Neoadjuvant immunotherapy has preliminarily been effective in multiple resectable cancers. However, its safety is still largely unknown.A systematic literature search was conducted in PubMed, Embase, Web of Science, and Cochrane Library up to February 28th, 2021. Pooled incidence and risk ratio (RR) of adverse events were calculated using the R software.vs. 76% (95% CI 47%-92%); P < 0.001] and high-grade  trAEs compared with neoadjuvant ICIs alone. The most common high-grade trAEs were lipase increased , colitis  and transaminitis  for neoadjuvant ICIs, and neutropenia , anemia  and AST increased  for neoadjuvant ICIs plus chemotherapy. Furthermore, the incidence rates of progressive disease while on treatment, treatment-related surgical delays and deaths were 6% , 3.2% (12 of 377 patients) and 0.47% (5 of 1075 patients), respectively.Twenty-eight studies involving 2863 patients were included. First, the incidence for all-grade treatment-related adverse events (trAEs) was 94% , with 43%  for high-grade trAEs. For different treatment groups, neoadjuvant immune checkpoint inhibitors (ICIs) plus chemotherapy was associated with a higher incidence of all-grade [99%  Compared with neoadjuvant ICIs alone, neoadjuvant ICIs plus chemotherapy had a higher incidence of trAEs. In addition, neoadjuvant immunotherapy had a low rate of progressive diseases, surgical delays and deaths.  Immune checkpoint inhibitors (ICIs) have demonstrated remarkable therapeutic efficacy in various advanced malignancies . In viewHowever, with the start of T cell-mediated antitumor immunity, ICIs could induce the infiltration of immune cells into normal tissues and unleash T cells with subsequent production of proinflammatory cytokines such as interleukin-2 and interleukin-7 . The incAlthough an increasing number of studies have reported the safety of neoadjuvant immunotherapy, there is still a lack of comprehensive understanding. Here, a meta-analysis was conducted to comprehensively assess the safety of neoadjuvant immunotherapy.A systematic literature search was conducted in PubMed, EMBASE, Web of Science and Cochrane Library up to February 28th, 2021. The search term was as follows: [neoadjuvant OR “Neoadjuvant Therapy” (Mesh)] AND  AND (cancer OR tumor OR carcinoma OR “Neoplasms” [Mesh] OR “Carcinoma” [Mesh]). Reviews, letters, editorials, comments, meeting abstracts and case reports were not included. Data from different treatment arms within the same study were extracted and reported separately. The references of relevant articles and reviews were also searched for additional eligible studies potentially overlooked.To be eligible, studies had to satisfy all the following inclusion criteria: (1) studies included cancer patients treated with neoadjuvant ICIs, neoadjuvant ICIs plus adjuvant ICIs, or neoadjuvant ICIs plus chemotherapy; (2) studies clearly reported the incidence of adverse events; (3) studies were published in English. When duplicate reports were identified, the one with a larger sample size and more detailed information was selected.Two authors (JX and YW) carried out the systematic literature search independently. If there were any disagreements, the study would be re-evaluated by a third investigator (YX).This meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .The title, first author, publication year, cancer type, drug, dose, phase of the trial, number of participants, and criteria for adverse events reported in each article were extracted. In addition, all-grade and high-grade (grade 3 or higher) adverse events data were also extracted separately. Data extraction was conducted independently by two investigators (JX and YW), and any discrepancies were resolved by discussing with a third author (YX).The Cochrane Collaboration’s tool was used to evaluate the quality and risk of bias of the included articles , includi2 statistic was used to quantify the inconsistency. The I2 cutoffs used to determine inconsistency were very low (< 25%), low (25% to < 50%), moderate (50% to < 75%), and large (> 75%). A fixed-effects model was adopted to pool the results if significant heterogeneity was not present (I2 < 50%). Otherwise, a random-effects model was used. Chi-squared test was used to compare the incidence of adverse events between different groups. In addition, the incidence rates of specific types of adverse events were also calculated in different groups. We focused on adverse events reported by at least 10% of the studies.The primary endpoint of this meta-analysis was to determine the overall incidence of adverse events in neoadjuvant immunotherapy. All-grade and high-grade adverse events were calculated respectively. In addition, subgroup analysis was conducted in different treatment groups to further explore the safety of different treatment modalities based on neoadjuvant immunotherapy. Rate consolidation was conducted using five methods . The method with results closest to the normal distribution was selected. Statistical heterogeneity in the included studies was assessed using Cochrane’s Q statistic, and IThe secondary objective was to compare the incidence of adverse events between the neoadjuvant ICIs plus chemotherapy group and the chemotherapy control group in the included randomized controlled trials (RCTs). Pooled risk ratio (RR) and 95% confidence intervals (CIs) were calculated. A fixed-effects model or a random-effects model was adopted due to the heterogeneity described above.In addition, the incidence of treatment-related events, including progressive diseases, surgical delays and deaths, were calculated to further evaluate the safety of neoadjuvant immunotherapy. The incidence rates of surgical delays and deaths were calculated by dividing the total number of surgical delays or deaths by the total number of patients in the relevant studies.P < 0.05 was considered statistically significant for all the analyses.All the analyses above were performed using R software, version 4.0.0  with the package Meta and the function of metaprop. A two-sided The systematic literature search brought up 5730 records, from which 68 potentially eligible studies were collected after screening the titles and abstracts. Ultimately, 28 studies were selected after reviewing the full text –37. The The characteristics of the 28 included studies were summarized in P < 0.001). However, there was no significant difference between neoadjuvant ICIs plus adjuvant ICIs and neoadjuvant ICIs (P = 0.780).Among the studies included, the overall incidence of trAEs was available in 16 studies. The overall incidence of all-grade trAEs was 94% . However, there was no significant difference between neoadjuvant ICIs plus adjuvant ICIs and neoadjuvant ICIs (P = 0.377).In addition, the overall incidence of high-grade trAEs was 43% .We compared the incidence of trAEs between the combined ICIs group and the single ICI group to further explore the influence of combined ICIs on trAEs in neoadjuvant immunotherapy. As shown in vs. 61% , P = 0.003] were higher in ≥ 3 cycles than in < 3 cycles, with no difference in high-grade trAEs between the two groups. In addition, in neoadjuvant ICIs plus chemotherapy, the incidence of high-grade trAEs was higher in ≥ 4 cycles than in < 4 cycles .To determine the impact of the number of cycles on the incidence of trAEs, the incidence of trAEs was compared between different ICI cycles. As shown in To compare the incidence of trAEs in immunotherapy between the neoadjuvant group and the advanced group, the incidence of trAEs in advanced cancer immunotherapy was obtained in two meta-analyses , 39, andTo further discover the distribution of adverse events, we focused on trAEs and irAEs which were reported by at least 10% of the included studies. As shown in In addition, concerning neoadjuvant ICIs, the most common all-grade irAEs were rash , transaminitis , and AST increased  , bilirubin, lipase, amylase increased, diarrhea, and colitis, which was similar to adverse events in advanced stage . BesidesMoreover, treatment-related specific events, including progressive diseases, surgical delays and deaths, are of great concern to doctors in neoadjuvant immunotherapy. The pooled incidence of progressive diseases was about 6%, considered relatively low. More importantly, in most cases, patients with progressive diseases during neoadjuvant immunotherapy could still be cured through timely surgery. Another interesting aspect is that neoadjuvant immunotherapy might be able to explore the mechanisms of pseudoprogression and hyperprogression reported before  due to tThe present study had several strengths. A major strength of this study was that we analyzed the safety of neoadjuvant immunotherapy from several aspects, including the overall incidence of trAEs, specific types of trAEs and irAEs, and several treatment-related events. In addition, various comparisons further clarified the pattern of adverse events in neoadjuvant immunotherapy.However, this study had several limitations. Firstly, the sample sizes of included studies varied significantly, which might explain the significant heterogeneity in some results. Secondly, the current number of studies was still insufficient to analyze safety in specific subgroups such as different cancers and anti-PD-(L)1 drugs. Therefore, further large-scale studies are required in the future, especially RCTs. In addition, some studies report the safety incompletely, especially the incidence of irAEs, which is important to understand the immunotoxicity in neoadjuvant immunotherapy. Furthermore, as treatment-related specific events such as surgical delays and deaths are not detailly reported in RCTs, the difference of safety between neoadjuvant immunotherapy and neoadjuvant chemotherapy is still largely unknown. We hope that the safety data will be reported more completely in future research, which is important in guiding clinical treatment and management.To the best of our knowledge, it is the first study to analyze the safety of neoadjuvant immunotherapy in multiple malignancies comprehensively. Several conclusions were reached in the study. First, compared with neoadjuvant ICIs alone, neoadjuvant ICIs plus chemotherapy resulted a higher incidence of trAEs. In addition, increased ICI cycles tended to have a higher incidence of trAEs. Furthermore, neoadjuvant immunotherapy had a low rate of progressive diseases, surgery delays, and deaths.The original contributions presented in the study are included in the article/LZ, JX, and YW designed the study. JX, YW, and YX performed the literature search, data extraction and quality assessment. YX and YQ performed statistical analysis, and XL and YS provided assistance. JX drafted the manuscript, and LZ revised the manuscript and had the right to grant on behalf of all authors. All the authors read and approved the final manuscript.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.


Comprehensive data are needed to monitor antibiotic prescribing and inform stewardship. We aimed to evaluate the current antibiotic prescribing patterns, including treatment switching and prolongation, in the paediatric primary care setting in Italy. This database study assessed antibiotic prescriptions retrieved from Pedianet, a paediatric primary care database, from 1 January 2012 to 31 December 2018. Descriptive analyses were stratified by diagnosis class, calendar year, and children’s age. Generalized linear Poisson regression was used to assess variation in the prescriptions. In total, 505,927 antibiotic prescriptions were included. From 2012 to 2018, the number of antibiotics per child decreased significantly by 4% yearly from 0.79 in 2012 to 0.62 in 2018. Amoxicillin prescriptions decreased with increasing children’s age, while macrolides and third-generation cephalosporins had the opposite trend. Prescriptions were associated with a diagnosis of upper respiratory infection in 23% of cases, followed by pharyngitis (21%), bronchitis and bronchiolitis (12%), and acute otitis media (12%). Eight percent of treatment episodes were prolonged or switched class, mostly represented by co-amoxiclav, macrolides, and third-generation cephalosporins. Our findings report an overall decrease in antibiotic prescriptions, but pre-schoolers are still receiving more than one antibiotic yearly, and broad-spectrum antibiotics prescription rates remain the highest. Italy is one of the countries with the highest rate of antibiotic prescribing for children in Europe ,2,3, as Studies have demonstrated that around 20 to 50% of antibiotic prescriptions are unnecessary or inappropriate ,7,8, witThis unnecessary exposure to antibiotics may subsequently increase the risk of serious drug side effects and costs . It has In order to understand the scope and the burden of antibiotic prescriptions in Italy, comprehensive data are needed. Despite several studies having been carried out in hospitals ,13,14,15Information captured in population datasets is valuable, as it will enable us to monitor antibiotic prescribing patterns. It will also assist in implementing antibiotic stewardship and informing policymakers.The primary aim of our study was to comprehensively explore the current antibiotic prescription patterns in the paediatric primary care setting in Italy over the years, by child’s age and by diagnosis. Secondly, we described the treatment switch and prolongation patterns between different antibiotic classes.In total, 157,915 children , for a total of 717,202 person-years of follow-up, were included. Overall, the median age was 7 years and most of the children resided in the north of Italy . The median number of children managed by each FP was 1144 (IQR: 640). In total, 505,927, prescriptions were considered. Descriptive data are presented in p = 0.004) from 0.80 prescriptions per child in a year (95% CI: 0.78–0.81) in 2012 to 0.64 (95% CI: 0.63–0.65) in 2018, with an annual 4% reduction   .The highest AIs were reported for children aged from 1 to 4 years, with values around 1.2; on average, for every year of increase in the patient’s age, the AI was reduced by 13% , from a maximum of 36% in 2012 for children <1 year of age to a minimum of 14% in 2014 for children aged 13 years for amoxicillin and from a maximum of 9% in 2012 for children aged 3 years to a minimum of 1% in 2018 for children aged 13 years for second-generation cephalosporins .An increasing trend with increasing age was noticed for co-amoxiclav, third-generation cephalosporins, and macrolides . Co-amoxiclav rates varied from a minimum of 28% in 2012 for children age <1 year to a maximum of 37% in 2017 for children aged 12 years; for second-generation cephalosporins, the rates varied from a minimum of 9% in 2018 in infants to a maximum of 18% in 2017 in 12-year-old children. In 2014, the macrolide rates were the highest with respect to the different calendar years in children aged 3–13 years .Males were at a higher risk of receiving co-amoxiclav and macrolides , and children residing in the centre and the south of Italy were at a higher risk of receiving co-amoxiclav and cephalosporins compared with children in the northern regions, who were at a higher risk of receiving amoxicillin .Of a total of 497,186 visits associated with an antibiotic prescription, 3595 visits were excluded because they had more than one diagnosis associated with the prescription.n = 113,518), followed by diagnoses of pharyngitis , bronchitis and bronchiolitis , and acute otitis media . In 84,006 prescriptions (16%), no diagnosis was associated with the antibiotic (Respiratory tract infections accounted for around 75% of the antibiotic prescriptions. Antibiotics were associated with a diagnosis of upper respiratory tract infection (URTI) in 23% of cases (tibiotic .Co-amoxiclav prescription rates varied from 51% for soft tissue infections (STI) to 24% for bronchitis and bronchiolitis. The amoxicillin prescription rate was 32% for pharyngitis, 30% for AOM, and 20% for URTI. Twenty-seven percent of prescriptions for urinary tract infections (UTIs) were for third-generation cephalosporins and J01XX class antibiotics  .The prescription rates for the different diagnosis classes stratified by calendar year and years of age are presented in Overall, 485,976 had just one diagnosis associated with one antibiotic prescription and 312 were excluded because the follow-up was shorter than 14 days. The 485,664 visits were grouped by 448,058 treatment episodes, and 1268 were excluded because they were associated with antibiotic prophylaxis.In total, 35,460 treatment switches or prolongations were included. Treatment episodes with at least one switch were 17,044, of which 453 had two or more switches. Treatment episodes with at least one prolonged antibiotic numbered 19,516. No difference in the diagnosis prevalence compared with overall prescriptions was noted .n = 5750 times, 31% of overall switches), especially for macrolides  and third-generation cephalosporins . The second most frequent class of antibiotics that was switched was macrolides , followed by amoxicillin . . .The antibiotics that were prolonged the most were co-amoxiclav , followed by third-generation cephalosporins (N = 3890) and macrolides (N = 3900), which each accounted for 10% of the overall treatment switches or prolongation .When stratified by diagnosis, the most frequent switch was for the combination of macrolides and co-amoxiclav for URTI, which accounted for 15% of the total switches for URTI.Overall, 3358 antibiotics were switched before the fourth day (early switch), while the rest were classified as late switches. Co-amoxiclav was switched with a third-generation cephalosporin in 123 cases of pharyngitis, which was the most common early switch overall. The antibiotic class that was switched the most after the fourth day was the macrolides, which were substituted with co-amoxiclav in 577 episodes of URTI .Overall, the mean day of treatment switch was the eighth (SD: 4.0), with variations based on the antibiotic class accounting for the switch and the diagnosis . In totaTo our knowledge, this is the first longitudinal study to comprehensively assess antibiotic prescribing patterns in paediatric primary care in Italy. Over the period 2012–2018, we observed a decrease in total antibiotic prescriptions. Male children and those residing in central or southern regions were at a higher risk of receiving an antibiotic. In the overall population, co-amoxiclav and amoxicillin prescription rates increased over the years, with co-amoxiclav increasing, and also with an increase in the child’s age, and amoxicillin decreasing with an increase in the child’s age. Children residing in northern Italy were at a higher risk of receiving amoxicillin than those in the central and southern regions. Co-amoxiclav was the most prescribed antibiotic for respiratory infections , accounting for half of the prescriptions. The prescribing patterns remained similar, with minimal variations between years of age over time. Overall, 8% of antibiotic treatment episodes were prolonged or switched in class, especially among co-amoxiclav, macrolides, and third-generation cephalosporins.Assessing the drivers for the overprescribing of broad-spectrum antibiotics in the community is crucial, especially nowadays, since antibiotic resistance is dangerously rising globally. Indeed, many ecological studies showed a population-level association between antibiotic use and resistance . GoossenIn line with previous studies ,5, ItaliSimilar to our previous study , amoxiciMost of the switches occurred on the eighth day after the initial antibiotic prescription. This indicates that treatment failure could be the cause of switching antibiotics, since if an adverse event occurs, the switch would likely be in the first days after the prescription. On the other hand, adverse events such as diarrhoea following high clavulanate dosages might appear around the fifth day after the treatment initiation, since the hypothesized changes in the microflora could take some time ,45. OtheSeveral limitations need to be addressed in our study. First, even though the study was based on individual-level patient data, we were not able to retrieve a diagnosis in 16% of cases. However, we believe that missing diagnoses in a few patients will not change the overall prescribing patterns in our study. Second, due to the nature of the infectious disease, some children had multiple treatment episodes, which could lead to close monitoring by their FPs. Thus, a 14-day screening period from the initial episode and any subsequent similar diagnosis treatment during the period was considered as a follow-up visit for the same episode. It might be argued that in some cases , the healing could be slower; however, we believe that this is an exception and not the rule. Moreover, by defining an episode, we were able to reduce the over-representativeness of complex patients. Third, since family paediatricians are the primary referrals for health-related matters as well as the gatekeepers for specialist referrals in the context of the NHS, we believe that loss to follow-up, in the case of treatment switching or prolongation, is rare. Indeed, most of the diagnoses considered consisted of uncomplicated community infections that are treated at the primary care level, where access to healthcare is free and granted by the NHS. Moreover, caregivers are discouraged from requiring further specialistic evaluations for uncomplicated community infections due to the long waiting lists and increased costs related to a specialist’s evaluation. However, we acknowledge this as a limitation. Fourth, most of the oral paediatric formulations are liquid formulations, and the dosage is based on the child’s body weight; thus, older children might require two packages to finish the antibiotic treatment course instead of one. According to our definition, if the second prescription was on a follow-up visit of the same treatment episode, it was reported as treatment prolongation. Fifth, data on allergies and vaccination status were not assessed. A change in these factors might have been the cause of the high broad-spectrum prescription rates or switches in therapy. Sixth, we were unable to determine the reasons for switching antibiotics in our current study. It is warranted to explore the reasons for switching antibiotics further.The Pedianet database was used as the source of the study. Pedianet is a national population database that contains anonymous patient-level data of more than 500,000 children since 2004, corresponding to around 4% of the annual paediatric population, who received healthcare from 161 family paediatricians (FPs) in Italy who were part of the Pedianet network.®)  in their professional practice. About 4500 family paediatricians use Junior Bit® in Italy and they are all potential candidates to be part of the Pedianet network [The network links FPs distributed throughout several Italian regions designated by the Italian NHS, including Friuli-Venezia Giulia, Liguria, Lombardia, Piemonte, Veneto, Lazio, Marche, Toscana, Abruzzo, Campania, Sardegna, and Sicilia, and who use the same software .Antibiotic index (AI): the number of antibiotic prescriptions per person-year.Treatment episode: all prescriptions occurring within 14 days of the first antibiotic prescription.Switch: treatment episode with a second prescription different in class from the first Early switch: the first switch occurring within 1–3 days of the first prescription Late switch: the first switch occurring within 4–14 days of the first prescription Treatment prolongation: treatment episode with a second prescription.Day of switching: difference in days between the date of the second prescription and the date of the first prescription in a switch.The following definitions were applied:The person-years of children included in the study were accumulated from either January 2012 or when the patients started receiving care. All patients were followed up until the last date of their care or the end of the study period in December 2018.The primary outcomes considered were: (i) antibiotic index, (ii) antibiotic prescriptions rat, (iii) time to switch/prolongation, (iv) early switch rate, (v) late switch rate, and (vi) prolongation rate. Switch and treatment prolongation were only assessed for prescriptions associated with one diagnosis amongst patients who had an antibiotic prescribed for a reason other than prophylaxis and for children with at least 14 days of follow-up after the prescription index date.Antibiotics were classified according to ATC codes as amoxicillin (J01CA04), co-amoxiclav (J01CR02), macrolides (J01FAx), third-generation cephalosporins (J01DCx), second-generation cephalosporins (J01DDx), J01XX (J01XXx), thiamphenicol (J01BA02), lincosamides (J01FFx), other aminoglycosides (J01Gx), and others (J01x). If two or more prescriptions with the same specific 7-digit ATC code were reported on the same visits for the same diagnosis, just one was considered for the purposes of the study.All diagnoses linked to the same visit (max = 3) were classified according to the diagnostic categories for common outpatient infections. For determination of the years of age, the date of birth was approximated to 1 January and the year of age was calculated as the difference between 1 January and the date of birth for all the calendar years considered.If two or more prescriptions occurred the same day, they were excluded from the analysis of Outcomes (iii), (iv), (v), and (vi) because the order of drug events could not be determined. Early and late switches were assessed for only the first switch in the treatment episode.The analysis had a mainly descriptive nature. Stratification was performed according to the diagnosis class, calendar year, and year of age . Categorical variables were expressed using numbers and percentages, and continuous variables were expressed as means and standard deviations (SD) or medians with interquartile ranges (IQR). Categorical variables were compared with a chi-squared test in a contingency table (r × c). Continuous variables were compared by a non-parametric Kruskal–Wallis rank sum test.To estimate the annual changes in antibiotic prescriptions, the Mann–Kendall test was performed to determine whether the time series had a monotonic upward or downward trend and, if significant, the generalized linear Poisson regression was fitted. The Poisson model was chosen over linear regression because of the use of count data for antibiotic prescriptions (non-negative integers) and the high number of zero values in the dependent variables. The dependent variable was a count of antibiotic prescriptions, while calendar year, sex, age , and geographical residency of the patient according to the ISTAT area were the covariates considered . The logp < 0.05 was considered statistically significant. The analysis was performed using R statistical software–v. 3.6.2  [A two-sided Austria) . Graphs Due to the retrospective nature of the study and the use of anonymized data, no ethical consent was required to conduct the study. The study protocol and access to the data were approved by the Internal Scientific Committee of So.Se.Pe. Srl, the legal owner of Pedianet.Our findings report an overall decrease in antibiotic prescriptions over the years, but pre-schoolers are still receiving more than one antibiotic yearly. Moreover, most of the prescribed antibiotic can be classified as broad-spectrum antibiotics  and little to no variation was noted over the years. Estimating the drivers of antibiotic prescriptions is essential to define the area of intervention for antibiotic stewardship in primary care, either to reduce prescriptions for infection that have mainly a viral cause, or to improve treatment selection . However


Listeria monocytogenes (Lm), a Gram-positive pathogenic bacterium responsible for listeriosis, one of the most dangerous foodborne zoonosis. After a brief summary of characteristics of VBNC bacteria, we highlight work on VBNC Lm in the environment and in agricultural and food industry settings, with particular emphasis on the impact of antimicrobial treatments. We subsequently discuss recent data suggesting that Lm can enter the VBNC state in the host, raising the possibility that VBNC forms contribute to the asymptomatic carriage of this pathogen in wildlife, livestock and even humans. We also consider the resuscitation and virulence potential of VBNC Lm and the danger posed by these bacteria to at-risk individuals, particularly pregnant women. Overall, we put forth the hypothesis that VBNC forms contribute to adaptation, persistence, and transmission of Lm between different ecological niches in the One-Health continuum, and suggest that screening for healthy carriers, using alternative techniques to culture-based enrichment methods, should better prevent listeriosis risks.Many bacterial species, including several pathogens, can enter a so-called “viable but non-culturable” (VBNC) state when subjected to stress. Bacteria in the VBNC state are metabolically active but have lost their ability to grow on standard culture media, which compromises their detection by conventional techniques based on bacterial division. Under certain conditions, VBNC bacteria can regain their growth capacity and, for pathogens, their virulence potential, through a process called resuscitation. Here, we review the current state of knowledge of the VBNC state of  Listeria monocytogenes (Lm) is a ubiquitous Gram-positive bacterium that contaminates food production lines and is pathogenic for humans and various animal species  foods, including uncooked vegetables and fresh-cut fruits (https://www.cdc.gov/listeria/outbreaks/index.html). species . This fa species . Lm is tfections . Listerifections , as wellfections . These rfections . The foot fruits . For exat fruits , while rLm to different ecosystems, in the environment , a variety of foods, and hosts of diverse species, makes it an important archetype of the “One-Health” approach to infectious diseases, which recognizes the interconnection between human health, animal health and environmental ecosystems. However, there is much to discover about the ecology of Lm, in terms of natural reservoirs and mechanisms of transmission. In particular, the potential for asymptomatic carriage of this pathogen has been less explored than its mechanisms of pathogenicity. However, healthy carriers play an important role as reservoirs and dispersion factors of Lm in the environment. As early as the 1960s, Lm is also found in many species of wild mammals, such as rats, hares, hedgehogs, badgers, deer, moose, otters, raccoons, foxes, bears, boars, monkeys  was less efficient in detecting healthy Lm carriers (an order of 5%), whereas full metagenomic studies using shotgun sequencing failed in detecting Lm DNA signal in the gut microbiota. This highlights the importance of using sensitive molecular techniques to detect healthy carriers. However, it should be remembered that these molecular biology-based approaches cannot determine whether the detected DNA is from live or dead bacteria.The remarkable adaptation of  monkeys  and even monkeys . Lm is aand cats . Oevermaand cats . While my cattle . Healthy studies . Howeverpulation . This stLm is known to be tolerant to many physical and physicochemical stresses, allowing it to survive in a wide range of environmental conditions  is also a key factor in the survival of this non-spore-forming bacterium following stress. This property was first described 20 years ago for Lm present in water . When conditions become favorable for regrowth, the spore is able to germinate to regenerate a vegetative bacterium , antimicrobial compounds  or host factors , metabolically inactive and resistant to many environmental perturbations . For clarity, we provide a table defining these terms , and actinobacteria . Because of their non-culturable nature and resuscitation capacity, VBNC bacteria are a public health problem. For species responsible for food poisoning, VBNC forms escape quality controls in food industries, as detection of contamination is most often based on methods dependent on bacterial growth in conventional enrichment media.Since its discovery in 1982, the VBNC state has been described for more than 100 species, two-thirds of which are pathogenic species . This liMycobacterium tuberculosis (Mtb). Human infection with Mtb produces an active form of the disease, tuberculosis (TB), one of the top ten causes of death worldwide, as well as an asymptomatic form called latent tuberculosis infection (LTBI)  . Matheman (LTBI) , withoutdormancy . Reactivs luteus  and lates luteus  are invos luteus .Lm into the VBNC state in a natural environment was first suggested by Lm in sterilized seawater in the laboratory , a medium used to study the VBNC state of Campylobacter jejuni  and be transferred into food products.More recently, a study showed that tap water was also a highly deprived medium in which surfaces . Thus, aThe studies mentioned in this section are listed in Lm contamination. Because of the health risks of such contamination, many studies have addressed this problem by modeling the contamination by VBNC Lm in reconstituted systems . The main technical difficulty is to correctly differentiate dead from VBNC cells in complex matrices. Works have examined the persistence of Lm in urban or agricultural effluents, after inoculation of bacteria into sewage sludge , a method combining the use of an amplification inhibitor, such as propidium monoazide (PMA) or ethidium monoazide (EMA), and qPCR  could be a vector for VBNC contamination. Lm contamination of a PWW was modeled in the laboratory, either by inoculating a PWW of shredded lettuce with a cocktail of six Lm strains  rather than viability   . Thus, aactA gene is kept inactive, vacuolar Listeria all cease to be culturable on agar media and can propagate during host cell mitosis . As mentioned above, a PCR-based approach was much more effective in detecting Lm in the feces of healthy humans than metataxonomics or metagenomics   . HoweverLm resuscitation in the host was first questioned by the experiments of Lm VBNC remained avirulent in vitro, in tests of infection of human colon cells (HT-29), and in vivo, in a murine listeriosis model . In agreement with this, recombinant Lmo0186 and Lmo2522 proteins have a muralytic activity on crude preparations of Lm cell walls, as well as on (NAG)3 MUF, an artificial polymer commonly used as a substrate for lysozyme  can be long, particularly when associated with pregnancy [up to 3 months,  . RPL is defined as three or more consecutive pregnancy losses before 20 weeks’ gestation. Its etiology is often unclear and may be multifactorial   but miscr plates . In addital loss . Experimg months . These omacaques . It seemactorial . A role actorial , but morLm is so far not described as a pathogen causing subclinical silent infections, but we cannot exclude that this bacterium hides in the organism for long periods of time, especially in a VBNC state within an intracellular vacuole  a more systematic sampling process in interconnected systems, which are animal and plant production systems (farms and their immediate environment), food production systems (food processing/packaging sites and food stores), and medical and hospital systems , (ii) the use of diagnostic methods without growth-based enrichment steps, to detect the presence of unculturable Lm, and (iii) whole-genome sequencing of isolated strains and comparative genomics, in order to determine which strains are most likely to enter the VBNC state, their ability to circulate, and in which ecosystem.The spread of human infectious diseases emerging from animal reservoirs, such as avian influenza caused by the H5N1 virus and Covid-19 caused by the SARS-CoV-2 virus, have brought to the forefront the fact that zoonotic diseases represent a major public health risk. It has also become clear that the asymptomatic carriage plays a key role in the transmission of these dangerous pathogens. Because of its ubiquity in the environment, animals, and humans, which are the three pillars of the One Health cts them Figure 2Lm contamination are all based on the growth capacity of this pathogen via the determination of CFU, both in the United States, with a zero-tolerance policy, and in Europe, with slightly less strict criteria for certain categories of RTE foods considered low risk (EC Regulation 2073/2005). These standards do not take into account the potential risk related to the presence of VBNC forms. As eradication of VBNC bacteria is difficult, their presence should be taken into account in risk assessment modeling and health regulation issues. In the context of VBNC bacteria in the host, many questions arise. For example, what are the physiological similarities and differences between bacteria in the VBNC state in an environmental niche and in a host? Are there strain-specific variations, with hypervirulent clones also more prone to silently persist as VBNC forms in the host, compared to hypovirulent clones? Do VBNC Lm survive asymptomatically in a host while being silently excreted through fecal or uterine discharges until the host dies, or can they reactivate in the same individual when the immune system is weakened, which would be consistent with latent listeriosis ? What are the reservoir hosts (including protozoa)? Answering these questions will require clinically relevant animal models and overcoming the problem of obtaining tissue samples from asymptomatic hosts. Additionally, in medicine, as in the agri-food industry, several challenges remain to be addressed, first of all, the development of sensitive, easy-to-use and inexpensive techniques to facilitate large-scale detection and tracing of VBNC Lm. Detection techniques based on PCR, DNA sequencing, or mass spectrometry, are improving and their development is crucial, as is the development of techniques demonstrating the viability of these non-culturable microorganisms. Second, it is necessary to identify the factors affecting the recovery and regrowth of VBNC cells. Finally, the pathogenic potential of VBNC forms must be properly assessed.In the context of the food industry, safety standards concerning teriosis Figure 2AL, HB: Writing–original draft preparation. AL, EM, HB: Writing–review and editing. AL, EM: Table preparation. HB: Figure preparation. EM, HB: Funding acquisition. HB: Supervision and Conceptualization. All authors contributed to the article and approved the submitted version.This work was funded by grants from ANR PERMALI (N°ANR-20-CE35-0001-01) and iXcore Foundation (2015) to HB, and INRAE-MICA division (AAP 2019-2020) to EM.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.


Antimicrobial misuse is common in low-income and middle-income countries (LMICs), and this practice is a driver of antibiotic resistance. We compared community-based antibiotic access and use practices across communities in LMICs to identify contextually specific targets for interventions to improve antibiotic use practices.We did quantitative and qualitative assessments of antibiotic access and use in six LMICs across Africa  and Asia  over a 2·5-year study period . We did quantitative assessments of community antibiotic access and use through supplier mapping, customer exit interviews, and household surveys. These quantitative assessments were triangulated with qualitative drug supplier and consumer interviews and discussions.Vietnam and Bangladesh had the largest proportions of non-licensed antibiotic dispensing points. For mild illness, drug stores were the most common point of contact when seeking antibiotics in most countries, except South Africa and Mozambique, where public facilities were most common. Self-medication with antibiotics was found to be widespread in Vietnam (55·2% of antibiotics dispensed without prescription), Bangladesh (45·7%), and Ghana (36·1%), but less so in Mozambique (8·0%), South Africa (1·2%), and Thailand (3·9%). Self-medication was considered to be less time consuming, cheaper, and overall, more convenient than accessing them through health-care facilities. Factors determining where treatment was sought often involved relevant policies, trust in the supplier and the drug, disease severity, and whether the antibiotic was intended for a child. Confusion regarding how to identify oral antibiotics was revealed in both Africa and Asia.Contextual complexities and differences between countries with different incomes, policy frameworks, and cultural norms were revealed. These contextual differences render a single strategy inadequate and instead necessitate context-tailored, integrated intervention packages to improve antibiotic use in LMICs as part of global efforts to combat antibiotic resistance.Wellcome Trust and Volkswagen Foundation. Antibiotic resistance has created an urge for global efforts to facilitate appropriate use of antibiotics. Global reports on antibiotic resistance have shown that studies are needed in low-income and middle-income countries (LMICs), where local data are scarce and resistance is widespread.A systematic reviewEvidence before this studyCommunity-based antibiotic access and use has received less attention than prescription behaviours in formal health-care settings. We have little understanding of antibiotic use in the community, which is considered a key driver of antibiotic resistance. We searched PubMed and Google Scholar for articles published between Jan 1, 2000, and Jan 12, 2021, and we restricted our search to articles published in English that reported factors associated with antibiotic use in low-income and middle-income countries (LMICs) using the search terms “antibiotic”, “self-medication”, and “low- and middle-income countries”. We found three systematic reviews covering 85 studies on antibiotic dispensing without a prescription worldwide, of which 59 were published in LMICs, 34 studied factors affecting self-medication with antimicrobials in LMICs, and 15 investigated factors affecting self-medication with antibiotics in LMICs. These studies revealed a high prevalence of self-medication. Accessibility, affordability, health-care facility conditions, and health-seeking behaviour were found to be the main determinants of self-medication in studied LMICs. However, there is considerable heterogeneity in the reviewed studies due to an absence of standardised criteria for survey data collection. The reviewed studies were limited to self-reporting on questionnaires, focused on either the supply or the demand side in households, and provided few insights regarding community practices to guide future interventions. Furthermore, most studies focused on Asia with little research on misuse of antibiotics in Africa, and there is a scarcity of comprehensive and contextual studies considering local complexities to guide future research and support the development of evidence-based interventions in promoting appropriate antibiotic use across different LMICs.Added value of this studyTo fill these research gaps, we used a comparative mixed-method approach to compare community-based antibiotic access and use practices across communities in six LMICs in Asia  and Africa  to allow for comparison by national income status and identify supply and demand drivers. Within the existing INDEPTH surveillance network, our study was powered to identify the contextual complexities and differences between countries with different income levels, policy frameworks, and cultural norms. The gained insight in antibiotic practices in different LMICs could inform the design of context-adjusted models for interventions to effectively address the usage of antibiotics and contain antibiotic resistance in these resource-constrained settings.Implications of all the available evidenceOur study found common themes reported in previous studies in LMICs, but also revealed the diversity and complexity of locally specific sociocultural determinants affecting antibiotic access and use among studied communities in six Asian and African LMICs. The identified factors will serve as targets for development of context-tailored interventions to effectively address the misuse of antibiotics and contain antibiotic resistance in LMICs.To fill these research gaps, we aimed to use a comparative approach to assess access and use practices across communities in six LMICs in Asia  and Africa  to allow for comparison by national income status and identify key drivers through both qualitative and quantitative measures.INDEPTH surveillance network, a global network of health and demographic surveillance sites, this study (the ABACUS project) was done in seven communities in six LMICs in Africa  and Asia . For each study site, all possible purchase or dispensing points for antibiotics (antibiotic suppliers) were included, comprising any formal or informal antibiotic supplier from public hospital pharmacies to street vendors. Country scans for the health-care system and antibiotic legal framework are provided in the Operating from the existing We used a mixed-methods approach with in-depth interviews and focus group discussions among drug suppliers and consumers, household surveys, and exit interviews among customers who purchased antibiotics. The study was done between July 1, 2016, and Dec 31, 2018, in four parts. Part 1 consisted of mapping of suppliers and inventories, and part 2 consisted of preparatory in-depth interviews and focus group discussions with suppliers (16 in-depth interviews per site) and community members (16 in-depth interviews and six focus group discussions per site). Part 3 involved quantitative longitudinal household surveys of Health and Demographic Surveillance Sites in two rounds, during a 1-year period (1100 households per site with 5–10% loss to follow-up). This number was adjusted for South Africa (600 households per site) and Mozambique (650 households per site) due to higher costs of fieldwork. Simultaneously, community antibiotic supply was quantified through standardised customer exit interviews at four timepoints over a 1-year study period  and synchronised across sites. In part 4, to explain any potential discrepancies between results from the household survey and the customer exit survey, further explanatory in-depth interviews and focus group discussions with selected groups (five in-depth interviews and two focus group discussions each site) were done.Antibiotic suppliers aged 18 years or older were eligible for qualitative interviews (in-depth interviews and focus group discussions). Ideally, each interviewee was affiliated with a unique antibiotic supplier, but if fewer than 16 eligible antibiotic suppliers could be identified at each site, a maximum of two employees from the same supplier could participate in the in-depth interviews but not in the same focus group discussion. Similarly, community members aged 18 years or older who were willing to share their experience and attitudes toward medicines were eligible to participate in qualitative interviews (in-depth interviews and focus group discussions). Participants from different age categories and of different sexes were involved to achieve a more informative view. Different targeted populations included men and women aged 18–60 years, men and women older than 60 years, and mothers who cared for children younger than 5 years, due to a higher frequency of health-care seeking. Two participants from the same household were avoided. Community members who participated in in-depth interviews were excluded from focus group discussion and vice versa. For the household surveys, households that already participated in an ongoing health and demographic surveillance system and had previously provided consent were eligible to participate in this survey. An adult household representative (18 years or older) needed to provide additional written informed consent before the household could be included in the survey. Other details on inclusion criteria and rationale for participant selection were published previously.The study was approved by the Oxford University Tropical Research Ethics Committee (31-15), and the local ethical committees of each participating study site.Research Electronic Data Capture. Data were extracted, cleaned, and analysed in R version 3.3.3. Descriptive data were described as mean (SD), median (IQR), and proportion (95% CI). The analysis and presentation of the quantitative data followed the STROBE guidelines for reporting observational studies.Data were collected using tablets and stored using a web-based application: For each of the studied communities, through observed metrics, we aimed to map all formal and informal suppliers of antibiotics, inventory antibiotic resources, and determine community antibiotic exposure and the appropriateness of the antibiotics supplied. Through reported experiences, we aimed to assess demographic, socioeconomic, and cultural characteristics, the accessibility of health care, and frequency of antibiotic intake and characterise the practices involved in antibiotic demand, access, and use .A thematic analytical approach was used on the English language summaries of each in-depth interview and focus group discussion for each study site. Because all the interviews and discussions were done in local languages, these summaries were made to facilitate centralised comparative analysis. The summaries were produced when the transcript was coded following a standardised format. The analysis was based on a-priori themes from the various topics that had been covered during the in-depth interviews and focus group discussions, with additional themes and subthemes emerging inductively during the reading and analysis process. Verification of the data and themes was done by members of the research team with skills in qualitative analysis . This verification involved validating and assessing the trustworthiness of the qualitative data using the four constructs of credibility, transferability, dependability, and conformability.Through reported experiences, we aimed to investigate health-care concepts and behaviours and socioeconomic and cultural characteristics associated with practices of antibiotic access and use in each studied context.The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Supplier mapping showed that Asian sites had a higher density of antibiotic suppliers than African sites, with the highest density found in urban Thailand (5 per 1000 inhabitants) and the lowest in rural South Africa “If I want to buy Yaa Chud [poly-pharmaceutical packs containing antibiotics], I just walk to the grocery shop nearby.” Perception of health-care facilities“It's not difficult to get to the hospital, the biggest problem is the delay to be seen, and sometimes the lack of pills at the hospital, what compels us to buy at the private pharmacy where [antibiotics] are so expensive and we do not have money to pay.” “The treatment is for free but people are hesitant to use it. Instead they want something that they will pay for.” “I would like to take treatment in government hospital, but I don't get proper service there. They don't do their assigned duty, neglect the patients.” Trust in supplier“When I have mild symptoms, I always go to the drug store first. I don't even know the name [of the medications] and their effect because I trust them completely. I only go to the hospital when I get more severe illness.” Convenience of access“I prefer buying medicine from a nearby drug store than going to the hospital. At the pharmacy, they ask the symptoms before they give the drugs and they are very effective.” “Most people don't like going to the hospital. When they visit the hospital, they will sit there for a very long time before they are attended to, so patients may end up dying with the illness they took to the hospital. So they will move straight to the drug store to buy medicines to cure their illness.” Perceived disease severity“Our family members did not like to go see a doctor, except when we got a serious sickness. If someone has a fever or cough, we buy Yaa Chud [non-prescribed poly-pharmaceutical packs containing antibiotics] from the drug store.” “I only go to the hospital when I get more severe illness.” Children are a priority“If children are sick, it is a must to bring them to hospital.” Perception of antibioticsMisperception of antibiotics' function“Antibiotics are powerful drugs, which are used for serious illnesses. When paracetamol does not work for cough and cold then antibiotics should be used.” “Recently, febrile illness could not be cured without antibiotics, and I have to spend thousands BDT [Bangladesh currency] every month for buying antibiotics”. “People are not dying like in the past; it means they are using antibiotics in a good way.” Confusion of antibiotic with anti-inflammatory“We don't know them by name. They have two colours.” “There are different types of antibiotics. We have the red and yellow [amoxicillin] that is used for stomach pains and wounds. There is a different type that is red it is also used for waist pains.”  “I know some [antibiotics] such as ‘Amox’ [amoxicillin], ‘Tiffy’ [paracetamol], ‘Panadol’ [paracetamol], ‘anpha choay’ [anpha-chymotripsin].” Limited knowledge of antibiotic resistance“I find that illness resolves after consuming antibiotics very quickly, but I think it creates another disease in our body and gives rise to side-effects. People are not aware of this properly.” “Recently, many people in my village have had common cold or flu due to the changing season. The disease has been more severe and difficult to treat as it has become resistant to flu medicines. Higher doses of antibiotics were, therefore, needed to recover.” PracticeSelf-treatment linked to previous experience“If you have previously used an antibiotic, you just go and buy one from the drug store. Recently, I bought some antibiotics (Amoxicillin), because I knew what I wanted [so] I just went to the drug store and asked for it.” Taking incomplete courses“I won't deny it, sometimes I also do that [laughing], I take pills and when I see I am better, especially if the pills are bitter, I stop taking them, I quit.” “They prescribe drug for me to buy some medicine, but I couldn't buy all the medicine, so I bought some of the medicine, and 2 weeks later, I bought the rest of the medicine that time all my feet got swollen.” Shared with others or save for future use“I share medicines with my neighbours and I usually ask them for Napa [paracetamol], peridon [domperidone], and antibiotics for dysentery.” “We keep them so that we can use them again when we get ill.” From in-depth interviews and focus group discussions, factors determining where treatment was sought for a household often involved policy-associated determinants and a balance between trust in the supplier and the drug, convenience of access, disease severity, and whether or not the consumer was a child . Where rIn household surveys, most respondents said they chose where to access antibiotics for mild illness on the basis of how convenient the location was to access, except Mozambique, where trust was the most common reason for choosing a public health-care facility . For sevIn Bangladesh, participants in focus group discussions shared that public health services at the district level or lower are free of charge but are considered low standard, with few types of antibiotics available. Therefore, community members mainly rely on nearby drug sellers  for mild conditions. In Ghana, the main and first approached sources for antibiotics were licensed chemical sellers  and drug peddlers . Findings from focus group discussions suggested that delays in receiving care at hospitals encouraged people to seek medicine from the licensed chemical sellers rather than from public health-care providers . This coThe largest proportion of households reporting use of antibiotics in the previous month in the first household survey was in Bangladesh (498 [49·4%] of 1009), followed by Vietnam (416 [45·0%] of 925), Ghana (728 [37·4%] of 1947), Thailand (294 [27·9%] of 1053), Mozambique (161 [25·2%] of 639), and South Africa  and consumers (household surveys) could not always distinguish antibiotics from other commonly sold medicines like painkillers  or antipyretics. Antibiotics were often identified by colour and shape, such as red and green capsules or pink elongated pills. The wide variety in names and appearances contributed to potential misidentification . MedicatAcross all sites, it was commonly reported in focus group discussions that antibiotics were used for similar conditions as found in the quantitative results. Participants in Bangladesh, Mozambique, and Vietnam thought antibiotics were medications for most diseases, including infections that were probably non-bacterial. In African sites, malaria and sexually transmitted infections were more commonly indicated conditions. Regardless of perceived treatable conditions, antibiotics were considered to be powerful and life-saving medicine across both continents .Access-group antibiotics were predominant in almost all sites (ranging from 3664 [50·7%] of 7222 defined daily doses in Vietnam to 1764 [84·6%] of 2052 defined daily doses in South Africa), except Bangladesh, where the Watch-group antibiotics accounted for 7414 (74·6%) of 9934 defined daily doses . No ReseQualitative findings from in-depth interviews and focus group discussions with community members showed that antibiotic use without prescription was a common practice in almost all sites except South Africa. It was commonly reported to be based on previous treatment experience.Community members were not aware that antibiotic courses should be completed and that antibiotics can be harmful to their body. In low-income countries, lack of money forces community members to buy incomplete courses of treatment. Even when people could afford a full course, they still bought a few pills to see if they worked . In South Africa where patients received full courses prescribed by health-care workers, patients did not always complete their courses, because they did not feel the need to take more pills after they recovered. Unused medicines were shared with others or saved for future use in almost all sites but could be returned and refunded in Bangladesh, Ghana, and Vietnam .Our results showed that a large proportion of antibiotics were acquired without a prescription across study sites in low-income and lower middle-income countries. 2875 (35·0%) of the 8214 customers interviewed across all six countries had purchased antibiotics without a prescription. This finding aligned with those from the household survey, in which 2290 (37·0%) of 6190 households reported self-medication with antibiotics. Despite the legal requirements to dispense antibiotics only with prescription, non-compliance was found to be widespread in the low-income and lower middle-income countries, but not in the two upper middle-income countries. This non-compliance could be a focus of future interventions, which will require context-adjusted models for optimal effect. In rural South Africa, for example, little health care was available and a clear governance structure that made antibiotics accessible only through prescription was adhered to, limiting access. Additional factors also limited access in South Africa, including ability to reach and to pay.Although prescription-only regulations for antibiotics might not be practical for many LMICs,30Confusion regarding how to identify oral antibiotics was revealed in both Africa and Asia. This issue needs to be considered before other issues of demand are addressed.Despite the richness of the data, this study has several limitations. First, the study was done in individual rural or urban and suburban communities in LMICs and is thus not fully representative of misuse of antibiotics in each country. Second, we might not have captured all antibiotic use, especially without prescriptions, by household members due to poor recall, self-perceived misuse of antibiotics, or lack of awareness that pills taken were antibiotics. Third, our stratified sampling resulted in an imbalanced proportion of supplier types owing to the small number of formal suppliers versus informal suppliers in some sites. Nevertheless, the collected information covers all types of suppliers that the community members are exposed to. Finally, we experienced challenges in approaching non-licensed antibiotics suppliers, which predominated in Asia, due to their refusal to participate in several study components. We might therefore have underestimated the proportion of inappropriate antibiotic use.This study showed that antibiotic use differed markedly between study sites in LMICs and that access generally appeared to be more restricted in African than Asian locations. In Asian sites, antibiotics were widely available through a high density of both formal and informal antibiotic suppliers, and we found more dispensing without a prescription relative to African sites. The associated contextual factors will serve as targets for development of context-tailored, integrated intervention packages that target both the supply and demand side, to improve antibiotic use in these settings, and will contribute to global efforts to combat antibiotic resistance.Data may be made available according to data sharing policy of the local partners in the six participating countries upon request to the corresponding author.


Reports analyzing the impact of pediatric antimicrobial stewardship programs (ASP) over long periods of time are lacking. We thus report our ASP experience in a pediatric tertiary referral center over a long-term period from 2011 to 2018. Our ASP was implemented in 2011. The program was based primarily on guideline development with key stakeholders, engaging and educating providers, followed by prospective audit with feedback (PAF). Monitored antibiotics included meropenem, piperacillin–tazobactam, and cefepime, followed by the addition of ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, levofloxacin, linezolid, and vancomycin at various time points. Specifically, the program did not implemented the core strategy of formulary restriction with prior authorization. Process- and outcome-related ASP measures were analyzed. We saw a 32% decrease in overall antibiotic utilization, a 51% decrease in the utilization of antibiotics undergoing PAF, and a 72% reduction in the use of broad-spectrum antibiotics such as meropenem. There was a concomitant increase in organism susceptibility and a reduction in yearly drug purchasing costs of over USD 560,000 from baseline without changes in sepsis-related mortality. Our study highlights that a pediatric ASP based primarily on the principles of guideline development and PAF can improve antibiotic utilization and institutional bacterial susceptibilities without a detrimental impact on patient outcomes by changing the culture of antimicrobial utilization within the institution. Antibiotics are amongst the most prescribed medications to hospitalized children, with a large majority of the use thought to be inappropriate and unnecessary ,2. AntibWe implemented a pediatric ASP in a free-standing tertiary care children’s hospital in 2011. In this study, we describe the process and core strategies applied and the outcome measures since the initiation of the program. We hypothesized that education, guideline implementation, and PAF would lead to a sustained long-term improvement in our institution. To the best of our knowledge, we report the longest duration of sustained impact of a pediatric ASP to date.Institutional overall antimicrobial utilization decreased by 32% and the utilization of antibiotics undergoing targeted PAF by ASP team members decreased by 51% from program inception through 2018 . The absp = 0.002). This suggests some of the reduction in utilization was driven by prescribers within the institution in addition to those directly promoted by the ASP program.Quarterly broad-spectrum antibiotic  utilization decreased by 72% from ASP initiation through 2018 with an absolute reduction of 60 DOT/1000 PD. Although broad-spectrum antibiotics were primarily targeted by our ASP and had greater relative reduction, the absolute reduction in overall antibiotic utilization seen in P. aeruginosa susceptibility to meropenem over time at our institution. This includes one additional year compared to our previous publication [lication . Our prolication .Sepsis-related mortality has not significantly changed at our institution since the creation of the ASP, illustrating that there were not significant adverse consequences to utilizing narrower-spectrum antibiotics .Antimicrobial purchasing costs also decreased on a yearly basis, with a decrease of over USD 569,000 in yearly spending from initiation of the ASP through the first quarter of 2019 . The chaAn ASP was started in June 2011 at Le Bonheur Children’s Hospital, a large tertiary referral hospital in Memphis, TN with a 1 FTE pediatric pharmacist and a 0.5 FTE pediatric ID physician (position shared by two people). Another 0.5 FTE pharmacist was added in 2014. The ASP initially focused on the development of empiric antibiotic use guidelines for critical care units as those were the sites of most broad-spectrum antimicrobial use. The ASP’s guidelines for the initiation of empiric antimicrobials in critical care units were implemented throughout the 2012 calendar year as previously described . In 2013Guideline development followed by PAF for targeted antibiotics was the cornerstone strategy of initiatives for our ASP. We previously reported on guideline creation for linezolid use and urinary tract infection treatment ,15. TablP. aeruginosa susceptibilities (excluding cystic fibrosis patients), sepsis-related mortality, and institutional pharmacy antibiotic purchasing costs. These measures were selected from recommended measures for tracking an ASP in the pediatric inpatient setting [This was a retrospective analysis of process and outcome measures related to the ASP efforts at our institution from October 2011 through 31 December 2018. The main process measure was institutional antibiotic days of therapy (DOT)/1000 patient days (PD) for all antibiotics and antibiotics monitored by ASP via PAF. Our outcome measures included institutional  setting ,10.p < 0.05 for statistical analyses. Statistical tests were performed using the IBM Statistical Package for Social Sciences software version 27 . Antibiotic purchasing data were collected on a yearly basis for all antibiotics, and these were provided for descriptive purposes. There was a change in wholesaler at the end of 2014 and beginning of 2015, and accurate purchasing data could not be obtained during that transition. Antibiotic utilization data were collected as antibiotic DOT/1000 PD for targeted antibiotics and all antibiotics within our institution. Mortality was reported as the percentage of sepsis deaths. Utilization and mortality data were obtained from the Pediatric Health Information System Database for our institution. Pearson and Spearman’s correlations were used to assess quarterly or yearly trends from the 3rd quarter of 2011 to the 4th quarter of 2018, for parametric and nonparametric data, respectively. Chi-squared test compared susceptibilities among years. Significance was set at Our study describes the long-term impact of an ASP in a free-standing children’s hospital. The strategy of restriction with prior authorization was not utilized at any time since the inception of the program. Instead, the approach focused on guideline development and provider education along with PAF with re-education as needed. Like our study, PAF was the most frequently used ASP core component strategy reported in a review of pediatric ASPs . HoweverAlthough a change in prescription practices is a well-reported outcome of ASPs, safety, reduction in health care costs, and change in resistance patterns have been less frequently described in detail. In a recent systematic review of outcomes of pediatric ASPs globally, only 14.2% of the included studies 16/113) quantified cost savings related to the intervention . Most st quantifiP. aeruginosa as depicted above  in addition to ASP interventions. Lastly, our rise in the m agents . This maTo conclude, we report our process and outcome metrics of our ASP in a free-standing children’s hospital. Our long-term reductions in antibiotic use show that a successful pediatric ASP is possible without the adoption of the core strategy of formulary restriction with prior authorization. Empowering physicians with education and guidelines followed by PAF loops can lead to a sustained culture change for antimicrobial utilization without compromising patient outcomes, as well as an improvement in resistance patterns and costs.


Results showed that 7% NaCl solution caused the highest chloride content in 0–5 mm depth, whilst the w/b ratio of 0.25 curbed the chloride penetration within 10 mm even for concrete subjected to dry–wet cyclic immersion for 360 d. Subsequently, a respond surface model (RSM) basing on polynomials was constructed to visually evaluate the effect of PF dosage and FA content. Results clarified that a cubic model was more precise and PF dosage and FA content turned out to have the positive facilitation to chloride resistance. The positive effect of PF however is not consistent and commensurate for concrete with varied fly ash content. Finally, a fuzzy logic based nonlinear model accommodating all seven influencing factors was verified to be proper and adaptive in predicting chloride content.Herein, the paper reports an experimental investigation lasting one year on the chloride resistance of polypropylene fiber (PF) reinforced concrete with fly ash (FA). Four influential factors at four levels were studied, viz. water to binder ratio (w/b) , PF dosage , FA content  and concentration of NaCl solution . Dry–wet cyclic immersion and long-term soaking were taken into consideration in addition to the aforementioned factors. A L16(4 The chloride ion is a typical aggressive substance that triggers the corrosion process of steel reinforcement and further causes deterioration, shortening the service life of structures. In the marine environment, those chlorides dissolve in water and ingress to the interior of concrete via a myriad of skeleton and connected pores. To this end, some active mineral additives are blended into concrete to modify the pore structure and connectivity. Among those mineral additives, environmentally friendly and cost-effective industrial byproducts including slag and fly ash are noteworthy. It was reported that the slag powder promoted cement hydration and curbed creep and drying shrinkage, resulting in an enhanced resistance of concrete to chloride penetration ,2. The aBy descending the porosity and ascending the tortuosity, fibers can preserve the impermeable nature of concrete in an uncracked state ,11,12,13However, the authors of previously published articles seldom studied the integrated effect of fly ash and PF on the chloride penetration of normal and high strength concrete subjected to two environmental factors of dry–wet cyclic immersion and long-term soaking at various corroding concentrations. The novelty of this research is to concurrently conduct in-depth research and model the combined influence of four main influential factors of w/b ratio, fly ash content, PF dosage and soaking concentration of NaCl solution, and one supplementary variable of the environmental condition on the chloride resistance of concrete. In this study, the sequence of significance of different independent variables was identified using range analysis; the optimal condition of each variable was determined. Furthermore, another interesting point was located using respond surface methodology (RSM) to predict the chloride content of PF reinforced concrete with fly ash. By RSM, the effect of fly ash and PF at various levels was visually portrayed.At last, the diffusion coefficient of chloride ion traditionally is set as a function of corrosive time and penetration depth and is thereby used to predict service life of structures. Nevertheless, the chloride penetration is a multifactorial problem, with influential factors sourcing from the nature of concrete and the exposure environment of the structure. In this study, the former is represented by w/b ratio, the PF dosage, and the fly ash content, whereas the latter is embodied by the concentration of NaCl solution and the distinct corrosive conditions (dry–wet cyclic immersion or long-term soaking). Conventional models of diffusion coefficient subjected to the identified function structure failed to accommodate all aforementioned factors at various levels even though complex mathematical calculations were involved, such as ,21. Furt3 and Blaine specific surface area of 3250 cm2/g. The low lime grade I fly ash was sourced from the Hengyun power station. Its density is 2.2 g/cm3 and the specific surface area is 2897 cm2/g. The physical properties of cement and chemical composition of cement and fly ash are given in The cement is P.O 42.5R, with density of 3.1 g/cm3 and the crushing index of 10.1%. The fineness modulus and apparent density of river sand are 2.7 and 2480 kg/m3, respectively. The sieving curve of aggregates is depicted by Dry and clean granite and river sand are used as coarse and fine aggregates, respectively. The size fraction of granite is 5–25 mm, with the apparent density of 2540 kg/mThe PF was purchased from a local company , with main properties exhibited in 4 = 256 specific experiments. Instead, the L16(44) orthogonal table that covered four factors with four levels was used, with details given in The mix proportion of reference concrete was designed by the unit volume method as per the Chinese national standard “Specification for mix proportion design of ordinary concrete” . Four tyRaw materials as per NaCl solutions at four concentrations as per P—content of chloride ion dissolved in water, %; G—weight of samples, g; V1—amount of water used to immerse samples, ml; V2—amount of filtrate extracted in each titration, mL; and V3—amount of silver nitrate consumed by each titration, mL.Samples reached designed curing days  were taken out from the solution and then underwent a series of processes including drilling, slicing, polishing, and chemical titration to determine the level of chloride ion in identified depth , with specific procedures as followings. First, core samples with diameter of 20 mm were drilled from cubic samples by a vertical drilling machine . Then, slices were cut from core samples by a low-speed diamond cutting machine , with the thickness of slices controlled by a digital adjustable ruler. Three kinds of thickness were used: 2 mm for the first layer, 3 mm for the second layer, and 5 mm for the third and subsequent layers, accurate to 0.01 mm. Next, slices were crushed and ground into powder in a porcelain mortar. Powder passing through the 0.63 mm sieve was oven-dried under 105 ± 5 °C for 2 h and then transferred to a sealed dryer to cool down to room temperature. At length, 2 g powder was weighted by an analytical balance with a sensitivity of 0.0001 g and then placed in a triangular flask. The flask then was filled with 20 mL distilled water, followed by a vibration lasting 1–2 min. The clean sample after 24 h placing was filtered and moved to another triangular flask. After adding two drops of phenolphthalein, the above prepared filtrate presented a color of light red that was then neutralized to colorless with dilute sulfuric acid. 10 drops of potassium chromate were added as an indicator, followed by the titration of silver nitrate solution until the dark red color was achieved. The amount of silver nitrate used was recorded and then the content of chloride ion dissolved in water was calculated by Equation (1) as per ; x is the first influential factor, viz. the PF dosage [vol.%]; y is the second influential factor, viz. the fly ash content [wt %]; and a to j are undetermined coefficients.The RSM is a statistical method that establishes a functional relationship between influential factors and the respond by the multiregression. In this study, both the quadratic (Equation (4)) and cubic (Equation (5)) polynomials were used to construct the RSM.ANFIS is herein used to construct the mathematical model in predicting the content of chloride ions in identified depths of concrete with varied constituents and in different corrosive conditions. A typical ANFIS contains four specific components, viz. the fuzzification of original input data, the establishment of fuzzy rules, the fuzzy reasoning, and the defuzzification of output data see . It is nx is the independent variable and  is the matrix of parameters.ix (I = 1 to 7) means the input value of each variable and  are the parameters.The specific building process of the ANFIS is illustrated by R2), and the mean absolute error (MAE) were used as criteria in evaluating the performance of the established mathematical model, with specific mathematical expressions available in Equations (8)–(10). Statistically, a smaller RSME or MAE (0 is the best) and a larger R-squared value (1 is the best) means a better prediction performance of the model.x, As suggested by , three sThe dry–wet cyclic environment accelerated the corrosion because the concentration gradient, the temperature gradient, and the humidity gradient concurrently coexisted in the interior of concrete. A severe corrosion was also observed on galvanized steel when a wet–dry cyclic environment applied to it . Chlorid2O3. CaCl2. 10H2O) that retarded the process of further diffusion and decreased aqueous ratios . It means each input variable has two specific MFs used for fuzzification. A total of 400 data sets sourced from the experimental study and other published articles ,21,25,26The basic information of trained ANFIS is as followings. Parameters of gbellmf and linear are given by Number of nodes: 294Number of linear parameters: 1024Number of nonlinear parameters: 42Total number of parameters: 1066Number of training data pairs: 360Number of checking data pairs: 120Number of fuzzy rules: 128R-squared value is calculated as 0.89. The value is higher than 0.7 that is usually set as a benchmark in engineering [The other 40 sets were used in testing, and the performance of testing is depicted by ineering , which mThe paper reported an experimental study lasting one-year on chloride penetration of PF reinforced concrete with fly ash and constructed some mathematical models basing on the RSM and fuzzy logic system. For a comprehensive understanding of the penetration and diffusion of chloride ions in concrete, three interior factors—w/b ratio, PF dosage, and FA content—and two external environment factors—NaCl concentration and curing condition—were selected. The concrete with a w/b ratio of 0.25 restrains the chloride penetration within 10 mm even considering a 360 d dry–wet cyclic immersion, attributing to the dense structure of concrete and the sinking effect of CSH gel on chloride ions. Relying on the filling effect and the pozzolanic reaction, FA content is positive related to the chloride resistance. However, the effect of PF is more prominent at the low dosage because PF potentially triggers postcracking due to a higher flexural creep coefficient. The concentration of NaCl solution is the most significant influential factor for all specimens because a higher concentration means a corresponding higher content of free chloride ions that potentially penetrate into the concrete via the mass transfer process. Relative to the long-term soaking situation, the dry–wet cyclic immersion causes a more severe influence on concrete because chloride ions in the surrounding solution can invade into the interior through the coupled action of the diffusion by the concentration gradient and the convection sourced from the humidity gradient. When it comes to the models, the cubic-polynomial-based RSM proves to be a better model in visualizing the effect of PF dosage and fly ash content on chloride content. The increase of either PF or fly ash shifts chloride content toward a lower level, whereas the impetus of PF is more prominent for concrete with lower content fly ash. The ANFIS that contains 128 IF-THEN rules is instead verified to be a precise model in modeling chloride content that is concurrently affected by seven influential factors.