time kill assays. Finally, combination behaviours were analysed for potential cross correlations.We collected 500 E. Out of the 4 antibiotic combinations screened none exhibited synergistic effects on any of the 254 strains. On the contrary all 4 exhibited important antagonistic effects on several isolates. Specifically, the combinations of AMP-GEN and CTX-GEN were antagonistic in 1.97% and 1.18% of strains respectively. Similarly, the combinations of AMP-TOB were antagonistic on 0.78% of all strains. PCA analysis revealed that an important factor on the responses to the combination treatments was the choice of a specific aminoglycoside over another. Subsequent cross correlation analysis revealed that the interaction profiles of combinations including the same aminoglycoside are significantly correlated (Spearman's cross correlation test p<0.001).The findings of this study elucidate potential risks of the common combination treatment for blood stream infections. They also demonstrate, previously unquantified metrics on how antibiotics in combination therapies are not interchangeable with others of the same class. Finally, they reiterate the need for case-by-case testing of antibiotic interactions in a clinical setting.This work was funded by grants to DIA from the Swedish Research Council, the Wallenberg foundation and the Swedish Strategic Research Foundation. The combination of β-lactam/aminoglycoside antibiotics has been widely used for the treatment of Blood Stream infections (BSIs). However, reports on the merits of this empirical treatment are conflicting. Several studies report no benefit of the drug combination compared to β-lactam monotherapy, with some reporting even greater efficacy of β-lactam monotherapy altogether. In contrast, other studies and metanalyses report an association between the use of this combination and improved clinical outcome. Conflicting are also the reports that study antibiotic resistance prevention, with some stating that combination therapy better protects against the rise of resistant infections and others finding no improved effect. There have been sporadic attempts to systematically quantify the effects of this drug combination, but the number of clinical isolates tested is insufficient to discern large scale trends.coli strains isolated from blood of patients with suspicion of a BSI. For most strains we detected an additive interaction between the antibiotics used in combination. Alarmingly in a fraction of our isolates we discovered, previously overlooked, significant levels of antagonism between some antibiotics. Specifically, the combination of AMP-GEN was antagonistic in approximately 2% of strains followed by the combinations of CTX-GEN and AMP-TOB that were antagonistic in approximately 1% of strains. A principle-component analysis on the interaction profile of the strains tested revealed that the interaction of the 2 antibiotics in these combinations largely depends on the choice of the aminoglycoside used.In this study, we set out to investigate the combinatorial efficacy of the most common antibiotic combination used in the treatment of Blood stream infections. We chose to test 4 such combinations: Gentamicin (GEN) or Tobramycin (TOB) combined with Ampicillin (AMP) or Cefotaxime (CTX). To this end, we performed the largest to date systematic quantification of antibiotic interactions in clinical isolates. Our study encompasses 254 patient samples of E. Our findings raise concerns in the common empirical practice of combination therapy for treating BSIs. In combination therapy schemes, synergy between the antibiotics used is preferred and antagonism between them is to be avoided. Although the vast majority of strains exhibited an additive (neither positive or negative) interaction between the antibiotics used, a significant number of them demonstrated antagonism with one isolate even exhibiting a suppressive effect in one combination. Not only is it concerning to detect antagonistic behaviours between antibiotics in clinical strains, but perhaps of more important note is that not a single case of antibiotic synergy was found. Finally, our data implies that the common practice of using antibiotics within the same class interchangeably might not be ideal, since behaviours in combination therapies seem to change depending on which antibiotics of the same class are used.Alt-text: Unlabelled box,de novo evolution of resistance, as is the case in tuberculosis (TB) infections that are treated with several different antibiotics.,A single antibiotic is often sufficient to treat the majority of acute infections caused by susceptible bacteria. However, depending on the bacterial species and the site of infection, some infections are treated with combinations of multiple antibiotics.,,Under that premise, empirical antibiotic treatments find extensive use in severe cases of Blood Stream Infections (BSIs), like sepsis and septic shock.,,BSIs in general pose a therapeutic challenge, which is further magnified by the rise of antimicrobial resistance.E. coli is the most commonly responsible for BSIs.E. coli primary infections can result in bacteremia or sepsis including surgical site infections,,31Of all potential pathogens coli blood stream infection, the first line treatment guidelines in Europe the US and specifically in Sweden, dictate as a first course of action the use of a broad spectrum combination of an aminoglycoside with a β-lactam.,36On a suspected E. Although β-lactam/aminoglycoside combinations are extensively used, there is an important parameter of this combination therapy that is often left unchecked. That is the effect the interactions between the drugs have on the overall treatment efficacy. Ideally the drugs used in the same treatment should synergize to a combined inhibition greater than the sum of each individual effect (antibiotic synergy). In some cases, however the drugs antagonize each other to a combined inhibition that is less than the sum of the individual effects (antibiotic antagonism). The differences between synergistic and antagonistic cases are important to take into account, especially since reports on whether or not β-lactam/aminoglycoside combinations result in better clinical outcome are conflicting.in vitro of one of the most commonly prescribed antibiotic combinations consisting of a β-lactam combined with an aminoglycoside for treatment of BSIs. We set out to systematically quantify the interactions of the β-lactam/aminoglycoside combination, against an extensive collection of BSI E. coli clinical pathogens. We quantified in vitro the combined antibacterial effects of two commonly used aminoglycosides, Gentamycin and Tobramycin (GEN and TOB), and two β-lactams, Ampicillin and Cefotaxime (AMP and CTX), for 254 E. coli BSI clinical strains. We then characterized the interaction of every antibiotic combination as synergistic, antagonistic or additive. A majority of the strains showed additive interactions, no strains demonstrated a synergistic response and, unexpectedly, a substantial fraction of the strains exhibited an antagonistic response. These findings demonstrate the potential risks of β-lactam/ aminoglycoside combinations and the need for isolate-specific testing to identify cases of antagonism in which the efficacy of the antibiotic combination might be reduced.In this study, we aimed to characterize the antibiotic interaction profiles TM, product number: 225230) and incubated overnight at 37°C. A single colony was picked from every plate and incubated in 1 ml Mueller-Hinton broth  overnight and incubated at 37°C, 195 rpm orbital shaking.All strains (Supplementary file 1) were isolated from purified clinical samples and frozen as purified single clones in 10% DMSO in LB solution. Clinical strains were acquired from Uppsala University Hospital, and were isolated from patients with positive blood cultures admitted in the hospitals ICU. The demographic of the hospital spans from urban to suburban and rural. the strain collection is composed of strains isolated from positive blood cultures of suspected bacteremia cases (from 2014-2017 to provide a sufficiently large strain collection), as part of standard care . The collection was provided from Uppsala University Hospital and screened and no power calculations were performed as the entire available strain population was screened. For all subsequent experiment strains were streaked from frozen stock on Mueller-Hinton agar plates  on the same day of use according to manufacturer's guidelines. AMP, CTX, GEN and TOB were all ordered from Sigma-Aldrich .5 cells). Plates were incubated overnight at 37°C with no shaking. Before measuring growth on the plates, the medium in all wells was resuspended. Inhibition of growth was measured by means of optical density measurements at 540 nm using a Multiscan FC Type 357 (Thermo Fisher Scientific). MICs were called on the well containing the lowest concentration of antibiotics sharing the same OD as control wells with no bacteria. Interaction testing was performed using CombiANT on susceptible strains, an antibiotic diffusion based method that quantifies antibiotic interactions in bacterial cultures as described in previous work.Minimum Inhibitory Concentrations (MICs) were calculated using the broth microdilution method. A two-fold serial dilution of AMP, GEN, CTX or TOB was prepared in 96-well microtiter plates in triplicates. Overnight cultures prepared in broth as described above were inoculated in a final volume of 180 μL in each well . At every time point  20 μL of bacterial suspension was sampled from each tube. The sample was then diluted and plated on agar plates in 5 μL droplets according to Miles and Misra spotting technique.Triplicate overnight cultures were grown from three separate single colonies in 2 ml LB. Overnight cultures were diluted in 4 different 10 ml tubes containing 2 ml Mueller-Hinton broth (MHB)to an approximate of 8 × 10Statistical analysis was performed in Graphpad Prism 9 version 9.1.1 for Windows. Principal component analysis was performed on a standardized scale. The input was the FIC indices of all 4 interactions for every strain. Principal components were picked by performing a parallel Monte Carlo analysis on randomized data.The funding bodies had no say in data analysis, collection, interpretation, or decision to publish.E. coli blood stream infections admitted to Uppsala University Hospital on suspicion of infection between January 1st 2014 and December 31st 2017. Two β-lactams, AMP and CTX, were screened for interactions with two commonly used aminoglycosides, GEN and TOB. Out of the 500 strains screened, 254 (50.8%) were identified as susceptible to all four antibiotics and analysed further.The study material consisted of 500 isolates from patients with In the combinations of GEN with either β-lactam, no synergy between the antibiotics was found in any of the strains . Both coIn the combinations with TOB, no antibiotic pairs were shown to have a synergistic effect against any of the strains tested . SimilarAs is evident from The loadings for AMP-TOB and CTX-TOB are clustered together , indicating a possible correlation between the interaction profile of these two antibiotic combinations. The loadings for the interaction profiles of AMP-GEN and CTX-GEN suggested a similar correlation. These observations were verified by examining the cross correlations between the interaction profiles of the four antibiotic combinations. As illustrated in We continued by further investigating the cases where an antibiotic combination proved to be antagonistic against a strain. We performed time kill assays to confirm by a different assay the effect of the combination compared to each drug individually . In the In the strains where CTX-GEN had an antagonistic profile, the effects of the combination on survival were again overwhelmingly smaller than the theoretical additive interaction. One strain (DA63192) exhibited an additive profile between the two antibiotics until the 8h time point, where the combination of the antibiotics had a substantially smaller effect than the theoretical additive. In all cases the antibiotic combination failed to prevent resurgence, and the populations recovered after 24 hours. Similar to the two previous combinations, strains identified as having an antagonistic interaction with the AMP-TOB combination exhibited overwhelmingly higher survival when treated with the combination than the theoretical additive model predicted. In both cases, the populations recovered after 24 h.,in vitro.,,,52Even though antibiotic combination therapies are commonly used, clinical evidence demonstrating the beneficial effects of this treatment approach is lacking,in vitro knowledge of antibiotic interaction profiles and with a lack of explicit clinical guidelines.A key contributing factor to this discrepancy is that contrary to monotherapy schemes, combination treatments are prescribed empirically. As soon as antibiotic susceptibility data become available, treatment is usually refined  or switched.in vitro diagnostics and clinical outcome. However, systematizing interactions testing to that scale would require suitable clinical tests that quantify antibiotic interactions. Such tests should allow for the same systematic case-by-case-testing performed for monotherapy schemes.Data driven guidelines for combination therapy could bridge the gap between E. coli, we found that interaction profiles of the same antibiotic combinations varied significantly across strains, with responses to combinations ranging from synergistic additivity to substantial antagonism. This finding, also illustrated by different analyses and meta analyses of clinical data, as well as in vitro studies,,,in vitro responses to antibiotic combinations, antagonistic interactions would remain undetected. Approximately 3% of our screened strains exhibited clinically antagonistic interactions. That is a potential risk with this specific combination choice as it could potentially reduce treatment efficacy and be a contributing factor to the underperformance of clinical combination treatments. It should also be noted that of the 4 combinations tested, none had a significant synergistic effect against any of the 254 strains.For the specific case of β-lactam + aminoglycoside combination there are conflicting reports on whether β-lactam monotherapy is better or worse than the combination,E. coli blood stream infection strains.In empirical antibiotic combination treatments, antibiotics of the same class are often used interchangeably according to regional preferences.,,,In this study, we focused on strains that are susceptible to both antibiotic classes for two reasons. Firstly, in a clinical setting, after appropriate susceptibility testing, treatment is modified to exclude antibiotics to which a strain is resistant to. Secondly, single or double resistant strains would reach levels of clinical resistance to antibiotics with a multitude of different resistance mechanisms and mutations.E. coli. Especially since of the multiple β-lactam-aminoglycoside combinations tested here, not one exhibited a synergistic interaction. However, in multiple cases the combinations had antagonistic and even suppressive effects. The detection of antagonistic interactions between drugs used in treatment for sepsis raises a valid concern on this clinical practice, and it motivates the need for strain-specific interaction testing to avoid antibiotic combinations that might reduce the efficacy of treatment Having data driven recommendations for the use of in vitro testing of antibiotic combinations could prove to be beneficial clinical practice. Other combination schemes might be identified to show less risk and perhaps exhibit in vitro synergy. Finally, these findings also reveal a potential clinical trade-off that needs to be considered between the beneficial effect of increased coverage of antibiotic combinations versus the risk of antibiotic antagonism that can reduce treatment efficiency.Taking the findings of this study into account is a discouraging indication of the suitability of this empirical treatment for BSI Dan I. Andersson and Nikos Fatsis-Kavalopoulos are involved in patent application SE 2050304-1 relating to the CombiANT method. Remaining author has no declaration of interest to disclose.


In total, 7/13 (53.8%) units showed a ≥10% increase. Overall, 615 days on antibiotics per 1000 patients were saved. Interrupted time-series analysis established a declining trend in DOT/1000 patient-days relative to the pre-intervention trend (p = 0.002); a monthly decrease rate of 28.96 DOT/1000 patient-days . The intervention had no impact on antibiotic choice. Antibiotic use was successfully reduced in Greek NICUs using a “low-hanging fruit” approach. In resource-limited settings, similar targeted stewardship interventions can be applied.Antibiotics are commonly prescribed in Neonatal Intensive Care Units (NICU), where stewardship interventions are challenging. Lowering antibiotic consumption is desperately needed in Greece, a country with high antibiotic resistance rates. We sought to assess the effectiveness of a low-cost and -resource intervention to reduce antibiotic use in Greek NICUs implementing a “low-hanging fruit” approach. A prospective quasi-experimental study was conducted in 15/17 public NICUs in Greece (9/2016–06/2019). The intervention selected was discontinuation of antibiotics within 5 days for neonates with gestational age ≥ 37 weeks, no documented signs or symptoms of sepsis, CRP ≤ 10 mg/L and negative cultures within 3 days of antibiotic initiation. Impact was evaluated by the percentage of discontinued regimens by day 5, length of therapy (LOT) and stay. Trends of antibiotic consumption were assessed with days of therapy (DOT) per 1000 patient-days. Overall, there was a 9% increase ( Antibiotic resistance is considered one of the most serious threats to global public health and is associated with increased morbidity, mortality and healthcare costs. Leading public health organizations around the world agree that among other actions, antimicrobial stewardship interventions can lead to the reduction of antibiotic use, a key driver in the evolution of resistance to different classes of antimicrobials .Among European nations, Greece ranks highly in terms of antibiotic consumption and rates of antimicrobial resistance ,3. The lAntibiotics are the most commonly prescribed medications in Neonatal Intensive Care Units (NICUs) due to several predisposing factors associated with this group of patients, such as natural susceptibility to infections, prematurity, and birth-related complications, as well as postpartum events . VariatiInitiating antibiotic therapy in neonates is often driven by personal judgement rather than identifying or excluding infection following practice guidelines , indicatThe primary aim of this study was to assess the effectiveness of a structured intervention in order to reduce duration of antimicrobial use for culture-negative early-onset sepsis across Greek NICUs. The intervention was based on a “low-hanging fruit” approach of antibiotic stewardship practices, so that participating units could enroll patients using the existing medical personnel and no additional financial resources. A positive outcome could be an indication that low-cost initiatives can have a significant impact on prudent antibiotic use in different settings such as NICUs.A total of 1025 cases of neonates that met the inclusion criteria were identified; 507 in the pre- and 518 in the post-intervention period. Demographic characteristics and risk factors of neonates included in both study periods are listed on p = 0.003) in the number of neonates that received antibiotics for five or less days in the post intervention period; from 52.5% (266/507) in the pre- to 61.5% (319/518) in the post-intervention period. Examining the changes in each unit separately  to 7.5 days (IQR 6–10) across the units . Thirteeparately , there wparately . Overallp = 0.535). However, in the post-intervention period, a decline trend was observed in the DOT/1000 patient-days relative to the pre-intervention trend (p = 0.002), leading to a monthly decrease rate of 28.96 days of therapy/1000 patient-days   .The interventions did not seem to alter the prescribing patterns in the units with regard to antibiotics selected for the treatment of EOS. For the cases that empiric treatment was discontinued within 5 days, ampicillin and aminoglycosides constituted more than 90% of the DOTs administered on the first day and throughout the course, both before and after the interventions.p < 0.001, p = 0.043). In four units the median LOS decreased by 1 to 3 days, and in one unit the median LOS increased by 1 day.Regarding the median length of stay in the seven units that discontinued antibiotics early, in two units there was a statistically significant decrease by 2 days  compared to one death among 309 cases (3.2 deaths/1000 cases) in the post-intervention period.p = 0.003).Antibiotics are frequently prescribed in neonates for suspected EOS, even though the real risk is low. In view of antibiotics’ adverse outcomes and increased risk for infection with multidrug-resistant pathogens, antimicrobial stewardship in the NICU is important. In a country with high overall antimicrobial use and resistance rates, stewardship initiatives are needed immediately, and at the same time they need to be adapted to work in a resource-limited healthcare system. The main study findings indicate that an antibiotic stewardship intervention using a “low-hanging fruit approach” can reduce the length of antibiotic therapy in low-risk neonates treated for possible EOS in Greek NICUs. A simple intervention, in which data on antibiotic use were shared with medical staff and a goal was established to stop antibiotic therapy by day 5, led to an overall reduction of 615 antibiotic days/1000 neonates within 15 months. Interrupted time series analysis revealed a declining trend in antibiotic consumption in the post-intervention period. Participating units that increased discontinued antibiotic regimens by day 5 showed a moderate but statistically significant increase in the actual number of neonates that received ≤5 days of antibiotics in the post intervention period from 52.5% before to 61.6% after the intervention , participating NICUs were asked to report the first 15 antibiotic regimens given each month, including those given within the 72 h of life for presumed early-onset sepsis (EOS), in order to explore possible targets for improvement. After reviewing these data, a group of neonates that were given prolonged antibiotic courses without sufficient risk factors for infection was identified. Neonates with the following characteristics formed the target group for the intervention: gestational age ≥37 weeks, no evidence of clinical sepsis, CRP ≤10 mg/L during the first 72 h of life and negative cultures obtained within the first 3 days of antibiotic administration.In April 2018, participating NICUs received a complete analysis of the data collected and agreed to set the goal of antibiotic discontinuation within 5 days for neonates fulfilling the characteristics mentioned above. Two units decided not to move into the intervention phase. During the post-intervention period (April 2018–June 2019), data collection was adjusted to capture all cases treated for possible EOS. There were no other exclusion criteria, nor was it expected for participating units to change their practice or conduct specific lab exams at set time points. Additionally, it was up to the physicians’ clinical judgement to decide if they would stop the antibiotics.To evaluate the impact of the intervention for each unit, the length of therapy (LOT) (the number of days the neonate was receiving at least one antibiotic) was calculated, as well as length of stay (LOS) for the selected cases and the percentage of discontinued antibiotic regimens by day 5. Additionally, the mean length of therapy was calculated and used to estimate the gain or loss of antibiotic days by multiplying the difference of the means before and after the intervention with the number of neonates that met the criteria in the post-intervention period. The network’s overall change in consumption was expressed per 1000 neonates for the post-intervention period. Days of therapy (DOT) per 1000 patient-days were used to assess trends of antibiotic consumption. DOT was defined as the aggregate sum of the days of exposure to each antibiotic on a 7-day follow-up from the initiation of empiric treatment. Each antibiotic for each day administered contributed by 1. For example, a neonate that was on ampicillin and gentamicin for 5 days would have an LOT of 5 days, but a DOT of 10, as each of the two antibiotics was given for 5 days. Death before discharge was also followed up through medical records. Our manuscript follows SQUIRE 2.0 guidelines .Categorical data are presented in absolute and relative (%) frequencies, while continuous data are presented with mean, standard deviation, median and interquartile range (IQR). Chi-square tests of independence were used to compare demographic characteristics before and after the intervention period, as well as the Mann–Whitney test, since normality of continuous data did not hold (tested with histograms). The Mann–Whitney test was also performed to compare the length of stay of neonates pre- and post-intervention . Interrupted time series analysis was used to establish whether there was a change in trend of antibiotic consumption after the implementation of the intervention. Results are presented as β-coefficients and 95% confidence intervals of the antibiotic use rate change. Statistical significance (a) was set to 5%. All analyses were performed with STATA v.13.Protecting neonates from prolonged and unnecessary antimicrobial exposure constitutes a public health priority. Fully developed stewardship interventions require multiple resources in terms of personnel and financial support making them difficult to implement. Adapting stewardship practices to local needs improves outcome and encourages practicing teams to participate. Following a “low-hanging fruit approach”, a significant reduction in antibiotic use in a large network of NICUs was achieved. Implementing similar low-cost and low-resource actions could be successful in settings of high antibiotic consumption.


Appropriate rehydration has always been significant in treating diarrhoeal diseases in children. Irrational antibiotic use among diarrhoeal children has remained a major public health concern. Information regarding antibiotic use in young infants suffering from diarrhoea is very limited and a unique aspect of research. We aimed to investigate the prevalence of antibiotic use in the community among 2–6 months infants with diarrhoeal illnesses and having different nutritional status.We investigated a total of 5279 infants aged 2–6 months at Dhaka hospital, International Centre for Diarrhoeal Disease Research, Bangladesh, between September 2018 and June 2019. Among them, 257 infants were suffering from severe acute malnutrition (SAM). History of taking antibiotics was ascertained by direct observation of a prescription by a physician, the bottle of antibiotic or asking the caregiver about the name of antibiotic or its price that is very close to the usual market price of an antibiotic.P value = 0.003) compared to infants with SAM and use of antibiotics increased with age . Commonly used antibiotics were azithromycin (13.3%), ciprofloxacin (7.7%), erythromycin (7.7%) and metronidazole (2.6%). The proportion of receiving ciprofloxacin was significantly lower in infants with SAM compared to their non‐SAM counterparts .Overall, 52% of infants received antibiotics before hospital admission. Non‐SAM infants had higher odds of receiving antibiotics (adjusted odds ratio [aOR] = 1.52, 95% confidence interval: 1.18, 1.97, The study underscores the excessive use of antibiotics among diarrhoeal infants, which is already a major public health concern in low‐ and middle‐income countries. Irrational use of antibiotics in the management of diarrhoeal diseases has been a long‐standing public health burden globally including South Asian countries.Antimicrobial resistance is increasing rapidly in South Asian regions, which is associated with the excessive use of antibiotics.There is limited information regarding antibiotic exposure in young Bangladeshi infants suffering from acute watery diarrhoea.A large proportion of young infants are being exposed to antibiotics at a very early age who were suffering from diarrhoea.Antibiotic consumption was higher in infants without severe acute malnutrition in contrast to those with severe acute malnutrition.Consumption of macrolides and fluoroquinolones was considerably elevated amidst this vulnerable age group.Diarrhoea, which is recognised as a global burden, causes 440 000 child deaths world‐wide and is one of the leading causes of death among children aged less than 5 years.In a low socio‐economic setting, financial conditions and scarcity of diagnostic resources combined with the huge burden of the disease make it difficult for the patients to find out the causative organisms. As a result, antimicrobials are prescribed empirically, which is not justified.Severe acute malnutrition (SAM) is recognised as a major global public health burden that threatens the lives of more than 14 million children under 5 years of age world‐wide.Data were extracted from the screening data set of a study entitled ‘Pilot of a Prebiotic and Probiotic Trial in Young Infants with Severe Acute Malnutrition’ (NCT03666572). A total of 5279 infants, aged 2–6 months, were screened between September 2018 and June 2019 when the infants were brought at Dhaka Hospital, International Centre for Diarrhoeal Disease Research, Bangladesh, for management of diarrhoeal illnesses. Dhaka hospital admits diarrhoeal children to the out‐patient department, short stay unit, longer stay unit and intensive care unit as required by the clinical condition of the patients. All infants were admitted to the hospital irrespective of their dehydration status and later sent to different units of the hospital after assessment and data collection. The patients were sick enough to warrant admission to the respective units of the hospital as required.A total of 257 infants were suffering from SAM in our study. SAM was defined in less than 6 months of age by using World Health Organization (WHO) classification criteria. Weight‐for‐length less than −3 Z‐score and presence of bilateral pitting oedema were the two identifying points.The participants' caregivers were asked if the infants consumed antibiotics in order to treat their present diarrhoeal disease for which they are seeking medical attention. The history of antibiotic usage was assessed by verification of a prescription by a registered physician, or showing the bottle that contained the antibiotic or asking the caregiver about the name of antibiotic or its price that is very close to the usual market price of an antibiotic. Morbidity and nutritional status were evaluated by physicians and trained health‐care workers immediately upon arrival of the patients at the hospital. Different clinical parameters were assessed clinically like septic shock, jaundice, Down's syndrome, presence of Anuria by a registered physician at the time of screening. Screening data were analysed anonymously, and this activity has the approval from both the Research Review Committee and the Ethical Review Committee of International Centre for Diarrhoeal Disease Research, Bangladesh.Severe acute malnutrition (SAM): Weight‐for‐length Z‐score value less than −3 SD of WHO growth standards or presence of bilateral pedal oedema.Not severe acute malnutrition (non‐SAM): Weight‐for‐length Z‐score value greater than − 3 SD of WHO growth standards.2 test or Fisher's exact test were applied for comparison between the categorical variables. Student's t‐test was used to compare the continuous variables. Cochran–Mantel–Haenszel test was performed to determine the age‐specific prevalence of antibiotic use in children with and without SAM. The strength of association was determined by calculating the odds ratios (ORs) and their 95% confidence intervals (CIs). A probability of <0.05 was considered statistically significant. We have performed multivariable logistic regression analysis to investigate the relationship between antibiotic use and the nutritional status of the children.Data were analysed using SPSS for Windows . χP value < 0.001; 58.8 cm vs. 60.6 cm, P‐value < 0.001). Infants with bilateral pedal oedema were not exclusively breastfed compared to infants without oedema, and the difference was statistically significant . In this study, more than half (52%) of the young infants received antibiotics before hospital admission. The use of antibiotics was higher among infants without SAM (52.5%) compared to infants with SAM (42%), and the difference was statistically significant   had SAM. Of the 257 infants, 99 (38.5%) had bilateral pedal oedema. The mean (SD) age of the participants was 4.24 1.14) months. 41.2% were female, and there was no difference between children with SAM and non‐SAM in terms of sex. The average weight and length were significantly lower in SAM infants compared to non‐SAM infants , and non‐SAM infants had higher odds of receiving antibiotics  before hospital admission compared to infants with SAM. , ciprofloxacin , erythromycin  and metronidazole  (Table S1). Among all the antibiotics used by the study participants, ceftriaxone (n = 53) and cefixime (n = 53) had 1% prevalence, whereas flucloxacillin, levofloxacin, meropenem, amoxicillin, ampicillin, amikacin, gentamicin, clarithromycin, doxycycline, ceftazidime, cefuroxime, cefradine, cefaclor, cefpodoxime, ceftibuten, co‐trimoxazole, pivmecillinam, trimethoprim had less than 1% prevalence. Around 19% of the caregivers could not mention the name of the antibiotic received by the infants, and only 1% of young infants received more than one antibiotic. The proportion of ciprofloxacin use prior to hospital admission was more in the non‐SAM group in comparison to malnourished group (7.97% vs. 2.7%), which was statistically significant .Commonly used antibiotics in infants were azithromycin . The prevalence of antibiotic use in non‐SAM infants follows a similar trend but was different in SAM children at 5 and 6 months of age. The prevalence of antibiotic use in SAM infants increased with age up to 4 months of age, then decreased at 5 months, but again increased at 6 months of age and the differences were statistically significant .Figure The study results show the practice of antibiotic prescribing to infants suffering from diarrhoeal diseases prior to their hospital admission. In a low‐to‐middle income country like Bangladesh, the increased use of antibiotics in such young infants is striking. Among 5279 infants, more than half the participants were prescribed antibiotics. There can be multiple determinants of the higher use of antibiotics in a country like Bangladesh. In low‐income countries, medicines are available to the general population from diversified sources including hospitals, local pharmacies, licensed drug stores, roadside drug shops and unskilled practitioners or traditional healers.We observed antibiotic intake was significantly more in non‐SAM infants in contrast to the malnourished group. Malnutrition itself is a critical condition that makes it difficult for the children to recover and is a bad prognostic factor.Different forms of malnutrition are associated with socio‐economic inequality.Antimicrobials can be used dependably for cases with bloody diarrhoea suspecting probable shigellosis and cholera‐like illness. If amoebiasis or giardiasis has been confirmed by investigation, it is indicated to be treated by metronidazole.In our study population, antibiotic consumption has been increasing with age, which needs further evaluation. We can theorise that as rotaviral diarrhoeal episodes tend to increase with age in children under 1 year of age, this factor could be contributing to this finding.The percentage of exclusively breastfed (EBF) infants was 21% in our study, which is relatively lower than the country prevalence of 65%.For the pressing need to refine antibiotic use and fight AMR, WHO has adopted a global action plan on AMR (GAP) in collaboration with Food and Agriculture Organisation of the United Nations and World Organisation for Animal Health (OIE) at the World Health Assembly in 2015.Lessons can be learnt from different environments across the globe about the potential adverse effects of AMR. Different studies from Mexico, Brazil, China, Thailand and France have reported increased mortality rate of intensive care unit patients which is associated with AMR.Our study has some limitations. Data were collected from the screening records, which held limited information restricting us to further analyse the possible relationship of antibiotic consumption with different variables, for example; socio‐economic status, birth history of the infants describing prematurity and low birthweight, previous hospital admission, the prescribed antibiotic was from a registered physician or local drug dispenser. The data derived were in a cross‐sectional manner, which limited us in investigating the trend of antibiotic use with age among young infants, which would have been possible if we possessed longitudinal data. Inadequate morbidity information confined us from investigating the rationality of antibiotic use in these younger infants, such as the history or clinical evidence related to watery or bloody diarrhoea, dehydration status. We did not follow‐up the participants as this was a cross‐sectional study and we consider it as a limitation. However, this does not affect the primary objective of the study, which is to investigate the prevalence of antibiotic use among diarrhoeal infants in this particular age group before seeking facility‐based management.The study illustrates the higher trend of antibiotic use among young infants, which is rather alarming. This further begets the question of what should be done to control this rampant use of antibiotics in diarrhoeal management. Excessive antibiotic exposure may bring about further complications, for example, AMR; a worrisome threat that is slowly spreading over the world. Policymakers of the country also need to step up and formulate new policy and guideline to control this irrational antibiotic prescribing practice in Bangladesh and elsewhere.Table S1. Six most frequently consumed antibiotics; overall and in infants with and without SAMClick here for additional data file.


Tensile strength of concrete is the basic property when estimating the cracking resistance of the structure and when analysing fracture processes in concrete. The most common way of testing tensile strength is the Brazilian method. It has been noticed that the shape and size of specimens influence the tensile splitting strength. The experiments were performed to investigate the impact of cylinder’s length on tensile concrete strength received in the Brazilian method. During the experiment the tensile concrete strength was tested on two different sizes cylindrical specimens: 150 mm × 150 mm and 150 mm × 300 mm. Experiments were performed in two stages, with two types of maximum aggregate size: 16 mm and 22 mm. The software “Statistica” was used to perform the broad scale statistical analysis. When comparing test results for shorter and longer specimens, the increase of tensile splitting strength tested on shorter cylinders was observed (approximately 5%). However, when performing deeper statistical analysis, it has been found that the length effect was not sensitive to the strength of the cement matrix and the type of aggregate but was influenced by the aggregate size. Further experiments are needed in order to perform a multi-parameter statistical analysis of scale effect when testing the splitting tensile strength of concrete. Concrete is considered as a quasi-brittle material. Tensile strength of concrete is much lower than its compressive strength. Although the compressive strength of concrete is of paramount importance when designing concrete structures, the tensile concrete strength cannot be neglected in design. The tensile concrete strength is the basic property when estimating cracking resistance of structure and when analysing fracture processes in concrete. Furthermore, the tensile strength plays the important role not only in case of serviceability states but also in some cases of ultimate states, for example flexural capacity of unreinforced and slightly reinforced members, shear capacity in members without shear reinforcement, bond failure and minimum reinforcement. Some aspects of the utilization of concrete tensile strength in the design of concrete structures have been discussed in the CEB Bulletin No. 237 .Concrete is a composite material. The bulk of concrete is made up of fine and coarse aggregates (crushed or gravel) and therefore concrete properties are significantly influenced by the aggregate type and granulation ,3,4. HowAll these conditions influence concrete quality and necessitate experimental testing of concrete properties and evaluating test results using statistical methods. Tests should be performed at standard conditions and on standardized specimens. When designing concrete structure on the basis of rules given in constructional standards like Eurocode 2 , AmericaThe modes of concrete failure in compression are subject to a size effect. In general, smaller specimens exhibits greater stress at failure than larger ones. Some findings reported in the literature suggest that size effect exists also when testing other properties of concrete like tensile strength ,14,15,16The basic methodology to obtain concrete tensile strength should be a uniaxial tensile test. The proper assessment of the uniaxial tensile strength of concrete is considerably affected by the testing technique. The specimen configuration and the local stress concentration, the type of the gripping system and the testing machine stiffness are important factors which affect the obtained results . Even whThe uniaxial tensile test is conducted on rectangular prisms of a cross section 100 mm × 100 mm or 150 mm × 150 mm and the length equals two times the specimen’s width. The flexural tensile test can be performed using a beam specimen in three point bending test and four point bending test. The tensile strength obtained in the three-point bending test is 13% higher compared to the strength from the four point bending test. Therefore, the loading arrangement by two concentrated forces is recommended in the code . In the The concrete splitting tensile strength is calculated from Equation (1).When designing concrete structures, the uniaxial tensile stress should be used. Noting that the values obtained of the Brazilian splitting method are higher than those got from the uniaxial tensile test, the conversion factor is recommended in Eurocode 2 , ,L16N ,H22N ,L22N ,H16G (higher strength concrete with maximum granite aggregate size 16 mm),L16G (lower strength concrete with maximum granite aggregate size 16 mm).The experimental investigation was performed to analyse the influence of a cylinder’s length on the concrete splitting tensile strength. During the experiment the tensile concrete strength was tested on two different sizes cylindrical specimens. Cylinders were characterized by the same diameter 150 mm and varied different length 300 mm and 150 mm. Six concrete mixtures were designed for forming the specimens. The additional variable parameters during the experiment were: two types of aggregate , two maximum sizes of aggregate (16 mm and 22 mm), and two cement-water ratios (C/W = 1.8 and C/W = 2.6). Taking into account the changing parameters of the mixtures, the following symbols for series of tested specimens were applied:The main aim of the experiment was to evaluate the impact of cylinder’s length on tensile concrete strength received in the Brazilian method. When changing other parameters of concrete mixtures, it was also possible to analyse the influence of the concrete matrix strength and the type of aggregate on tensile strength when testing specimens of different length.The experiments were performed in two stages. In the first stage concrete mixtures were based on the gravel and the granite aggregate, both with the maximum size 16 mm and in the second stage the gravel aggregate of the maximum size 22 mm was used. The compositions of concrete mixtures are shown in The composition of the aggregate grading is the important task when designing concrete mixtures. In the performed experiment, the proportion of aggregate granulation was very similar in all concrete mixtures, both with gravel and granite aggregate and the grading curves were located between the upper and the lower limit curve.The strength properties of cement matrix and aggregates were evaluated in additional tests. The strength of hardened cement mortar was tested on beams 40 mm × 40 mm × 160 mm in size. The tests were carried out in accordance with the code  in a thrThe strength of aggregate was determined on the basis of the aggregate crushing test according to the Polish recommendations . StandarConcrete compressive strength was tested in the uniaxial compression test on cylindrical specimens 150 mm × 300 mm, according to the procedure given in the code . Strengtct,sp15/30—tensile splitting strength tested on cylinder 300 mm long, fct,sp15/15—tensile splitting strength tested on cylinder 150 mm long.The tensile splitting strength of concrete was tested in the Brazilian method . Two types of cylinders were used during the test: the cylindrical specimens 150 mm in diameter and 300 mm in length, and the cylindrical specimen 150 mm in diameter and 150 mm in length. The obtained test results are presented in A higher splitting tensile strength of concrete was noted for specimens with nominal dimensions of 150 mm × 150 mm compared to specimens 150 mm × 300 mm for concretes contained natural gravel and crushed granite with the maximum aggregate size 16 mm which were tested in the first stage of experiment. The increase of tensile strength for concretes with the gravel aggregate was 6.6% for the stronger concrete (C/W = 2.6) and 15.9% for the weaker gravel concrete (C/W = 1.8). In case of concretes with the granite aggregate, the strength increase was 2.9% for the lower C/W and 11.7% for the higher C/W. However, this tendency was not confirmed for specimens tested in the second stage of the experiment which were made of concrete with the gravel aggregate of the maximum size 22 mm. In case of those specimens the splitting tensile strength for shorter cylinders were lower compared to longer cylinders. The tensile strength decrease was 4% and 7.5% for stronger concrete (C/W = 2.6) and weaker concrete (C/W = 1.8), respectively.higher tensile strength was obtained for concretes with granite aggregate, although the lower aggregate strength of gravel mix was measured ; it can be concluded that the shape of aggregate grains (gravel or crushed) is of importance,the influence of maximum aggregate size in concretes with gravel aggregate was not clearly observed,the increase of the cement matrix strength caused the increase of the tensile strength.When analysing tensile strength of concrete together with other changing parameters—the strength of cement matrix and the aggregate crushing see , the folThese conclusions were suspected but when investigating length effect, the unique trend cannot be deduced from the obtained test results. It can be concluded that strength properties of aggregate and cement matrix are not significant when investigating the impact of the specimen’s length on the tensile strength of concrete. When analysing the influence of the maximum aggregate size on the tensile strength it was difficult to find coherent conclusions. Therefore, more advanced statistical analysis of test results was performed.critical).The software Statsoft’s “Statistica”  was used to perform the broad scale statistical analysis of the length effect on testing tensile splitting strength of concrete. In the first step of the analysis, the normality Shapiro-Wilk test of the obtained tensile strength results was performed. Two samples were analysed separately: the first one consisted of all test results for longer cylinders and the second one for all test results for shorter cylinders . The norTwo regression functions were generated (Equations (4) and (5)), as shown in The regression function for tensile strength of experimental results for longer cylinders:T—total number of calculation and forecast periods,n—number of forecast periods,i—the actual value of the variable in the period i,Yip—the predicted value of the variable in the period i.YThe Mean Absolute Percentage Error (MAPE) was calculated in order to examine the fit between the plotted functions and the test results, from the following formula (Equation (6)):ct15/15 and fct15/30.T—total number of calculation and forecast periods,n—number of forecast periods,i—the predicted value of the variable fct15/15 in the period i,Yip—the predicted value of the variable fct15/30 in the period i.YThe obtained MAPE was 2.8% for tensile strength of longer cylinders and 1.4% for tensile strength of short cylinders. It has been concluded that a good fit was obtained between the generated regression functions and the test data in both cases. The regression functions for longer and shorter cylinders described by Equations (4) and (5) were used to investigate the length effect. The Mean Percentage Error (MPE) was calculated (Equation (7)) in order to analyse the relation between predicted values of the variables fThe MPE between the regression functions generated for longer and shorter specimens was +5.53%. Therefore, it can be concluded that approximately 5% higher tensile splitting strength is obtained when testing concrete strength on cylindrical specimens 150 mm long comparing to tensile splitting strength obtained on cylinders 300 mm long.The role of other parameters at analysing length effect was examined. It appeared that the flexural strength of cement matrix and the aggregate crushing were not important whereas the maximum aggregate was found to be the significant parameter which also influenced the length effect. Therefore, this parameter was taken into account in the next step of the statistical analysis.max = 16 mm and Dmax = 22 mm. Two samples of data containing test results for longer and shorter cylinders were created in every group. Therefore, four samples were analysed:max = 16 mm ,test results of tensile strength tested on cylinder 300 mm long for concretes with Dmax = 16 mm ,test results of tensile strength tested on cylinder 150 mm long for concretes with Dmax = 22 mm (series H22N and L22N),test results of tensile strength tested on cylinder 300 mm long for concretes with Dmax = 22 mm (series H22N and L22N).test results of tensile strength tested on cylinder 150 mm long for concretes with DAll test results were separated into two groups in accordance with the maximum aggregate size DThe Shapiro-Wilk test was applied to investigate the normality of the data distribution. The normal distribution was confirmed for all samples .max = 16 mm:for tensile strength values of shorter cylinders, DThe regression functions were generated (Equations (8)–(11)):max = 16 mm:for tensile strength values of longer cylinders, Dmax = 22 mm:for tensile strength values of shorter cylinders, Dmax = 22 mm:for tensile strength values of longer cylinders, DThe comparison of regression functions for two groups of samples is presented in max = 16 mm,MAPE = 1.41%, for tensile strength values of shorter cylinders, Dmax = 16 mm,MAPE = 2.58%, for tensile strength values of longer cylinders, Dmax = 22 mm,MAPE = 1.96%, for tensile strength values of shorter cylinders, Dmax = 22 mm.MAPE = 1.38%. for tensile strength values of longer cylinders, DThe mean absolute percentage error MAPE was calculated from Equation (7) to investigate the generated functions:max = 16 mm,MPE = + 8.74%, for tensile strength tested on cylinders made of concrete with aggregate Dmax = 22 mm.MPE = −6.21%, for tensile strength tested on cylinders made of concrete with aggregate DLow values of MAPE were obtained and therefore the functions were recognized as a well-fitted approximation of experimental results. The Equations (8)–(11) were applied in the comparative analysis. When comparing regression functions generated for shorter and longer cylinders, the mean percentage error MPE was calculated from Equation (7) in case of two separate statistical samples:max = 16 mm and “–“ in case of Dmax = 22 mm), a very interesting and unsuspected observation can be made.When comparing line plots presented in max = 16 mm , the higher strength was obtain when testing tensile strength on cylinders 150 mm long. The length effect has appeared very similar as in the case when investigating all test results together, although a little bit higher MPE was calculated when analysing the limited test data (without test results of concrete with Dmax = 22 mm). On the contrary, the opposite relation occurred when separating test results for specimens made of concrete with the aggregate Dmax = 22 mm (series H22N and L22N). The higher tensile splitting test was obtained for cylinders 300 mm long. All test results for shorter cylinders did not exceed the results for long cylinders and it found reflection in a minus sign of the calculated MPE.In case of tensile splitting strength of concrete with aggregate of DThese findings indicate that all changing parameters should be taken into consideration and should be examined during the statistical analysis of test data. In the performed research, the maximum aggregate size appeared to be the additional parameter which also played the significant role when analysing length effect at testing tensile splitting strength.max = 16 mm and Dmax = 22 mm.Recapitulating, the effect of the specimen’s length on the tensile splitting strength was observed. However, the performed investigation did not allow to determine a general quantitative relation between tensile splitting strength tested on cylinder 150 mm long and on cylinder 300 mm long, mainly because different conclusions were drown when analysing test data separately for concrete specimens with different maximum size DConcrete, like other composite materials, consists of fractions with different stiffness. Given that normal strength concrete is a material made up of stiff aggregate particles embedded in a softer hydrated cement matrix, therefore it cannot be used without activating the tensile capacity. Furthermore, compressive failure of concrete is, in reality, a tensile failure perpendicular to the direction of the principal compressive stress. Both compressive as well as tensile failure are subject to a size effect where smaller specimens exhibit higher stresses at failure than larger ones. This effect can be clearly observed when testing strength properties of concrete on different size specimens. The size effect is primarily associated with the utilization of the concrete tensile strength. The most important cases when tensile strength is the necessary design parameter are specifically addressed in the CEB Bulletin no. 237 , among tThe proper assessment of the uniaxial tensile strength of concrete is of the paramount importance when designing concrete structures and analysing the failure processes in members made of concrete. The existing test data on testing concrete properties reported in the literature  offer eTensile strength of concrete is mostly tested in splitting tensile test. Therefore, the designers have to calculate the uniaxial tensile strength using the simple relation given in Eurocode 2 , (EquatiThe performed experimental investigation has shown that the influence of the length of cylindrical specimens on the tensile splitting strength of concrete is an important experimental topic worth to deal with and allowed to indicate the course for further research. The analysis of length effect requires experiments which should be performed on normal and high strength concretes with different type and granulation of aggregate and on more numerous samples. The advanced multi-parameter statistical analysis of the obtained experimental results would help to evaluate the length effect with taking into account not only the size of the specimen but also other parameters, like the maximum aggregate size.


Diagnostic work-up of IgG4-related disease (IgG4-RD) pleural involvement is a complex task, as there is a broad spectrum of differential diagnoses to consider. We report the case of a patient presenting with relapsing pleural effusion, discussing the main challenges for achievement of a definite diagnosis. A 63-year-old man was admitted for pleural effusion prevalent on the ride side, initially labeled as idiopathic non-specific pleuritis, based on tissue evaluation after a medical thoracoscopy. He was started on steroids with initial improvement, but a later CT scan showed a relapse of pleural effusion associated with diffuse pleural thickening; a subsequent surgical pleural biopsy revealed features suggestive for IgG4-RD, with a marked increase of IgG4 positive plasma cells. High IgG4 serum levels were also found. The present case underlines the importance of increasing awareness of this potential condition among physicians in order to properly guide the diagnostic work-up, as it is likely that IgG4-RD accounts for a proportion of patients with pleural effusions, labeled as idiopathic. In particular, in patients with unexplained pleural effusion, IgG4-RD should be included among differential diagnoses when lymphoplasmacytic infiltration is observed, and a multidisciplinary interaction between clinicians and pathologists appears crucial for an accurate diagnosis and an appropriate management. IgG4-related disease (IgG4-RD) is a chronic, systemic fibro-inflammatory disorder characterized by lymphoplasmacytic infiltration rich in IgG4-positive plasma cells, storiform fibrosis, obliterative phlebitis and often, but not always, elevated serum IgG4 levels ,2.IgG4-RD, firstly described in the pancreas, can virtually affect any organ, including kidneys, retroperitoneum, aorta, lymph nodes, skin, as well as lungs and pleura. Thoracic involvement occurs in up to 50% of patients with heterogenic features, such as mediastinal lymphadenopathy, lung consolidations, pericarditis and pleural manifestations ,4. In paDiagnostic work-up of IgG4-RD thoracic involvement may be challenging, as there is a broad spectrum of differential diagnoses to consider, including multisystemic diseases and conditions with similar clinical-radiological appearance or with elevated IgG4 tissue levels.Therefore, a multidisciplinary interaction between clinicians from different specialties and pathologists appears essential for an accurate diagnosis. In this context, the role of interventional pulmonologists and thoracic surgeons is crucial to obtain adequate pleural samples, in order to allow pathologists to detect the peculiar histological features, often unevenly distributed.Herein, we report the case of a patient presenting with relapsing pleural effusion and we discuss the main challenges of this complex diagnostic work-up.A 63-year-old man was admitted to our Respiratory Disease Unit at the University Hospital—Ancona, for a 6-month exertional dyspnea and bilateral pleural effusion prevalent on the ride side, detected on chest computed tomography (CT).He was former smoker without occupational exposure to asbestos. His medical history was remarkable for asymptomatic brain aneurysm, blood hypertension, multiple lumbar disc herniation. On admission to our unit, physical examination, oxygen saturation on room air, heart rate and blood pressure were normal, whilst breathing sound was suppressed at the third right lower lung fields.The patient first underwent a repeated CT scan that allowed us to rule out a pulmonary embolism and confirmed moderate right pleural effusion with parietal and visceral pleural thickening, in the absence of significant parenchymal abnormalities . ThoraciA subsequent medical thoracoscopy (MT) revealed the presence of yellow pleural fluid  and parietal pleura hyperemia with fibrotic plaques . Ten pleAn extensive diagnostic work-up, including echocardiogram, abdominal angiography CT scan, autoimmune, viral, and bacterial serology, failed to detect any potential known cause of NSP and blood tests were normal, except for a mild elevation of C-reactive protein. Thus, the patient was diagnosed with idiopathic NSP and therapy was started with steroids  for a surgical pleural biopsy. Histological examination revealed a diffuse fibrosing pleuritis, with occasional hyaline features, fibrinous exudate, and a dense lymphocytic and plasma cell inflammation. Interestingly, inflammatory infiltrate was diffuse but with irregular distribution throughout the histological specimen. Immunoperoxidase stains showed a marked increase of IgG4 positive plasma cells (up to 50/HPF) with an IgG4 disease . High Ig disease . Based oAfter exclusion of systemic involvement of IgG4 disease, steroid therapy was started  with a complete and stable resolution of pleural effusion, improvement of respiratory symptoms and a progressive reduction of IgG4 serum levels, returned within normal limits (64 mg/dL).The present case report describes a relatively uncommon cause of relapsing exudative monolateral pleuritis, finally diagnosed with IgG4-RD, but initially labeled as idiopathic NSP, underlining the challenging diagnostic work-up of this condition. IgG4-RD is an increasingly recognized fibro-inflammatory disorder, firstly described in association with autoimmune pancreatitis, salivary and lacrimal glands inflammation, formerly known as Mikulicz’s disease . ClinicaThoracic involvement, indeed, includes interstitial lung disease, inflammatory pseudotumours, fibrosing mediastinitis, lymphadenopathy and pleural disease ,4. With Overall, IgG4-RD pleural disease occurs more frequently in men 78%), the mean age is 63.5 ± 14.7 years old and an exudative pattern is reported in almost all cases %, the me. Cell blWith reference to serology, it is important to underline that high IgG4 levels (>135 mg/dL), significantly contribute to the achievement of a definite diagnosis in a suggestive context, but their absence does not allow us to exclude this condition. IgG4 levels within normal limits were detected in approximately 50% of biopsy-proven, clinically active IgG4-RD patients, while, on the contrary, high concentrations were found in 5% of healthy subjects . These dTherefore, in this context, histopathological assessment plays a crucial role in order to improve the diagnostic confidence, as differential diagnosis ranges from other multisystem diseases , to those with similar clinico-radiological features  or to conditions with local and systemic inflammation with elevated IgG4 tissue levels  . FurtherThoracoscopic findings at endoscopic view are usually not specific, including pleural hyperemia, pleural thickening, hyalinized white plaques, expression of collagen fibers deposit and small nodular lesions . The maiIgG4-RD lung involvement may occur either in isolation or as part of a multisystem process, and, due to its significant prevalence, all patients with an established diagnosis of extra thoracic disease should undergo proper chest imaging. In an elegant study by Corcoran and colleagues [Therefore, an accurate assessment of any potential organ involvement is essential for a proper management, in order to avoid irreversible damages. Steroid therapy remains the first line approach, with dosing varying according to local practice, but 0.5–1 mg/kg daily of oral prednisone is commonly used. Assuming a favorable response, this can be gradually tapered over 3–6 months. In resistant/relapsing cases, additional immunomodulatory medications such as rituximab, azathioprine and mycophenolate can be used, although there is no robust evidence to guide treatment choice in this field .Pleural involvement in IgG4-RD can occur in isolation, or association with pulmonary or extrapulmonary manifestations, and its prevalence appears to increase over time, althougth it remains an under-recognized condition. It is important to increase awareness of this potential condition and its varying manifestations among physicians from different specialties in order to properly guide the diagnostic work-up, as it is likely that IgG4-RD accounts for a proportion of patients with pleural effusions labeled as idiopathic. In particular, in patients with unexplained pleural effusion, IgG4-RD should be included among differential diagnoses when lymphoplasmacytic infiltration is observed, and a multidisciplinary interaction between clinicians and pathologists appears crucial for an accurate diagnosis and appropriate management. Further studies on large cohorts are needed to assess true prevalence, clinical evolution, and response to treatment of pleural IgG4-RD.