C-Met tyrosine kinase receptor plays an important role under normal and pathological conditions. In tumor cells’ overexpression or incorrect activation of c-Met, this leads to stimulation of proliferation, survival and increase of motile activity. This receptor is also described as a marker of cancer initiating cells. The latest research shows that the c-Met receptor has an influence on the development of resistance to targeted cancer treatment. High c-Met expression and activation in renal cell carcinomas is associated with the progression of the disease and poor survival of patients. C-Met receptor has become a therapeutic target in kidney cancer. However, the therapies used so far using c-Met tyrosine kinase inhibitors demonstrate resistance to treatment. On the other hand, the c-Met pathway may act as an alternative target pathway in tumors that are resistant to other therapies. Combination treatment together with c-Met inhibitor reduces tumor growth, vascularization and pro-metastatic behavior and results in suppressed mesenchymal phenotype and vascular endothelial growth factor (VEGF) secretion. Recently, it has been shown that the acquirement of mesenchymal phenotype or lack of cell differentiation might be related to the presence of the c-Met receptor and is consequently responsible for therapy resistance. This review presents the results from recent studies identifying c-Met as an important factor in renal carcinomas being responsible for tumor growth, progression and metastasis, indicating the role of c-Met in resistance to antitumor therapy and demonstrating the pivotal role of c-Met in supporting mesenchymal cell phenotype. MET. The activation of the c-Met receptor through its ligand, hepatocyte growth factor (HGF), also known as the scatter factor (SF), leads to the stimulation of various biological effects. Under normal conditions, this receptor takes part in embryogenesis, development of organs, differentiation of i.a. muscular and nerve cells, as well as regeneration of the liver  amplification or gain of chromosome 7. Foretinib demonstrated activity in patients with pRCC with median PFS 9.3 and a high response rate in patients with germline MET mutations [Lastly, foretinib, a multikinase inhibitor targeting c-Met, VEGF, AXL, RON and Tie-2, was tested on patients with papillary renal cell carcinoma. pRCC is often characterized by mutations or amplification in the utations . DespiteAnti-angiogenic therapies of RCC, based on TKIs, such as sunitinib or axitinib show significant benefits in vasculature reduction and decrease of tumor size, especially in ccRCC patients. However, despite the initial good response, relapse occurs due to the acquisition of resistance, resulting in a shift to alternative pathways that will support and promote angiogenesis. The c-Met/HGF signaling pathway plays a significant role in tumor and endothelial cells where it acts as a potent pro-angiogenic trigger. New TKIs, which target not only VEGFR, however also the c-Met receptor, appear to be a response to the problem of resistance, showing a similar tolerance and toxicity as first-line TKIs, yet a superior PFS response and efficiency .Developing resistance to treatment that limits the success of cancer therapy is one of the most difficult problems of the contemporary oncology. Despite significant progress in the use of chemotherapy over the past 40 years, most chemotherapeutic regimens have failed to provide satisfactory therapeutic outcomes due to relapse caused by resistance to treatment. Understanding the molecular mechanisms that cause resistance to a therapy is of great importance for the development of alternative therapies and the improvement of clinical benefits. Most of the mechanisms of resistance include, among others, secondary mutations of inhibited pathways or amplifications of growth factor receptors. However, it seems that the resistance acquisition process is more complex and dependent on a number of changes in the tumor cell itself and in the tumor microenvironment.The acquisition of therapeutic resistance and progression to distant metastatic disease might be a result of the epithelial to mesenchymal transition (EMT) process.The epithelial to mesenchymal transition was observed for the first time in the context of embryonic development, where it regulates key morphogenetic steps such as gastrulation and neural crest formation . EMT is The c-Met receptor has been postulated as an essential factor responsible for the functional cancer stem cell (CSC) phenotype in some tumors and as a CSC factor, it is believed to be responsible for therapy resistance . The results obtained by our team and other research groups have shown that the c-Met receptor is also important in the process of cell differentiation and resistance to chemotherapy for some tumors. In the biology of rhabdomyosarcoma, one of the most common tumors found in the soft tissues of children, the c-Met receptor is very important for the process of proliferation, survival and resistance to chemotherapy ,87,88,89The silencing of the c-Met receptor results in changes of the phenotype in other types of tumor cells. Similar research has shown that the silencing of the c-Met receptor in cervical cancer cells with epithelial origin induced the increase in the level of E-cadherin . DecreasInteresting results also came from research on lung cancer where the reinstitution of the E-cadherin expression increased the cancer cells’ sensitivity to the cytostatic drug, gefitinib . SummariHowever, the role of the c-Met receptor in the acquisition of mesenchymal phenotype is not well understood for kidney cancer. Our group has already shown that in an in vitro model of clear cell renal cell carcinoma cells and in the normal human renal cell line HEK293, increased level and phosphorylation of c-Met correlates with the acquisition of mesenchymal cell characteristics, i.e. an increase in the migration activity, the suppression of E-cadherin and upregulated β-catenin and vimentin . The incThe presence of the c-Met receptor is crucial not only for the high level of proliferation and initialization of tumor growth in in vivo conditions, however is also a significant factor in sustaining the undifferentiated/mesenchymal phenotype of cancer cells .MET gene mutations lead to c-Met activation and stimulation of various pathological effects, such as proliferation, survival, angiogenesis and increased motile activity. In addition, this receptor is described as a marker of cancer initiating cells. All this leads to tumor growth and progression.The c-Met receptor plays a significant role in healthy kidney and RCC development. Paracrine or autocrine HGF stimulation, EMT or MET gene causes constitutive activation of the c-Met protein, which correlates with poor survival rates. The c-Met receptor becomes one of the main factors involved in acquiring resistance to targeted therapies used in RCC. Mechanisms underlying therapy resistance consist of the compensation of blocking VEGFRs by activating other receptors involved in the regulation of angiogenesis such as the c-Met. Therefore, combination treatment of anti-angiogenic drugs with c-Met inhibitor reduces tumor growth, vascularization and VEGF secretion. Moreover, the role of the c-Met in tumor progression is extremely significant through its impact on sustaining the undifferentiated mesenchymal phenotype of cancer cells, which consequently is responsible for therapy resistance. Here we showed that c-Met level is upregulated in RCC tumor tissue compared to normal kidney and increases during RCC progression. Frequent VHL mutation and hypoxia lead to increased c-Met expression in clear cell RCC. In papillary RCC, missense mutation of the Taken together, c-Met is an important factor involved in the acquisition of resistance to targeted therapies, sustaining mesenchymal, undifferentiated phenotype, and increased renal carcinomas’ growth, progression and invasiveness.


Escherichia coli resistance to amoxicillin (AMX) and cefotaxime (CTX) from positive blood culture based on MALDI-TOF MS. Potential sparing of broad-spectrum antibiotics was also evaluated. We tested 103 E. coli-positive blood cultures. Blood cultures were subculture 1-hour in antibiotic-free rich liquid media before further incubation with and without AMX for 2.5 h or CTX for 2 h. Protein extracts associated with an internal control were spotted on a MALDI-TOF target and spectra were analyzed with the MBT-ASTRA prototype software . Bacterial growth ratio was calculated from the AUC spectra obtained in the presence and absence of the antibiotic and compared to a threshold which classified E. coli as susceptible or resistant. Results were interpreted with MICs determined using agar dilution method as reference technique. MBT-ASTRA recognized 95% and 84% of the AMX- and CTX-susceptible isolates, respectively. Overall, quantitative analysis of mass spectra allows susceptibility testing within 4 hours after the positivity of blood culture with E. coli. At the first report of positive blood culture, MALDI-TOF MS would then provide the prescribers with the bacterial identification and the susceptibility to AMX and CTX, thus limiting the use of broad-spectrum compounds.Antibiotic resistance is a major public health problem requiring the early optimization of antibiotic prescriptions. Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) has been shown to accurately identify bacteria from positive blood culture. Here, we developed a rapid detection of  Enterobacteriaceae limits treatment options [i.e. third-generation cephalosporins, carbapenems) and to ensure the timely appropriate antibiotherapy. Hence, rapid antibiotic testing is an area of intense research.In recent decades, antibiotic resistance has become a major public health problem requiring the optimization of antibiotic prescriptions. However, the current methods for the determination of bacterial resistance require time. Indeed, after bacterial identification, antibiotic testing needs from 7 h for automated systems with fast-growing species  to 24 h  options . The anti) the sub-species identification or typing (ii) and the detection of particular β-lactamase-based resistance [e.g. impermeability, target mutations, antibiotic modification, active efflux).Recently, Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) has become a valuable tool in clinical laboratories for rapid microorganism identification, from colonies or directly from positive blood vials samples , 5. MALDsistance –8. HowevKlebsiella pneumoniae to meropenem can be determined within 1 hour with the new method named MALDI Biotyper-Antibiotic Susceptibility Test Rapid Assay (MBT-ASTRA) [A preliminary study showed that the susceptibility of T-ASTRA) . This teE. coli to amoxicillin and cefotaxime directly from positive blood cultures. Our goal was (i) to simultaneously report the bacterial species and the therapeutic options to the clinician (ii) to evaluate the potential sparing of broad-spectrum antibiotics.A recent analysis compared cost-effectiveness of rapid laboratory testing for the diagnosis of bloodstream infections . It founi) technique development on blood vials (negative after 5 days of incubation at 35°C) inoculated with clinical E. coli isolates (set 1), (ii) implementation to E. coli-positive blood cultures of patients (set 2). All the isolates were identified as E. coli by MALDI-TOF MS Microflex LT with a log score value ≥ 2 according to the manufacturer's recommendations . The susceptibility to AMX and CTX was determined by agar dilution method according to the CLSI recommendations [We carried out the study in 2 steps for both amoxicillin (AMX) and cefotaxime (CTX):  between 2011 and 2016 and stored at the Centre de Ressources Biologiques Filière Microbiologique, Besançon . E. coli isolates to AMX and CTX. We seeded blood-containing vials  with E. coli isolates for a final load of 106 CFU. Vials were incubated at 35°C in an automated blood culture system  until its detection.We used 123 non-duplicate clinical isolates of E. coli (We included 103 patients (age > 18 years) hospitalized in the University Hospital of Besançon between April 2017 and November 2017 with a blood culture positive with E. coli .E. coli-containing blood culture were transferred in a pre-warmed Brain Heart Infusion (BHI) broth and incubated at 37°C for 1 h under agitation (180 rpm). Bacterial suspension was adjusted to 1 McFarland with BHI and further incubated with or without antibiotic . One μl of the E. coli protein extracts were deposited in duplicate on a MALDI-TOF target. Spots were overlaid with 1 μl of HCCA matrix solution  and air dried  that contains an E. coli reference strain along with RNase A and myoglobin.We acquired the spectra with a MALDI-TOF MS MicroFlex LT  in the linear positive ion mode, with a laser frequency of 60 Hz. Parameter settings for Microflex LT were: ion source 1: 20 kV, ion source 2: 18.5 kV, lens: 6 kV, pulsed ion extraction: 100 ns. The MALDI-TOF analysis used spectra of medium molecular masses ranging from m/z 2000–10000. Thus, when normalized with a remaining internal control, the antibiotic-susceptible isolate AUC is reduced when incubated in the presence of this antibiotic (www.openepi.com).Spectra were analyzed with the MBT-ASTRA prototype software  which estimates the bacterial growth. The incubation of an isolate with an active antibiotic reduces the intensity and the number of the peaks detected by MALDI-TOF MS. On the other hand, the incubation of an isolate with an inactive antibiotic will not modify the spectra . The aretibiotic . For eactibiotic . PrelimiE. coli-positive blood culture over the duration of the study (April to November 2017), we recorded demographic and clinical data, concomitant infectious diseases, prescribed probabilistic antibiotic treatment, prescribed antibiotic treatment after blood culture sampling and after information of the clinician of bacterial identification and resistance. These data were analyzed with regards to the MICs and MBT-ASTRA results. Since the present technique was under evaluation, the results had not been transmitted to the clinicians during this study.We compared antibiotic treatments received by the patients (first 48 h) with treatments that could have been prescribed if the resistance to AMX and CTX had been assessed directly from the positive blood vials. We collected retrospectively the data from medical records and from the pharmacy software. For each patient with a The study was approved by the ethical committee ‘Comité d’Etude Clinique’ of the Besançon hospital, Besançon, France.We evaluated the performance of MBT-ASTRA technique for the rapid AMX and CTX susceptibility testing. Using MIC determination as a gold standard, for the detection of resistance to AMX we found that 97% of the isolates (59 out of 61) were correctly classified by the MBT-ASTRA technique with a sensitivity of 100% and a specificity of 93% . Two AMXThe overall categorical agreement between MBT ASTRA method and the reference method was 97% (100 isolates out of 103) and 83% (85 isolates out of 103) for AMX and CTX, respectively . The MBTE. coli, de-escalation of antibiotic treatments was achieved for 16 patients. No adaptation was done for 39 patients. In 34 cases, either data on the antibiotic treatment were missing from medical charts or antibiotic sparing was impossible due to associated infections. Of the 16 cases where antibiotic de-escalation was performed, 11 isolates were AMX-susceptible, and 5 isolates were AMX-resistant. An early report with MBT-ASTRA would have saved 8 and 7 days of third-generation cephalosporins and piperacillin-tazobactam respectively. Among the 39 patients who had not been de-escalated, MBT-ASTRA would have saved 112, 40, and 12 days of third-generation cephalosporins, piperacillin-tazobactam, and carbapenems, respectively.Among 89 infections with a CTX-susceptible Enterobacteriaceae, Pseudomonas sp., and Acinetobacter sp.) were tested in combination with different antibiotic classes  [Mycobacteria tuberculosis was tested with rifampicin and izoniazid, respectively [i.e. antibiotic concentration and incubation time) is necessary for each species-antibiotic combination. Unfortunately, the antibiotic (ATB) concentrations to be tested (ATBMS-ASTRA) cannot be predicted from the susceptible clinical breakpoints (ATBS breakpoint). For instance, the ratio CTXMS-ASTRA/CTXS breakpoint varies from 2 to 20 depending on the bacterial species and studies [MBT ASTRA method indirectly estimates the growth of bacterial strains. The peak normalization combined with an internal quantitative control permits the comparison of the total intensity of the MALDI-TOF MS spectra. The peak intensity reflects the amount of bacterial proteins, which directly depends on the bacterial growth . Recentlcosides) , 14. Mycectively . The pro studies , 14. MorE. coli to AMX and CTX directly from positive blood culture vials. Early information of the susceptibility to these compounds is necessary to spare broad-spectrum antibiotics such as third-generation cephalosporins, piperacillin-tazobactam, and carbapenems. Despite many attempts, we failed to find optimal testing conditions for testing susceptibility to piperacillin-tazobactam with MBT ASTRA, possibly due to an initial plateau phase in the time-kill curves of piperacillin or to the instability of the antibiotic association [We present here a procedure for the rapid susceptibility testing of ociation , 14.E. coli (set 1) and further implemented on E. coli-positive blood cultures (set 2). The MBT- ASTRA assay testing AMX was highly sensitivity (100% and 98%) and reasonably specific (93% and 95%) with set 1 and 2, respectively. This contrasted with the assay testing CTX. Hence, while the sensibility and the specificity with set 1 was good , the sensibility and the specificity was poor using E. coli-positive blood cultures . The discrepancy between the sets is presumably due to the low proportion of CTX-resistant isolates in set 2. The correlation between the relative growth value and the MIC of CTX could be ameliorated with the extension of the incubation time but makes the test less attractive.MBT ASTRA method was first optimized using a set of sterile blood vials inoculated with i) determine the susceptibility of E. coli to AMX and (ii) identify CTX-susceptible E. coli directly from positive blood culture in less than 4 h, independently from the resistance mechanism. In the same workflow, species identification with MALDI-TOF MS and rapid antibiotic susceptibility testing with MBT-ASTRA can significantly reduce the time to re-evaluate the antibiotic therapy. Our data confirm that MBT-ASTRA approach is promising [Rapid antibiotic susceptibility testing is an area of intense interest –18. Hereromising –15. MALDWe also assessed the potential sparing of broad-spectrum antibiotics. The most important point to emphasize is that the MBT ASTRA concept would streamline the information transmitted to prescribers. Shortly after the detection of positive vials by automates, they would be informed of the pathogen identification together with its susceptibility to keystone antibiotics, which favors the prescription of the active antibiotic with the narrowest spectrum. However, the potential sparing of broad-spectrum antibiotics using MBT ASTRA remained to be demonstrated in real life and depends on the commitment of prescribers in antimicrobial stewardship policies.S1 Fig200 μl of E. coli-positive blood cultures were transferred in a pre-warmed BHI broth and incubated at 37°C for 1 hour. The density of the bacterial culture was adjusted to 1 McFarland with BHI and further incubated at 37°C with and without the antibiotic to test. After washing, formic acid, acetonitrile and internal control were added. The supernatant was spotted on a MALDI-TOF target and overlaid with HCCA . Bacterial growth ratio was calculated from the AUC spectra obtained in the presence and absence of the antibiotic and compared to a threshold which classified E. coli as susceptible or resistant.(DOC)Click here for additional data file.


It is debated whether transperitoneal membrane transport of larger (charged) molecules in peritoneal dialysis can be partially governed by the electrokinetic model. In this model, it is postulated that streaming potentials are generated across the capillary wall by forced filtration of an ionic solution, for example transcapillary ultrafiltration induced by osmotic forces as in peritoneal dialysis. We investigated the presence of streaming potentials in the process of transperitoneal transport in Peritoneal Dialysis (PD) patients by measuring ratios of dialysate concentrations of IgG2  and IgG4 (negative), both 150kD, under different conditions of transcapillary ultrafiltration.Adult PD patients randomly got two consecutive dwells of 120 min each, with either 2 L Physioneal 1.36% or 3.86% glucose dialysis fluid  as their first dwell. A blood sample was taken at the test start, and dialysate samples were taken at 5, 15, 30, 60 and 120 min. IgG2 and IgG4 concentrations were measured (ELISA) and ratios calculated.P = 0.007). At none of the time points and irrespective of glucose concentration, a significant difference was found between the IgG2/IgG4 ratios at any time point.In 10 patients , drained volume after 120 min was different between the 1.36%  and 3.86%  glucose dwells ; prospective randomized trial. Within the electrokinetic model, streaming potentials are generated across a filter by forced filtration of an ionic solution . The forIn peritoneal dialysis (PD) it is accepted that solute transport across the peritoneal membrane can be modelled by the three pore model , where tIf this electrokinetic model exists in the peritoneal membrane, it will alter our understanding of transperitoneal transport, potentially opening new opportunities to use alternative osmotic agents, develop protective strategies, or detect early changes in peritoneal membrane integrity. Furthermore, a better understanding of streaming potentials would lead to a better insight in the increased transperitoneal protein loss in PD over time.Our study therefore intends to investigate the hypothesis of the presence of (reversed) streaming potentials in the process of transperitoneal transport in PD patients by measuring dialysate IgG2/IgG4 concentration ratios, under different conditions of transcapillary ultrafiltration. These differences were induced by using non-hypertonic vs hypertonic glucose, and by measuring at different time points during the dwell, as transcapillary ultrafiltration is also decreasing with dwell time. If streaming potentials do play a role, these differences in transcapillary ultrafiltration will result in different IgG2/IgG4 ratios during the dwell, and also between the non-hypertonic and hypertonic exchanges.Consecutive adult patients on peritoneal dialysis in the Ghent University Hospital were asked consent for participation in this prospective randomized cross-over study until 10 patients had completed the study. Exclusion criteria were active infection, pregnancy, unstable hemodynamic condition precluding use of hypertonic glucose, peritonitis in the last 4 weeks preceding the study, and age below 18. Patients with malfunctioning catheters were excluded. Malfunctioning was assessed based on medical history. In addition, when on a routine Peritoneal Equilibration Test (PET) test residual volume was more than 15%, the patient was also excluded.The study was approved by the local Ethics Committee (Commissie voor Medische Ethiek - UZ Gent - Ref 2015/0075 - Belgian Registration Number B670201523397), and written informed consent was obtained from all participants.www.randomization.com.Study flow is depicted in Fig. A blood sample was taken from the patient before the test start, and dialysate samples were taken during each dwell at 5, 15, 30, 60 and 120 min via the peritoneal catheter. At each of these time points, the patient was first rolled from side to side to enhance optimal fluid mixing in the peritoneal cavity before 200 mL dialysate fluid was drained to diminish the impact of remaining fluid in the catheter’s dead volume. Of this sample, 30 mL was sampled and put on ice for transport to the laboratory, while the remaining 170 mL were re-instilled into the peritoneal cavity. After 120 min dwell time, dialysate fluid was drained completely, and the volume checked using gravity.Samples were immediately centrifuged at 4 °C during 10 min , after which the serum and dialysate were stored at − 80 °C until batch analysis. For all samples, IgG2 and IgG4 concentrations were estimated using commercially available ELISAs . Preliminary tests were performed to check the most optimal dilution of the samples for the respective ELISAs; for serum, dilutions were needed of 1/500,000 (IgG2) and 1/5000 (IgG4), and for dialysate fluid dilutions were 1/500 (IgG2) and 1/10  and 1/50 (IgG4 at time point 60 and 120 min).For each time point and in each patient, the ratio of IgG2 over IgG4 concentrations was calculated.The IgG2 and IgG4 assays detect both natural and recombinant human IgG2 and IgG4 respectively. No cross reactivity or interference of circulating factors of the immune system was detected. The curves obtained by serial dilutions of the dialysate was parallel to the standard curve.The limit of detection of resp. human IgG2 and IgG4 defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be resp. 0.25 ng/ml and 0.1 ng/ml (mean of 4 independent assays).The intra-assay coefficient of variation for serum samples was 3.6% for IgG2 and IgG4. The intra-assay coefficient of variation for dialysate samples was 4.3% for IgG2 and 5.9% for IgG4.Statistical analyses were performed with SPSS 23 (IBM©). To compare results from the 1.36% to 3.86% dwell, patients were considered their own control, so a paired non-parametric analysis was applied . For the evolution of IgG2/IgG4 over the dwell time, a repeated measures Friedman analysis was applied. We also constructed a general linear model for ratio of IgG2/IgG4, including patient identification, osmotic tonicity and time points as covariates, and interaction terms for tonicity and time points.No formal sample size calculation was performed, as this was deemed not reliable as no data were available to estimate effect size or standard deviation and patients served as their own controls.2 at the time of the study were included. Drained volume after 120 min was different between the 1.36% and the 3.86% glucose dwells: i.e. 1950  mL versus 2540  mL, respectively (P = 0.007).Ten patients , 65 ± 17 years of age, 20 ± 17 months on dialysis, and with a residual renal function of 9.7 ± 5.6 mL/min/1.73mSerum concentrations were 1.7  mg/mL for IgG2 and 1.3  mg/mL for IgG4.IgG2/IgG4 concentration ratios in dialysate are shown in Table p < 0.001, R2 = 0.022,), only patient identification but not use of hypertonic vs non-hypertonic bags or timepoint of dwell had an impact on IgG2/IgG4 ratio , as would be expected if such electrokinetic forces would be present, and this despite the fact that sufficient differences were obtained in transcapillary ultrafiltration by using two different osmotic strengths of glucose. Also, no difference could be observed between the transport ratios at different time points, in contrast to what would be expected if the electrokinetic model would apply, as transcapillary ultrafiltration decreases during the dwell.The electrokinetic model of transcapillary transport has been a subject of debate for a long time –14. PropIn peritoneal dialysis, there is an observation of higher transperitoneal protein transport in conditions of inflammation , 16. WheDifferent explanations can be forwarded to explain our negative findings.First, it might be that IgG2 and IgG4 are not suitable to test the  impact of the electrokinetic forces in peritoneal dialysis. Although they have a different charge, IgG2 and IgG4 both have a comparable molecular weight around 150kD, which is 3 fold higher than that of albumin. Also, albumin has a globular structure, whereas immunoglobulins have not, but can have different, more tube-like shapes. As such, the hindrance in transport purely based on sterical hindrance can be so big that eventual small charge effects as would be induced by the electrokinetic forces in the peritoneal capillary, are overruled. However, at the level of the glomerular basement membrane, IgG2/IgG4 ratio was found to be decreased in patients with glomerulonephritis as compared to healthy volunteers (nearly 3 fold) or patients with other causes of underlying kidney disease . This fiSecond, it might be that the electrokinetic model cannot be applicable to peritoneal membrane transport because of huge differences between anatomical structures surrounding the capillaries of the glomerular barrier versus those of the peritoneal membrane . WhereasThird, it might be that we did not achieve a sufficient gradient of solute drag and transcapillary ultrafiltration. However, we actually achieved a difference in ultrafiltration of 500 mL over a 2 h dwell, which is equivalent to a mean transcapillary ultrafiltration of around 4 mL/min. As transcapillary ultrafiltration is maximal at the beginning of the dwell, and decreases with duration of the dwell, the achieved values in the early stage of the dwell should be substantially higher than at the end. However, no difference in IgG2/IgG4 ratios at different time points was observed, neither with 1.36 or 3.86% glucose solutions.Last, our set up might lack sufficient power to detect a meaningful difference, and sensitivity of the approach might be too low to detect a meaningful effect, especially as many other interfering forces might be at play in the clinical setting. Also Imholz et al.  could noAlthough the electrokinetic hypothesis is appealing to help explain transcapillary transport in peritoneal dialysis, we failed to provide evidence for the existence of streaming potentials in peritoneal dialysis. More sophisticated exploration of this intriguing hypothesis is warranted.


Using cyclic voltammetry, an electrochemical functionalization of the GO layers with PDPA doped with H3PMo12O40 heteropolyanions takes place, as demonstrated by Raman scattering and FTIR spectroscopy. The presence of the amine groups in the molecular structure of the doped PDPA functionalized GO layers induces a chemical adsorption of PDITC on this platform, when the thiourea groups appear simultaneously with o-thiocarbamate groups. A chemical mechanism is proposed to take place at the interface of the GO sheets and the doped PDPA functionalized GO layers, respectively, with PDITC.Adsorption processes of 1,4-phenylene diisothiocyanate (PDITC) on two new platforms of the type graphene oxide (GO) sheets and GO layers functionalization with polydiphenylamine (PDPA) are studied by Raman scattering and photoluminescence (PL). An interaction in solid state phase of the two constituents, i.e. PDITC and GO sheets, and a deposition of PDITC onto the PDPA functionalized GO layers, respectively, by the drop casting method, were performed. In the first case, it is shown that interaction in solid state phase of GO with PDITC leads to an intercalation of the organic compound between GO sheets simultaneously with the appearance of the o-thiocarbamate groups, that induces: (i) an enhancement of the PDITC Raman lines situated in the 400–800 and 1000–1300 cm Applications of PDITC in the field of electrochemical immunosensors, for the detection of markers such as the epidermal growth factor receptor1 and Murine double minute 22 in the brain tissue or human plasma, have involved a chemical adsorption of PDITC onto the gold nanoparticles/electrodes surface modified with cysteamine. The high cost of the Au nanoparticles/electrodes is a major drawback for the manufacturing of such platforms at large-scale. In order to overcome this inconvenient, in the present work, the GO sheets and the GO layers functionalized with polydiphenylamine (PDPA) are characterized. In this work, the attention will be focalised on the understanding of the PDITC adsorption mechanism during the interaction in the solid-state phase of PDITC with the GO sheets and the deposition by drop casting method of PDITC onto the GO layers functionalized with PDPA. A study concerning the adsorption/interaction of the PDITC on/with GO sheets or their composites has not been reported until now. In this work, the main aim consists in the understanding of the adsorption mechanism of PDITC onto the GO sheets functionalized with PDPA in doped state. Thiocyanate adsorption was studied using Ag nanoparticles5, Au films modified with cysteamine6 and the Au-Pd core-shell nanoparticles7. Optical methods often used in evaluating PDITC adsorption on the metallic nanostructures include surface enhanced Raman scattering, IR spectroscopy and atomic force microscopy6.Organic compound 1,4-phenylene diisothiocyanate (PDITC) is one among from the often used coupling agents (cross-linkers) for biological applications performed in the presence of various surfaces modified with amines of the type ethylenediamineIn this work, the Raman scattering will also be used for assessing the PDITC adsorption on the GO sheets and the PDPA functionalized GO layers. The photosensitivity of PDITC will be highlighted by PL in all subsequent studies shown in this work, in order to take into account this process. The influence of GO on the PDITC photosensitivity will also be shown. A mechanism for the PDITC adsorption on the two structures, i.e. the GO sheets and the PDPA functionalized GO layers, will be reported as well.−1, assigned to the hexagonal rings breathing vibrational mode and the E2g phonon mode at the Brillouin zone centre8. Figure −1 spectral range and other five lines of low intensity localized in the 300–700 and 2000–2200 cm−1 spectral ranges. The PDITC Raman lines, peaked at 368, 434, 632–695, 1157, 1257, 1583, 1603 and 2080 cm−1, are assigned to the following vibrational modes: deformation of a p-substituted benzene ring9, bending deformation of the NCS bond10, asymmetric C-S stretching11, C-S bending11, C-H in benzene ring + C-C stretching + C-N stretching9, C=C + C-C stretching in benzene ring12, C-C stretching + C-H bending in benzene ring9 and C=N stretching5, respectively. The following differences are observed in the Raman spectra of the platelets of PDITC with 0, 1 and 2 wt.% GO  from 5.29  the ratio between the relative intensities of the Raman lines peaked at 1583, 1255–1257 and 1157 cm−1 (I1583/I1255–1257 and I1583/I1157) decrease from 0.71 and 1.1  from 8.82  an t.% Fig. ; (ii) a .29 Fig.  to 2.82–.29 Fig.  with inc1.1 Fig.  to 0.39 1.1 Fig.  or 0.34 1.1 Fig. , as the .82 Fig.  to 0.63 .82 Fig.  and 0.56.82 Fig. , when th −1 Fig.  to 1348  −1 Fig.  and 1352 −1 Fig.  occurs i−1 spectral ranges.All these changes indicate the presence of a steric hindrance effect that can be explained only if we accept that an interaction between PDITC and GO takes place according to Fig. −1, these being assigned to the following vibrational modes: C=C, OH groups from the GO sheet edges, alkoxy groups, H2O adsorption onto the GO sheets' surface, C=O groups belonging COOH and OH stretching8. The most intense IR bands of PDITC are peaked at 827 and 1489 cm−1, these being assigned to the vibrational modes of bending of C-H out of plane in benzene p-substituted and –C=N-benzene ring, respectively13. The interaction of PDITC with the GO sheets leads to the following changes in the IR spectra of the two constituents: (i) a decrease in the ratio between the absorbance of the two IR bands of PDITC peaked at 1489 and 827 cm−1 from 0.71 . The difference in the value of the PL band maximum shift is due to the small number of the isothiocyanate groups in the compounds resulted from the interaction of the GO sheets with PDITC. Summarizing all these results, we can conclude that: i) interaction in solid state phase of GO with PDITC leads to an intercalation of the organic compound between GO sheets, leading to the generation of new o-thiocarbamate functional groups, and ii) the manipulation of PDITC and its derivates must to be carried out in the absence of UV light in order to avoid occurrence of photochemical reactions.Additional information concerning the interaction of the GO sheets with PDITC is obtained by IR spectroscopy. Figure −3 M H3PMo12O40 and 1 M HCl in DMF:H2O with different concentrations of DPA, i.e.10−3  a down-shift of the Raman line from 833 to 815 cm−1 accompanied of a change of the ratio between the relative intensity of Raman lines localized at 1602–1609 and 1200–1202 cm−1 from 3.2 to 2.3; and (iii) a gradual decrease in the relative intensity of the D band of GO simultaneously with the appearance of the Raman lines peaked at 1283, 1329 and 1372 cm−1. These changes indicate: (i) the insertion of the H3PMo12O40 heteropolyanions onto the PDPA macromolecular chain, confirmed by the presence of the Raman lines situated in the 850–1100 cm−1 spectral range; (ii) a progressive coverage of the GO sheets with PDPA in the doped state by the intensity increase of Raman lines of the macromolecular compound and (iii) the covalent functionalization of the GO sheets with PDPA, highlighted by the new Raman line peaked at 1283 cm−1, that was also reported in the case of triphenylamine and its derivate compounds, this being assigned to the vibrational mode of large C-N stretches with associated C-C stretches18. The molecular structures of the GO sheets covalently functionalized with PDPA doped with the H3PMo12O40 heteropolyanions as well as of the macromolecular compound in doped state are shown in Fig. 3PMo12O40 heteropolyanions with the GO sheets. Additional information concerning the functionalization process is shown in Fig. 3PMo12O40 heteropolyanions. According to previous studies20, the IR bands belonging to H3PMo12O40 have the maxima peaked at 792, 870, 960 and 1065 cm−1. A careful analysis of Fig. −1 to 800 and 887 cm−1, while the last two IR bands attributed to the Mo=O and P-O vibrational modes are down-shifted from 960 and 1065 cm−1 to 945 and 1057 cm−1 20. The change of the position of these IR bands was correlated with the doping level induced by this heteropolyacid to conjugated polymers20. In this context, we note that this behaviour is similar to that reported in the case of the single-walled carbon nanotubes functionalized with PDPA doped with H3PMo12O40 heteropolyanions16. The IR bands belonging to PDPA are peaked at 692, 746, 1020, 1176–1202, 1313, 1466, 1491 and 1591 cm−1 and their assignment is shown in Table 1S21. As increasing the DPA concentration in the synthesis solution, an increasing in the absorbance of the IR band peaked at 1591 cm−1 is noted as a consequence of a greater weight of PDPA compared to the GO layer surface. As shown in our previous work, the ratio between the absorbance of the IR bands of standalone PDPA peaked at 698, 752 and 1028 cm−1 (A698/A1028 and A698/1028) is equal to 7.1 and 6.9, respectively21. These IR bands were assigned to the vibrational modes of inter-ring deformation, benzene ring deformation and A1 benzene21. According to Fig. −3 M to 5 10−3/10−2 M, one observes that: (i) the vibrational modes of inter-ring deformation, benzene ring deformation and A1 benzene are down-shifted at 692, 746 and 1020 cm−1; (ii) the values of the A692/A1020 and A746/1020 ratios are changed from 0.39 and 0.43 to 0.68/0.95 and 0.86/1.15, respectively and (iii) the ratio between the absorbances of the IR bands peaked at 945 and 1020 cm−1 is changed from 0.39 to 1.23 and 1.67. The higher absorbance of the IR bands assigned to the deformation vibrational modes of the benzene and quinoid rings as well as inter-rings highlights the significant steric hindrance effects induced by the covalent bonding of PDPA doped with H3PMo12O40 heteropolyanions onto the GO layers.Figure 0−3 Fig. , 5 10−3  −3 Fig.  and 10−22 M Fig. . A short2H5OH having the concentration 0.1 mg/ml, by the evaporation of 1 ml (a) and 2 ml (b) solvent. The GO layers electrochemical functionalized with PDPA in doped state were prepared using a semi-aqueous solution of 10−3 M DPA, 10−3 M H3PMo12O40 and 1 M HCl in DMF: H2O. In the two cases, the Raman spectra are dominated by the PDITC Raman lines. Other Raman lines observed in Fig. −1 belonging to the Mo-O-Mo vibrational modes of the H3PMo12O40 heteropolyanions and the GO D band. The absence of the Raman lines of the PDPA is difficult to be understood. In order to show an evidence for the presence of macromolecular compounds, Fig. 2, (ii) 11-Mercaptoundecanoic acid modified Au electrode22; and (iii) β-cyclodextrin – reduced graphene oxide-tetraetylene23. An optical image of the samples of the doped PDPA functionalized GO layers after the PDITC adsorption is shown in Figure 6c1 and 6c2.These images highlight the formation of a one-dimensional macrostructures as a consequence of the generation of the thiourea groups by the PDITC adsorption onto the doped PDPA functionalized GO layers. The presence of isothiocyanate groups onto the surface of the GO sheets and that of the GO layers functionalized by PDPA, which were intercalated/modified with PDITC, open new perspectives on the applications of these platforms in the immunosensors field for the detection of the cancer markers, for example.Figure 2H5OH having the concentration of 0.1 mg/ml, by the evaporation of 2 ml solvent onto the GO sheets functionalized with PDPA doped with H3PMo12O40 heteropolyanions. According to Fig. We note that samples resulted after the deposition of PDITC onto the GO layers functionalized by PDPA must be handled in the absence of UV light. In order to support this sentence, Fig. i)the interaction in solid state phase of PDITC with the GO sheets results in a chemical adsorption of organic compound onto the carbon nanostructure. Taking into account the changes reported in the Raman spectra of PDITC and the GO sheets, two resulting compounds were identified: one corresponding to the GO sheets intercalated with PDITC, exhibiting o-thiocarbamate groups, and a second in which the GO sheets are modified with PDITC having in the molecular structure both the o-thiocarbamate functional groups and an isothiocyanate terminated surface.ii)−3M H3PMo12O40 and 1 M HCl in DMF: H2O, when SPCE modified with GO was used as working electrode, leads to the GO layers covalently functionalized with PDPA in doped state;the electropolymerization of DPA in the presence of the semi-aqueous solution of 10iii)the adsorption of PDITC onto the GO layers covalently functionalized with PDPA in doped state involves the appearance of the thiourea groups simultaneously with those of the type o-thiocarbamate.iv)a photochemical process was reported in the case of the PDITC adsorption onto the GO sheets surface as well as the GO layers covalently functionalized with PDPA in doped state. These results indicate the necessity of the manipulation of these platforms in the absence of the UV light.In this work, we have reported new results concerning the adsorption of PDITC onto two platforms, i.e. GO sheets and GO layers covalently functionalized with PDPA in doped state. Our results have highlighted that:3PMo12O40 x H2O, HCl, graphite, dimethylformamide (DMF), C2H5OH, sodium nitrate, H2SO4, KMnO4 and H2O2 were used as received from Sigma-Aldrich. PDITC was purchased from Expert Trade. The GO sheets were synthetized according to ref.8.All chemical compounds, i.e. diphenylamine (DPA), H3PMo12O40 acts as an initiator for the growth of macromolecular chain of PDPA onto the GO sheets and a doping agent of PDPA. The GO layers functionalized with PDPA doped with H3PMo12O40 heteropolyanions were prepared by electrochemical polymerization of DPA onto the screen-printed carbon electrodes (SPCE) modified with GO purchased from DropSens. The cyclic voltammetry studies were carried out by the immersion of the SPCE into a solution consisting from DPA , 10−3 M H3PMo12O40 and 1 M HCl in semi-aqueous solution of DMF:H2O having the volumetric ratio of 1:1. The potential range was between +100 and +960 mV vs. Ag electrode and a sweep rate equal with 50 mV s−1 was used. The reported cyclic voltammograms using SPCE were recorded with a potentiostat/galvanostat, Voltalab 80 model, purchased from Radiometer Analytical.In the present work, HThe interaction in solid state phase of PDITC with the GO sheets was carried out in the absence of light by the mechanico-chemical reaction of the two constituents that were compressed non-hydrostatically at the pressure of 0.58 GPa, time of 5 minutes, resulting in platelets of PDITC with 1 and 2 wt.% GO.2H5OH having the concentration of 1 mg/ml.The PDITC deposition on the PDPA functionalized GO layers was performed by the drop casting method using a solution of PDPA in CRaman spectra of PDTIC, GO sheets, PDPA intercalated GO sheets and PDPA functionalized GO layers before and after the interaction with PDTIC were recorded under the excitation wavelength of 514 nm using a spectrophotometer Raman, T64000 model, from Horiba Jobin Yvon, endowed with an Ar laser.The PL spectra of PDTIC, PDPA intercalated GO sheets and PDPA functionalized GO layers before and after the interaction with PDTIC were recorded at room temperature, under the excitation wavelength of 375 nm and 275 nm, respectively, with a spectrophotometer Fluorolog-3.2.2.1, from Horiba Jobin Yvon.The IR spectra of GO sheets before and after the interaction with PDITC were recorded with a FTIR spectrophotometer, Vertex 80 model, from Bruker.Supplementary Information


Antibiotic misuse is considered one of the major causes of antimicrobial resistance.The study aims to investigate the practices of antibiotic misuse in the region of Hail, Saudi Arabia and the extent of people awareness of antibiotic resistance.Participants ≥18 years of age of both genders were recruit by convenience sampling from different public places.Out of 500 participants, 26% obtained their antibiotic without a prescription, 27% took antibiotics for unknown infections and only 34% completed antibiotic course. A total of 36.2% did not know about antibiotic resistance and its rise, but the majority were willing to learn. The results warrant further study into malpractice with a wider geographic area and sample size to generalize the results for the whole country. Antibiotics are a group of drugs that treat different bacterial infections. However, inappropriate use of antibiotics such as overuse (used when they are not needed) or use for the wrong reason can lead to antibiotic resistance (bacteria that are harder to treat). Patient knowledge of antibiotics is considered an important contributing factor for antibiotic misuse. Thus, this study was conducted to evaluate patient knowledge about antibiotic misuse and resistance among a population of Hail in the northern part of Saudi Arabia. Antibiotics have been around since the 1950s, helping people survive what could be fatal infections and ultimately live longer. Many antibiotic classes were discovered during the ‘golden era’ of the 1950s to 1970s . This inet al. that surveyed ∼15,485 participants across 19 European countries reported a consistent association between prescribed use and self-medication [Despite technological advancements and public awareness of such an epidemic, many practices remain resistant to change. For instance, in Europe, a study by Grigoryan dication . The pradication . In the dication . InapproWith AR on the rise, the United Nations has called for a global action plan to combat antimicrobial resistance. AR poses a major threat to human and animal health, crop production and development. The compounding effect of AR can be observed with the epidemic of newborn deaths  that are a result of infections that do not respond to current antibiotics, as well as the rise of the number of cases of untreatable bacterial infections . StrainsMany factors have been attributed to the rise in AR including overuse, misuse and the availability of few new antibiotics . OveruseIt is evident that AR is a multifactorial problem and coordinated efforts among various parties including patients is necessary to initiate headway in combating this deadly inevitable evolutionary phenomenon. Therefore, in this study, we aimed to investigate the practices of people who consume antibiotics and the extent of their awareness of AR in the region of Hail, Saudi Arabia. These data present one of the first reports of antibiotic use and AR awareness in the region of Hail, a small town in the northern region of Saudi Arabia.A submission including full project proposal was made to the Scientific Research Ethical Committee (SREC) at the University of Hail in the Kingdom of Saudi Arabia. The proposal was reviewed and unconditionally approved by the Committee. Approval number H-2016-046.To calculate the sample size of the study, the absolute error was estimated to be 5% and a 95% confidence level was used. Therefore, the sample size calculated for this study using the aforementioned information was 368 participants. In addition, the attrition rate  was 10%. Therefore, the required number of participants was 405.This is a cross-sectional questionnaire-based study that was carried out from October 2016 to February 2017. Participants were randomly selected by convenience sampling method at public places  all over the city of Hail.Participants consented to participate after they were given full details of the study and its intended aims. All participants were made aware that this study is for research purposes only and their participation was voluntary. They were not asked for their names or contact information, ensuring the privacy of survey respondents.The questionnaire was designed in English then translated to the local spoken language Arabic by proficient speakers of both languages and was revised to be suitable to the general population.The questionnaire used in the current study was developed to evaluate the participant's Knowledge, Attitude and Practice (KAP) regarding antibiotic usage and the related AR. In order to validate the questionnaire, a pilot run was performed using a group of 20 randomly selected individuals from each of the surveyed locations. The final version of the questionnaire used in this study was based on the results of the face validity and results of the pilot run.Inclusion: We included males and females ≥18 years of age. Exclusion: People who were less than 18 years old, those who did not consent to participate, and those who live out of the region were excluded from the study.The questionnaire contained nine questions divided into four sections. The first section collected demographic information of the participants. The second section contained two close-ended questions about accessibility to antibiotic therapy. The third section collected information about the practices of the participants with the antibiotic including: presence of an infection, duration of the use and methods of disposal of excess antibiotic. The last section collected information about AR awareness and their readiness to learn more about AR.All data collection forms were kept in a secure setting, only available to the principal investigator, and will be destroyed by the principal investigator after completion of the study, in accordance with the requirement of the SREC at the University of Hail.All data were analyzed using MS Excel 2016 by means of descriptive statistics Chi-square analysis was used to calculate the p-value. Results with p-values less than 0.05 (p < 0.05) were considered statistically significant.All 500 participants  completeWe relied on the participants to honestly self-report their access to antibiotics. We obtained 500 responses, some participants reported access to both prescribed and nonprescribed antibiotics. The latter refers to the practice when an antibiotic is accessed/dispensed by pharmacists in absence of a medical prescription. This is commonly practiced in many developing countries including Saudi Arabia as part of pharmacy malpractice that we described in an earlier study . NeverthTo further understand the relevance of obtaining an antibiotic in the region of Hail, we asked the same participants to honestly self-report whether their use of the antibiotic was for a known infection. Among the 500 participants, 218 (44%) participants reported that they used the antibiotic for a known infection, while 133 (27%) participants used the antibiotic without a known infection and 149 (30%) participants were not sure if they used the antibiotic for an infection. We categorized the participants who were not sure of their infection status and who used the antibiotic without a known infection as one category and compared them to the respondents who took the antibiotic for a known infection. This resulted in a total of 282 (57%) participants who used an antibiotic without a known infection, compared with 218 (44%) participants who used an antibiotic for a known infection  participants were aware of AR, 86 (17.2%) participants were not sure of AR and finally 181 (36.2%) did not know about AR and its rise A. The suThere is a rapid increase in the emergence of antibiotic-resistant bacteria that threatens the efficacy of antibiotics, and puts the lives of millions of people at risk. While there is a growing interest in alternative drug sources such as those of plant origin or drug repurposing ,14, therSupporting other findings that demonstrated lack of appropriate antibiotic use in Saudi Arabia, the survey respondents of the current study, demonstrating a given population in Hail, obtained their antibiotics without a prescription (∼26%) majorly from community pharmacies ,17. The According to the data obtained from our study, approximately 57% of respondents took an antibiotic without a known infection. This can lead to the unnecessary exposure of the bacteria to the drug, which can further increase the chances of acquiring resistance by gene mutation or horizontal gene transfer . RestricPredictors of correct antibiotic use and knowledge of antibiotics have been found to be affected by education and source of information in Europe ,21. The The first drawback of the current study is that it was conducted in one region of Saudi Arabia, which does not necessarily reflect the general knowledge and practice of the country. Another potential limitation is that we did not investigate the type of antibiotics or the infections to which they are being used for. Also, we did not investigate, if any, side effects following the unregulated use of antibiotics, as these questions were beyond the scope of the study. Finally, the majority of our sample population held a college degree; this may have overestimated the actual practices of antibiotic use and knowledge about AR in the region of Hail.Our study highlights the necessity and need to increase public knowledge about antibiotic use and awareness about AR in the region of Hail. Additionally, enforcing community pharmacies to adhere to policies established to restrict antibiotic dispensing unless prescribed, encouraging antibiotic stewardship among healthcare practitioners, together with increasing public knowledge of antibiotic use will collectively contribute to the global efforts to reduce the threat of AR.AR represents a real worldwide challenge. Both physician prescription practice and patient knowledge are the main contributing factors that drive AR. Several studies, including the current one, have described a low patient knowledge about AR in Saudi Arabia. Therefore, unless there is a widespread community awareness about the issue, antibiotic misuse and AR will remain significantly high in the region. Also, there is a need for change in physician prescription practice in the region including the implementation of strict policies that limit antibiotic prescriptions. However, such measures will need many years to become an effective practice. Thus, we believe that there will not be major changes in practice and knowledge in the next decade or so.Antibiotic misuse is widespread in the Hail region in the northern part of Saudi Arabia.Misuse of antibiotic leads to resistance, which represents a major threat to human health.Patient knowledge, attitude and practice are thought to contribute to antibiotic resistance.There is a low knowledge about antibiotic resistance in the region, but the majority are eager to learn.


Nurses are the main communication link for healthcare messages to hospitalized patients and also play a crucial role in preventing the emergence and spread antibiotic resistant bacteria through antibiotic stewardship and infection control programmes. This requires them to possess correct knowledge and attitudes towards antibiotic use and resistance. This study was carried out to identify the level of knowledge, attitude and practices on antibiotic use and antibiotic resistance among student nurses.A descriptive cross-sectional study was conducted using a pre-validated, self-administered questionnaire with closed and open ended questions, among 199 student nurses at a government nurses training school in Sri Lanka. Scores and proportions were analysed with non-parametric methods and thematic analysis was done for the qualitative data. The study cohort had a mean knowledge score of 71.9% (SD 14). However, close to 40% believed that taking antibiotics will help to prevent cold from worsening and make recovery faster. Infection control was identified as the main method that nurses can engage in preventing antibiotic resistance. While the knowledge among our study cohort appeared to be good, some misbeliefs were present. Our findings can be used in developing the nursing curricula on antibiotic use and resistance. Antibiotic resistance is one of the major public health threats faced by the global community. Nurses play a crucial role in combating antimicrobial resistance. They play key roles in antibiotic stewardship and infection control, two main preventive measure of antibiotic resistance , 2. TheyThis study was conducted at a Government Nurses’ Training School in the North Western Province of Sri Lanka in 2018. A convenience sampling method was used and all consenting students were recruited for the study. Age, sex, study stream for advanced level examination and the year of study were collected as independent variables.A pre-validated self-administered questionnaire with sections on knowledge (12 questions), beliefs and practices about the appropriate use of antibiotics, and antibiotic resistance was used. Mean and median scores were calculated and compared using Mann–Whitney U test and Kruskal–Wallis test as the marks were not normally distributed. Percentage giving correct answers were calculated and presented descriptively. For questions related to beliefs and practices, percentages for different categories of answers were calculated. These were compared across the study years using Chi square test. All statistical analysis were performed on SPSS Statistics (v22). A qualitative approach with thematic analysis was conducted to analyze data that were qualitative in nature. All data were manually inspected to identify common themes and proportions expressing similar ideas were calculated.Total of 199 nursing students were enrolled in this study and among them only 21 (10.5%) were males. All students had studied biological sciences for advanced level examination. Seventy-eight students (39%) were from 1st year, 61 (30.5%) from the 2nd year and 60 (30%) from the 3rd year. All had started their clinical training.Hundred and ninety-three students (97%) had heard about antibiotic resistance. Of them, 161 had defined it using their own terms. Their definitions could be categorized into several themes. The commonest them  of antibiotic resistance was “bacteria not being responsive or not answering to antibiotic treatment”. Forty (24.8%) participants defined antibiotic resistance as “bacteria resisting the activity of antibiotics”. Seventeen (10.6%) of the participants had defined antibiotic resistance as humans becoming resistant to antibiotics.Fifteen participants included a qualifier about antibiotic use in their definition of resistance. Nine stated that resistance occurs due to frequent use of antibiotics, five stated that it occurs due to long term use of antibiotics while one had stated that it occurs due to misuse of antibiotics.A unique finding in our study was that though a high percentage of the participants had heard the term antibiotic resistance qualitative analysis revealed that understanding is not accurate. Some defined antibiotic resistance as humans becoming resistant to antibiotics. Most available studies state that health science students are aware of the term antibiotic resistance , 5. HoweA total knowledge score percentage was calculated using 12 questions. Marks ranged from 100 to 33.3% with a mean score of 71.9% SD 14). Considering 72 as the average mark, 115 (57.8%) had above average marks. This compares well with studies done in other countries among health care students, professionals and prescribers [. ConsideThe mean and median knowledge scores among females were 71.5% (SD 14.4) and 75. While for males they were 75% (SD 9.8) and 75. Difference between the two sexes was not significant .The mean knowledge score among the 1st year students was 74.9 (SD 13.5) (median was 75). For the 2nd years, the mean knowledge score was 70.9 (SD 15.8) (median 75). The mean knowledge score among third year students was 69.0 (SD 12) (median 66.7). The difference was statistically significant . The highest knowledge score was found among first years (74.9%) while the lowest was found among the third years (69%). This interesting finding maybe explained by the longer exposure to actual clinical practice leading to cynicism [The question scoring the lowest percentage of correct answers was on genetic spread of resistance, while the scoring the highest percentage was the one on the activity of antibiotics on bacteria Table . These iAmong individual questions, 65.3%, 74.4% and 74.9% stated that antibiotics are not active against viruses, fungi and protozoa respectively. While 91.5% stated that antibiotics are active against bacteria, only 72.4% had awareness about the differences in spectrum activity of antibiotics. While these figures compare well with previous studies done among students of health sciences, this is a fundamental point that needs to be corrected if antibiotic misuse for infections are to be prevented [Seventy-four 37.2%) and 79 (39.7%) believed that taking antibiotics during a cold will prevent it from worsening or help them recover faster  stated that they would stop taking further treatment, 54 (27.1%) stated that they save the remaining antibiotics for another time and 103 (51.8%) stated that they would complete the full course of antibiotics. Furthermore, 124 (62.3%) of the study participants stated that they discard leftover antibiotics while 48 (24.1%) stated that they would give leftover antibiotics to friends. These are practices that need to be actively discouraged, both among health care professionals and the general public.One of the commonest instances where antibiotic misuse occurs, both over prescription and over the counter medication, is upper respiratory tract infections , 13. AmoWe found that antibiotic misuse for upper respiratory tract infections, due to mis-information or false attribution of reasoning is prevalent among the study cohort. Similar findings have been found in other studies too . HoweverIn relation to patient care related practices, only 78 (39.2%) said they always take culture before patient is given an antibiotic dose and 111 (55.8%) said they always check the expiry date before giving the antibiotic.This was assessed by the open-ended question “Write in your own word, in any language that you prefer two things that you as a nursing officer can do to minimize the emergence and spread of resistant organisms”. Hundred and seventy (85.4%) participants replied to this question which had spaces to write 2 answers.Of the 170, 21 had given two answers while the remaining 149 had given only one answer. The total number of possible actions to be taken identified by participants was 191. These could be categorized into give broad themes. Majority were in infection control , antibiotic stewardship 34, 17.8%) and health education  while some actions were those that can be taken at a personal level   (Table Our study was conducted at a single nurses training school in Sri Lanka. However, the curricula remain the same across all the government training schools. We did not inquire the source for the information they received, any formal training or look at the nursing school curricula. The term antibiotic though contextually used to denote antibacterials, maybe literally interpreted to include anti-fungal and anti-protozoal drugs too. This may have led to some misinterpretation of the questions on this section.However, the main strength in our study is that we included a qualitative component, that helps to explore the ideas prevalent among the study population, that would not have been revealed in a quantitative study.


It has been recently accepted that IgG4-related thyroiditis is comparable to the Hashimoto and Riedel thyroiditis and Graves disease which are rich in IgG4-secreting plasma cells. Many physicians believe that in IgG4-related thyroiditis, the thyroid is entirely enlarged and diffusely affected, which is similar to conventional thyroiditis, but rarely ever accompanied by pseudoneoplastic mass formation as in IgG4-related disease in the other organs. This report introduces another pattern of IgG4-related thyroiditis as mass-forming thyroiditis and presents the occurrence of IgG4-related regional lymphadenopathy as an unusual accompanying symptom.A 66-year-old woman presented with an approximately 2.5-cm mass in the right thyroidal lobe and regional lymph node swelling, which were preoperatively misinterpreted as thyroidal carcinoma. After lobectomy, histological examination was performed, revealing that the mass showed dense stromal fibrosis, lymphoplasmacytic infiltration, and effacement of thyroid follicles, while the background thyroidal tissue seemed to mimic lymphocytic thyroiditis without fibrosis. Immunohistochemistry revealed predominance of IgG4-secreting plasma cells among infiltrating lymphocytes independent of mass lesion or background tissue. In addition, the regional Delphian and paratracheal lymph nodes were swollen, histologically showing numerous IgG4-secreting plasma cell infiltrations in the interfollicular zone.IgG4-related mass-forming thyroiditis, which may be an extremely rare but recognizable pattern of IgG4-related thyroiditis, may be distinguishable from Hashimoto and Riedel thyroiditis, Graves disease, and thyroidal carcinoma. In addition, the regional IgG4-related lymphadenopathy, also possibly misdiagnosed as metastatic thyroidal carcinoma, may be a newly recognized manifestation of IgG4-related thyroiditis. IgG4-related disease is a spectrum of inflammatory disorders characterized by lymphocyte infiltrates that are rich in IgG4-secreting plasma cells . It is hA 66-year-old Japanese woman presented with a 5-month history of cough and sore throat. Clinical examination revealed a palpable elastic hard mass on the right side of the neck; ultrasonography and computed tomography revealed the mass to be in the lower pole of the right thyroidal lobe without extrathyroidal extension and the Delphian and paratracheal lymph nodes to be slightly enlarged. No other enlarged lymph nodes and metastatic lesions were detected in the body. Serum thyroid stimulating hormone  and anti-thyroid peroxidase antibody  levels were elevated. Serum free T3  and serum free T4  levels were slightly decreased; however, other laboratory data were normal, including thyroglobulin  and IgG4  levels. Fine needle aspiration of the thyroidal mass obtained follicular cell clusters containing less amounts of colloid, which were categorized as “atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS),” in a background slightly rich in lymphocytes and plasma cells . The patGross examination revealed that whitish, firm, somewhat nodular lesions were distributed into the upper and lower poles Fig.  and b. PAs shown in Fig. As shown in Fig. The Delphian and paratracheal lymph nodes were grossly swollen, measuring 7 mm and 11 mm, respectively (major axis). Histologically, lymph node architecture was reactively hyperplastic, and plasma cells, including IgG4-secreting cells, were infiltrated in great number, particularly in the interfollicular area of the inner cortex Figs.  and 2c.IgG4-related disease is a spectrum of inflammatory disorders characterized by lymphocyte infiltrates dominated by IgG4-secreting plasma cells , 2. EvenIgG4-related thyroiditis has not been definitely recognized as a separate category yet. It has been widely accepted that some cases of Hashimoto and Riedel thyroiditis can be recategorized into IgG4-related diseases because of lymphocyte infiltration rich in IgG4-secreting plasma cells and marked storiform fibrosis , 10, 20.In addition, the mass was preoperatively misinterpreted as thyroidal carcinoma in the present case. It is well known that Riedel thyroiditis is also frequently misdiagnosed because fibrosis, often extending beyond the thyroid, is marked so that the thyroid seems to be entirely occupied by thyroidal cancer . MeanwhiIgG4-related thyroiditis, like IgG4-related disease of the other organs, can be divided into two categories: the organ-specific type and the systemic type. Li et al. suggest that IgG4-related Hashimoto thyroiditis is compatible with the thyroid-specific type, whereas IgG4-related Riedel thyroiditis is a manifestation of the systemic type of IgG4-related disease . In the It has been widely accepted that in IgG4-related thyroiditis, the thyroid is entirely enlarged and diffusely affected, which is similar to that in conventional Hashimoto and Riedel thyroiditis and Graves disease, but rarely ever accompanied by pseudoneoplastic mass formation as in IgG4-related disease in the other organs. Mass-forming thyroiditis may be distinguishable from the other manifestations of IgG4-related thyroiditis that are already defined, and it can be clinically and radiologically misinterpreted as thyroidal carcinoma because it forms a somewhat circumscribed intrathyroidal mass. In addition, regional, not systemic, IgG4-related lymphadenopathy, also possibly misdiagnosed as metastatic thyroidal carcinoma, may be a newly recognized manifestation of IgG4-related thyroiditis.


Point prevalence surveys have been used in several studies to provide immediate and easily comparable information about antibiotic use and showed that about one third of hospitalised children had on ongoing antimicrobial prescription during their hospital admission. The aim of this study, as part of the Global Antimicrobial Resistance, Prescribing and Efficacy in Neonates and Children project, is to describe antimicrobial prescriptions among hospitalised children in four tertiary care hospitals in Italy to show if something has changed over the years.Four tertiary care Italian’s hospitals joined three Point Prevalence Surveys (PPSs) in three different period of the year. All children under 18 years of age with an ongoing antimicrobial prescription, admitted on the participating wards at 8 o’clock in the morning of the selecting day were enrolled.A total of 1412 patients (475 neonates and 937 children) were admitted in the days of three PPSs. Overall, among the total admitted patients, 565 patients (40%) had an ongoing antimicrobial prescription in the days of the survey A total of 718 antibiotics were administered in the 485 admitted children and 133 in neonates. The most common indications for antibiotic therapy in children was Lower respiratory tract infections , while in neonates were prophylaxis for medical problems , newborn prophylaxis for newborn risk factors  and prophylaxis for surgical disease .Based on our results, it appears that nothing has changed since the last PPS and that the quality improved targets, underlyined in previous studies, are always the same. Serial PPSs can be part of AMS strategies but they are not sufficient alone to produce changes in clinical practice. According to the World Health Organization (WHO)‘s statements, antimicrobial resistance (AMR) is one of the major contemporary threats to public health, both for epidemiological and economic reasons . SeveralThe aim of this study, as part of the Global Antimicrobial Resistance, Prescribing and Efficacy in Neonates and Children (GARPEC) project, was to compare antimicrobial prescriptions among hospitalised children in four tertiary care hospitals in Italy using the same methodology from previous pediatric PPS’s, to investigate if previous PPS studies had led to any change in clinical prescribing practice.The present study is part of the GARPEC, the global study that followed the European Antibiotic Resistance and Prescribing in European Children project (ARPEC) –6. Threet test and Mann-Whitney tests were used to compare variables between groups. The χ-square test and Fisher test were performed when appropriate.Statistical analysis was performed using SPSS . Metric data were tested for normal distribution. Results were expressed as mean  or median  as appropriate. Unpaired p < 0.005).A total of 1412 patients (475 neonates and 937 children) were admitted on the days of three PPSs were carried out. Overall 565 patients  received at least one antibiotic prescription on the days of the survey, with 51.8% (485/937) and 16.4% (80/475) in children and neonates, respectively. Of these, 34.6% (114/329) in the first PPS, 39.8% (220/552) in the second PPS, and 47.3% (231/531) in the third PPS; p = 0.199). Excluding antibiotics prescribed for prophylaxis, a statistically significantly higher proportion of antibiotics were prescribed in the second and in the third PPS (p < 0.001). About 41.9% of children received a combination of two or more antibiotics. The two most common indications for antibiotic prescribing were lower respiratory tract infection (LRTI) (34%) and prophylaxis for surgical disease and for medical problems (14.3 and 7.4% respectively). Overall, the most commonly prescribed antibiotics were the third generation cephalosporins (26.3%), penicillin with enzyme inhibitor (18.1%), and aminoglycosides (12.7%). The parenteral route of administration was used in 590/718 cases (82.2%). Almost half of patients received antibiotics reported as community acquired infections (CAI) , and only 24.9% (179/718 patients) as hospital acquired infections (HAI), and 22.1% (159/718 patients) as prophylaxis. In the cases of antibiotics administered for treatment , only 27.4% (154/560) of antibiotics were prescribed targeted treatment with pathogen and antimicrobial susceptibility test confirmed.A total of 718 antibiotics (an average of 1.48 antibiotic prescriptions per children) were administered in children (aged > 1 months) with no significant differences between three PPSs (p < 0.001). Prematurity was the most common reported underlying conditions in our study population . Regarding antibiotic prescription, a total of 133 antibiotics were administered (an average of 1.66 antibiotic prescriptions per neonate). The most commonly prescribed antibiotic, alone or in combination, was ampicillin or ampicillin and enzyme inhibitor (27.8%) followed by gentamycin (18.8%).Overall, 16% (80/475) neonates received at least one antibiotic in the days when PPSs were carried out. Overall, 80 neonates received at least one antibiotics in the days of three PPSs were carried out. Neonates with reported underlying conditions were more likely to receive antibiotic for treatment compare with those without underlying condition during the surveys , antibiotics were administered for treating HAIs in 21.3% (27/127), for CAIs in the 15.7% (20/127) and for prophylaxis in 63% (80/127). The parenteral route of administration was preferred in 128/133 cases (96.2%). The most common reasons for antibiotic treatment in neonates were prophylaxis for medical problems (26.3%), newborn prophylaxis for neonatal risk factors (21.8%), prophylaxis for surgical disease (11.3%), sepsis (11.3%), treatment for surgical disease (10.5%) and LRTI (9%). The two most commonly prescribed antibiotics for medical prophylaxis were  gentamycin  and ampicillin .Two other studies, using the PPSs methodology have been published in the literature in 2007 and in 2012, respectively . The percentage of patients with an antibiotic prescription, the number of antibiotics/treated patients, the percentage of antibiotics prescribed empirically and the characteristics of antibiotic use in children and neonates are reported in Table The present study provides an estimate of antibiotic use in children and neonates in four tertiary care Italian hospitals, in three different seasons, using a standardised PPS methodology. About 40 % of admitted patients received at least one antibiotic during their hospital stay; however, when new-borns are excluded, this percentage increases to more than 50%. These data are concerningly, very similar to the previous PPS studies conducted in 2007 and in 2012 in Italian tertiary care hospitals (Table PPS is an easy method to perform and monitor antibiotic use, with the main aim of raising awareness of the rationale of antibiotic prescriptions in the paediatric population. However, our study showed that PPSs by themselves are not sufficient to produce any changes in the clinical practice if they are not sustained by ASPs. Based on the present study, it appears that nothing has changed since the last PPSs , 5 and t


To elucidate the clinical features of strabismus associated with IgG4-related ophthalmic disease (IgG4-ROD).All of the four patients with IgG4-ROD showed marked enlargement of the extraocular muscles, however, two patients showed orthotropia with full ductions and versions. One patient showed a small angle of exotropia and hypertropia of less than 5 prism diopters. One remaining patient showed orthotropia, full ductions and versions despite marked enlargement of the extraocular muscles, then developed hypertropia up to 35 prism diopters with activation of inflammation, which promptly improved after treatment with oral steroids.IgG4-ROD usually shows normal ocular motility despite extraocular muscle enlargement, which is the key distinguishing feature from other orbital inflammatory diseases. Active flare-up with increased serum IgG4 levels may produce a large angle of eye deviation, but mostly respond well to steroid treatment. IgG4-related disease (IgG4-RD) is an immune-mediated systemic condition characterized by enlargement of affected organs caused by lymphoplasmacytic infiltration with a predominance of IgG4-positive plasma cells –3. IgG4-A retrospective review of ophthalmologic examinations and MR imaging findings was performed on four patients who visited the Department of Ophthalmology, Seoul National University Bundang Hospital between the years 2004 to 2014 and were confirmed to have extraocular muscles affected by IgG4-ROD. IgG4-ROD was diagnosed based on the diagnostic criteria for definite or probable IgG4-ROD proposed by Goto et al.  The diagOphthalmologic examinations of ductions and versions together with alternate prism cover tests at 6 cardinal gazes were performed. Computed tomography (CT) imaging was conducted using detector-row machines  with an intravenous nonionic contrast material . Axial and coronal images were reconstructed with 4 mm thickness at 3 mm intervals. Magnetic resonance (MR) imaging was conducted using a 1.5 Tesla system  or a 3 Tesla system  with a SENSE (sensitivity encoding) head coil. T1- and T2-weighted imaging were performed to evaluate the orbit including extraocular muscles. Abnormalities in orbital contents including the extraocular muscles were reviewed.All four patients with IgG4-RD showed marked enlargement of the extraocular muscles: horizontal muscles in all of them, vertical muscles in three of them, and the inferior oblique muscle in one of them Case 3) Table  Figs. 11-4. Howe Table  FA 58-year-old man was referred for ophthalmologic evaluation by the rheumatology department with the diagnosis of IgG4-RD after submandibular gland biopsy showing increased IgG4+ cells (> 200 cells/HPF). Serum IgG4 level was elevated to 1295.0 mg/dL  at the time of diagnosis.On examination, his uncorrected visual acuities were 20/20 OU. He had orthotropia at distance and at near in the primary position with the alternate prism and cover test Fig. . DuctionOrbit CT images showed enlargement of lymph nodes in both peribronchial areas and right level I/III, right superior rectus, right medial rectus, left lateral rectus, and left inferior rectus muscles Figs. , and infA 62-year-old woman presented with left upper eyelid swelling which developed 1 year ago. She also had experienced recurrent conjunctival injection for 3 years.On examination, her uncorrected visual acuities were 20/20 OU. She had orthotropia at distance and at near in the primary position with the alternate prism and cover test Fig. . DuctionOrbit CT showed a 2.5 cm sized enhancing mass in the left lacrimal gland and enlargement of the left lateral rectus muscle belly like a spindle shaped mass Figs. . AnterioA 66-year-old man was referred from the outside hospital for further evaluation of enlarged extraocular muscles which were incidentally found on CT during evaluation of sinusitis.On examination, his corrected visual acuities were 20/30 OU. Automatic refraction showed + 0.00 Dsph − 0.25 Dcyl x 110A OD and + 0.25 Dsph − 1.00 Dcyl x 75A OS. He showed 4 Δ of exotropia (XT) and 3 Δ of right hypertropia (RHT) in the primary position, XT 4 Δ and RHT 3 Δ in right gaze, XT 2 Δ and RHT 3 Δ in left gaze, XT 2Δ and RHT 4 Δ in upgaze, and RHT 1 Δ in downgaze. With either right or left head tilt, he showed XT 2 Δ and RHT 3 Δ. Ductions and versions were full Fig. . He had Orbit MR imaging showed enlargement of the left lacrimal gland, right medial rectus, right inferior rectus, right lateral rectus, and right inferior oblique with nodular components Figs. . Serum IA 74-year-old man presented with right exophthalmos which suddenly developed 15 days ago. On examination, his best corrected visual acuities were 20/30 OD and 20/100 OS. Automatic refraction showed + 1.00 Dsph − 0.50 Dcyl x 180A OD and − 2.00 Dsph − 0.50 Dcyl x 180A OS. Slit lamp examination showed left posterior capsular opacity. He had orthotropia at distance and at near in the primary position with the alternate prism and cover test Fig. . DuctionOrbit MR imaging showed an infiltrating mass involving both orbits, especially the lacrimal gland and both lateral rectus muscles, foramen rotundum, infraobital groove and foramen Figs. , trigemiThree years later, he presented with right facial edema Fig. , itchingOne month later after taking oral prednisolone 20 mg a day, diplopia resolved and he showed orthotropia at distance and at near in the primary position with alternate prism and cover test Fig. . DuctionOur four cases demonstrated the characteristic clinical features of eye motility associated with IgG4-ROD. Firstly, despite marked enlargement of extraocular muscles, patients maintained orthotropia with full ductions/versions. In other words, a distinctive disparity between structure (enlargement of extraocular muscles) and function (eye movement) may suggest IgG4-ROD. Thus, we can not exclude extraocular muscle involvement even though a patient with IgG4-RD does not have diplopia or eye movement abnormalities. Secondly, active flare-up of inflammation may cause a large angle of ocular deviation with underaction of the extraocular muscles as in idiopathic myositis. Similarly, the deviation as well as the underaction may promptly respond to oral steroid treatment. Thirdly, all of our patients showed enlargement of the lateral rectus muscle which is the least affected muscle in thyroid orbitopathy. Fourthly, trigeminal nerve enlargement was also observed, which is not usually involved in other orbital inflammatory conditions such as idiopathic orbital inflammation or thyroid orbitopathy. Lastly, serum IgG4 level increased with the aggravation of extraocular movement and decreased after steroid treatment and symptom relief.Serum levels of IgG4 varied widely between cases, and though it is one of the diagnostic criteria for IgG4-ROD, it can easily increase or decrease according to disease activity. Case 2 and early phase of case 4 showed normal levels of serum IgG4; however they were firmly diagnosed with IgG4-ROD, according to their typical imaging and histopathologic findings based on the diagnostic criteria for IgG4-ROD .There have been many reports of extraocular muscle involvement associated with IgG4-ROD, however, most of them did not mention the affected muscles or the clinical features of ocular motility problems –10. HigaSogabe et al.  did not There are many inflammatory conditions affecting the orbital structures such as thyroid orbitopathy, idiopathic orbital inflammation, sarcoidosis, tuberculosis, granulomatosis with polyangiitis, and IgG4-ROD . Among tIgG4-ROD usually shows normal ocular motility despite extraocular muscle enlargement, which is the key distinguishing feature from other orbital inflammatory diseases. The lateral rectus muscle is frequently involved and may also accompany trigeminal nerve enlargement. Some of them may develop a large angle of eye deviation with active flare-up of inflammation and increased serum IgG4 levels, but ocular motility mostly improves after steroid treatment.


To the Editor:We read with great interest the paper published in the Journal on November 19, 2019, by Abe et al. [p = 0.152) reflects this lack of linearity of the antibiotic therapy treatment effect. Furthermore, from a practical point of view, it is quite rare that the antibiotic therapy treatment effect is maximum since the first hour after administration.Nevertheless, to our opinion, some methodological issues deserve their results interpretation. From a statistical point of view, the categorization of the variable “time-to-antibiotic therapy” induces an information loss. Despite facilitating results interpretation, such categorization implies two consequences. First, it assumes that the treatment effect of antibiotic administration, from the 1st minute, if practically possible, to the 59th minute after diagnosis, is equivalent. Second, it would imply that the antibiotic therapy treatment effect is equivalent in all predefined categories, from 0–60 to 361–1440 min, which does not correspond to the reality, because the relationship between antibiotic therapy and mortality is not linear [Beyond this, to reach infected tissues, antibiotics need the restoration of a sufficient tissue perfusion pressure . In theiWe fully agree with the authors that the impact of earlier antibiotic therapy is greater for most severe septic patients, but as reminded in the SSC, the outcome of these patients is not only dependent on a sole therapy but more from a bundle of care . More thToshikazu AbeWe appreciate the consideration and comments from the SAMU de Paris regarding our study.Management of time data is one of the most important processes in “time to intervention” studies. We studied multiple different time intervals as we recorded time as a continuous variable; however, results with these values were not different from what we ultimately described. The relationship between time to antibiotic administration and mortality is not linear; therefore, we dealt with time data as a categorical variable. Hourly categorization is the most acceptable time interval used by clinicians. Because the number of patients receiving antibiotics after 361 min was small, we grouped those patients together. Our study did not mention causal inference, and it is a descriptive analysis using implementation science.We did not show mean blood pressure, but we controlled tissue perfusion pressure by using the Sequential Organ Failure Assessment (SOFA), which includes a cardiovascular score. We also stratified patients by the presence or absence of shock. However, we did not find any association between time to antibiotic and outcomes with adjustment of those variables.As you noted, the effect of antibiotics would be related to the time to administration and antibiotic sensitivity, concentration, and tissue perfusion. These variables may be even more important than time to administration. The lack of association between time to antibiotic administration and outcomes in our study may have been because of the lack of information about the variables. Other aspects of treatment may have differed among institutions, although we controlled for that using generalized estimating equations (GEE).We believe that the effect of time to administration will be significant only when the overall quality of care is excellent. As with the differences for door to balloon time for acute coronary syndrome noted in the research by Menees and colleagues  and the A more accurate diagnosis may allow for better antibiotic choices, which is related to the outcome of time to antibiotic administration , 9. Gene