Meta-analysis of randomized clinical trials (RCT) demonstrated several health benefits of fecal microbiota transplantation (FMT). However, there has been little comprehensive assessment of the strength and quality of evidence. We conducted an umbrella review to summarize the evidence of the association between FMT and health outcomes.PubMed, Embase, and Cochrane library databases were searched from inception to August 6, 2021. The random-effects model was applied to recalculate the effect estimates. We used AMSTAR 2 and GRADE to assess the methodological quality and to grade the evidence.A total of 7 meta-analyses comprising 26 RCTs  were included in the current umbrella review describing 45 unique associations. There were 22 statistically significant associations (49%) demonstrating beneficial outcomes of FMT for antibiotic resistance burden, functional constipation, inflammatory bowel disease, and C. difficile infection. FMT does not appear to be associated with positive outcomes in irritable bowel syndrome and metabolic syndrome. Eight significant associations (36%) were supported by moderate-quality evidence, nine associations (41%) were supported by low-quality evidence, and the remaining associations found to be significant were supported by very low-quality evidence.Although we found that FMT was positively associated with several outcomes, caution should be exercised in choosing this approach, given the insufficient number of primary studies, low methodological quality, and low quality of evidence. Further high-quality randomized controlled trials with long-term follow-up are needed to improve the strength and credibility of the evidence base. Accumulating evidence emphasizes the potential contribution of commensal gut microbiota in human health and various gastrointestinal diseases like inflammatory bowel disease (IBD) , irritab.FMT is an emerging therapeutic method that has become a research hotspot in biomedicine and clinical medicine . The provia both meta-analyses and network meta-analyses  on the efficacy and acceptability of FMT has been obtained analyses . Howeveranalyses . In thisThe systematic literature search was conducted in PubMed, Embase, and Cochrane Library from database inception to August 6, 2021, to identify meta-analyses of RCTs investigating the effect of FMT. The search strategy used a combination of the following terms: fecal microbiota transplantation  and meta-analysis . No restrictions or filters were applied for the search process. We also manually searched the cited references of the retrieved articles and reviews. Two authors (LYP and ZTT) independently conducted the literature search. Any disagreements were resolved by consultation with a third author (LNY). The detailed search strategy is provided in Systematic reviews with meta-analyses of RCTs were included. For multiple meta-analyses of the same result, we selected only one meta-analysis for each result to avoid including duplicate studies . In thisStudies were excluded if they were network meta-analyses, if they were systematic reviews without meta-analyses, if the full text of the meta-analysis was not available, or if the meta-analysis lacked data for summary estimates.P values, the number of participants and events, follow-up time, meta-analysis models used (fixed effects or random effects), and information on heterogeneity, small-study effects, funding, and conflict of interest. We also extracted any recorded subgroup analysis estimates.Two authors (ZTT and SJH) independently extracted data, and disagreements were resolved by consensus. From each meta-analysis, we extracted the first author, journal name, publication year, study design, type of comparison, interesting outcomes, and the number of included studies. We also extracted relative risk (RR) estimates, odds ratio (OR), 95% confidence intervals (CI) and corresponding .We used AMSTAR 2 (A Measurement Tool to Assess Systematic Reviews 2), a strict, validated, and reliable measurement tool, to assess the methodological quality of each meta-analysis . It consP-value was calculated from the 95% confidence interval of the collective effect estimate by using standard methods.We used the GRADE  assessment to evaluate the credibility of the evidence provided by each association in the meta-analysis . EvidencI2 statistics. Values < 50% indicate acceptable heterogeneity, values > 50% suggest moderate heterogeneity, and values > 75% are indicative of high heterogeneity , 8 adverse events, 8 lipid profile outcomes , 3 glycemic profile outcomes , 5 anthropometric measures , 4 stool measures , 3 FMT formulation outcomes , 4 FMT method outcomes , and 3 FMT donor outcomes (multi-donor and single-donor) p < 0.05). Nine (49%) of which were supported by low evidence, followed by moderate [8 associations (36%)] and very low evidence [5 associations (23%)] (Strength of evidence of the 45 associations assessed using GRADE found that 23 associations (51%) were supported by low evidence strength, while the remaining associations were supported by moderate [11 associations (24%)] and very low [7 associations (16%)], respectively. Four of these associations cannot be assessed (9%) , which is considered low evidence.Six associations of functional constipation were evaluated, of which four were supported by very low evidence, followed by one low evidence. The strength of evidence for safety cannot be assessed. Five associations are statistically significant, indicating the positive benefits of FMT on functional constipation.Six primary studies documented the impact of FMT on IBD involved 3 associations. Two associations had moderate-quality evidence, in this meta-analysis that found FMT was associated with increased clinical remission  and clinical response  in patients with IBD compared with placebo. The strength of evidence for safety cannot be assessed.via lower digestive tract  appears to be more effective than up digestive tract , and frozen feces  are more effective than fresh feces . There was no statistical difference in adverse events  between FMT and placebo, supported by moderate-quality evidence.Current literature also reports the contribution of FMT to UC. Six statistically significant associations were supported by low to moderate quality of evidence. FMT was associated with increased clinical remission  in patients with IBD compared with placebo. Single donor  and multiple donors  share the same validity. However, transplantation Treatment of CDI with FMT was evaluated in 8 primary studies involving 2 associations. This meta-analysis found that FMT was associated with increased clinical remission  in patients with IBD compared with placebo or vancomycin, supported by moderate-quality evidence. However, multiple infusions are more effective than single infusions .via colonoscopy was less effective than placebo .Among the 7 associations supported by very low to moderate-quality evidence, only 2 were statistically significant. One association had moderate-quality evidence found that FMT was not associated with clinical remission in patients with IBS compared with placebo . Another association with moderate-quality evidence suggested no difference in adverse events between placebo and FMT . Two significantly associated outcomes suggested that oral FMT capsules appeared to be more effective , while Metabolic syndrome refers to a pathological state in which proteins, fats, and carbohydrates are disordered, such as obesity and insulin resistance. After 2-6 months follow-up, HDL cholesterol  and LDL cholesterol  were the only FMT types associated with statistically significant in participants with overweight or obesity compared with placebo, supported by moderate evidence. However, the effect became insignificant after 12 months of follow-up.Of all 45 associations, 24 had acceptable heterogeneity (<50%), 14 had significant heterogeneity (>50%), and another 7 could not be assessed. Associations initially graded as very low to moderate quality retained the same rank when removing RCTs with significant heterogeneity.The publication bias and small study effects for each meta-analysis were evaluated by Egger tests. Unfortunately, all the associations included fewer than 10 primary studies. Therefore, the contribution of small-study effects to the quality of evidence was not assessed.This study is the first umbrella review that systematically assessed the role of FMT in several health outcomes by incorporating the evidence from the current meta-analysis of RCTs and evaluated the evidence by using well-recognized GRADE criteria. Our findings are valuable in the context of the lack of evidence-based support and standards for FMT strategies that inform clinicians and the general population. We used the random-effects model to recalculate each association for better comparison across outcomes. Moreover, we used standard approaches to assess the methodological quality of meta-analyses and the quality of evidence for each association. Furthermore, we performed sensitivity analyses and small study effects analyses to further strengthen the reliability of the results.The gut microbiota refers to the bacteria, viruses, parasites, and fungi colonizing the intestinal tract . The aduThe application of stool therapy can be traced back to ancient Chinese medicine nearly 1700 years ago . Eisema first reIn this umbrella review, we found 45 unique associations. Of these, 22 statistically significant associations (49%) were distributed across assessments of antibiotic resistance burden, functional constipation, IBD, and CDI. In contrast, few significant associations were found in metabolic syndrome and IBS. Evidence for statistically significant associations ranges from very low to moderate. None of the associations were supported by high-quality evidence. FMT was mostly successful in the initial phase . Although participants may frequently experience a plateau afterward, some effects persisted after longer follow-up . For safety, although 8 associations were assessed, 4 did not record specific effect estimates. Of the remaining 4 associations, 3 showed no statistically significant difference in adverse events between FMT and placebo. Furthermore, common adverse events, such as bloating, diarrhea, nausea, abdominal pain, and fever, were mild and resolved on their own.This umbrella review used the AMSTAR 2 tool to assess the methodological quality of meta-analysis and identified several potential flaws, like inaccurate assessments of risk of bias and heterogeneity, a lack of funding sources, and literature exclusion lists. These flaws lead to the low quality of the evidence from primary studies, thereby affecting the overall quality  of the meta-analysis. Insufficient information on randomization, allocation concealment and blinding are the main factors that downgrade the quality of evidence. This was followed by small sample size and significant heterogeneity. We did not find any convincing factors to upgrade the quality of evidence. Of note, future meta-analyses in this field should use AMSTAR 2 as an executive checklist to ensure high-quality evidence. Additionally, the risk of bias for most of the associations was rated as very severe according to GRADE criteria. These associations were ultimately rated at high risk of bias in part because of incomplete blinding, which may have affected treatment assignments and outcome measures between groups. The limited number of primary studies and participants was also a major contributor to the high risk of bias.Our review yielded several key messages of high interest to clinicians and patients, especially those contemplating FMT strategies. It is important to emphasize that despite multiple preclinical evidence supporting the health benefits of FMT, evidence of clear and sustainable clinical benefits is still lacking. Furthermore, although this novel approach seems safe and easy to implement, we should be cautious because the long-term effects are still unknown or unrecognized. Moreover, as an emerging medical therapeutic strategy, FMT is not yet a standardized treatment method. The protocols vary according to local procedures. Uniform standards not established on fecal formulation, transplantation method and frequency may be the reason for the inconsistent results. Therefore, well-designed studies are strongly needed to investigate the long-term efficacy and safety outcomes of FMT.Potential limitations should be considered when interpreting the results of our work. First, we used pre-established tools to assess the quality of meta-analysis, which relies on complete data in the primary study. Although the two authors conducted the assessment back-to-back, subjectivity was inevitable. Second, we did not include meta-analyses of observational studies that may have had a longer follow-up. Most of the RCTs included in our analysis were limited to short-term follow-up and relatively small sample sizes. Therefore, follow-up assessments of continued beneficial effects after cessation of FMT are lacking. Third, Insufficient number of primary studies and methodological flaws may limit the true understanding of FMT. Fourth, we used DerSimonian and Laird’s random-effects method to calculate the aggregate hazard ratio and the corresponding 95% CI to ensure comparability with the previous meta-analysis. However, future meta-analyses should use the Hartung-Knapp method, which can better reflect the uncertainty of the differences between studies, expressed with a wider confidence interval. Fifth, existing meta-analyses documented fewer adverse events, which prevented us from systematically assessing the safety of FMT.This umbrella review found beneficial associations of FMT with several health outcomes. Although our work highlights the significance of the use of FMT by public health authorities in some diseases, the quality of evidence is less convincing due to insufficient numbers of primary studies and low methodological quality. Continued research into the therapeutic effects of FMT is important. In addition to large-scale RCTs, well-designed long-term follow-up protocols must also be considered to evaluate longer-term efficacy and safety.NL and YL conducted the literature search, study selection and data extraction and analysis. TZ and JS contributed to the data interpretation, writing, and editing of the manuscript. All authors have read and agreed to the published version of the 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.


Clostridioides difficile infection (rCDI), with proposed mechanisms including restoration of the microbiota and microbiota-mediated functions, such as bile acid (BA) metabolism. This study reports a quantitative and sensitive assay for targeted metabolomic assessment, and the application of the assay to profile BA composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from 113 participant stool samples from 27 RBX2660-treated rCDI participants in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision. Furthermore, the assay enabled the observation of primary BAs as the dominant BA species at baseline in stool samples from clinical trial participants, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. After RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs that sustained through 24 months post-RBX2660. Taken together, we describe a robust assay that demonstrates altered BA metabolism in rCDI patients treated with RBX2660 administration.Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce recurrent  Clostridioides difficile infection (rCDI) is a significant public health concern with insufficiently effective treatment options. While antibiotic treatment remains the standard of care for rCDI, antibiotics fail to rectify the root cause of CDI recurrence—disruption of healthy gut microbiota, known as microbiome dysbiosis—and recurrence after antibiotics remains high. rCDI-associated dysbiosis is characterized by significant changes in the composition and diversity of bacterial microbiota. Consequently, each successive CDI recurrence after antibiotic treatment increases the chance for subsequent recurrence [Recurrent currence .C. difficile [C. difficile spore germination and vegetative growth [Alteration of the fecal composition of bile acids (BAs) is one of the functional mechanisms by which dysbiosis may promote CDI recurrence ,3. The pifficile . In rCDIe growth ,6. Thus,To address the dysbiosis in rCDI patients and thereby reduce recurrence, several investigational microbiota-based therapeutics are in clinical development, including RBX2660—a stabilized, microbiota-based investigational live biotherapeutic . In seveTo assess the functional impact of the microbiome changes observed with RBX2660 treatment, this study aimed to measure changes in fecal BA compositions from participants in the PUNCH CD2 Phase 2 trial. Building on a few studies that have focused on analytic rigor of fecal BA measurement ,13,14,15BA standards and d4 deuterated internal standards were obtained from Cayman Chemical or Steraloid . BAs in Clinical trial participants voluntarily provided fecal samples per consent requirements (NCT02299570). The institutional review board/research ethics board (IRB/REB) at each participating center approved the study protocol. Wet fecal samples were diluted with saline to a final concentration of 0.3 g/mL. The suspension was mixed thoroughly using a mechanical homogenizer. The homogenate was gauze-filtered to remove insoluble and oversized matter if necessary, aliquoted into 96-well plates and stored in the freezer (−50 °C) prior to further processing.A 20× working stock solution (in methanol) of the BA standard mixture was made and used for a 10-point calibration curve with the dynamic range of 10–5000 ng/mL. Another 20× working stock solution (in methanol) was prepared for quality controls and used for a 4-point curve with target concentrations of 30, 60, 300 and 3000 ng/mL. Internal standards, d4-UDCA and d4-LCA were prepared at concentration of 2500 ng/mL, whereas d4-GCDCA, d4-CA, d4-GCA, d4-TCA, and d4-DCA were prepared at 250 ng/mL in 0.1% formic acid and methanol and mixed in equal volumes to create the internal standard mixture. This solution also served as the extraction buffer to precipitate proteins in the biological matrix.For standard curve samples, the BA standard mixture was diluted 20× in saline and further diluted 5× in extraction buffer. A 5 µL volume was injected into liquid chromatography tandem mass spectrometry (LC-MS/MS) system for quantification.The control stool (“blank” matrix) from a normal sample contains varying levels of BAs, amongst other extensive matrix related interferences, which required mathematical correction for assessment of spiked analyte concentrations. While the use of a control stool as matrix could induce further error in the assay, choosing an unrelated background matrix (like a buffer or plasma) is unfavorable.For quality control (QC) samples, 10 µL of control stool homogenate was diluted with 180 µL saline. The mixture was spiked with 10 µL of 20× working stock solution. To extract the BAs, 800 µL of the internal standard mixture was added to the sample. For QC samples, the final concentration of BAs detected was the sum of the endogenous BAs in the control stool plus the spiked concentrations from the QC working stock solution. For blank QC samples, 10 µL of control stool homogenate was diluted with 190 µL saline. BAs were directly extracted by addition 800 µL of the internal standard mixture. The mixtures were vortexed for at least 3 min. After centrifugation, 200 µL of the mixture was taken and 5 µL was injected to the LCMS.For test samples, 10 µL of fecal homogenate was diluted with 190 µL saline. BAs were directly extracted by addition of 800 µL of the internal standard mixture. The mixture was vortexed for at least 3 min. After centrifugation, 200 µL of the mixture was taken and 5 µL was injected into LC-MS/MS. A 20× dilution of test samples was selected so that the fecal homogenates can be easily and reliably drawn using a reasonably high volume (minimum 10 µL) to maximize pipetting accuracy.A dual column setup was designed to maximize throughput by allowing one column to be used while the other was being regenerated.–Ultra-performance liquid chromatography (UPLC) Column: Waters ethylene bridge hybrid (BEH) C18 1.7 µm, 2.1 mm × 150 mm Column–Mobile Phase A: 20 mM Ammonium Acetate 0.1% Formic Acid in Water–Mobile Phase B: 20 mM Ammonium Acetate 0.1% Formic Acid in 1:3 Methanol:Acetonitrile–Gradient: 35% B for 5 min, 35−100% B in 20 min, 5 min re-equilibration–Flow Rate: 0.2 mL/minLC Method (30 min/sample):The LC was connected to a QTRAP 5500 MS/MS instrument . Using individual BA standards MS/MS conditions were optimized for Multiple Reaction Monitoring (MRM) detection by infusion of the BA standard. With the use of the ammonium acetate in the mobile phase, improved sensitivity and selectivity was obtained as the BAs formed ammonium adduct precursor ions . LCA, IsAccuracy and recovery in fecal matrix of the quality control samples were corrected with endogenous concentration of BAs of the blank matrix. The total volume of extraction was less than 1 mL to allow use of deep-well 96 well plates. Freeze–thaw stability was assessed by fully thawing the matrix at room temperature for 1 h followed by overnight refreezing at −50 °C. Assay characterization using standard controls, control stool, and quality control samples included assessment of sensitivity (limit of detection and quantitation assessment), linearity , accuracy (intra- and inter-day experiment % accuracy), precision (Intra- and inter-day experiment %CV), and freeze–thaw stability.To demonstrate proof-of-concept in assessing BA compositions in clinical samples, the assay was performed on fecal samples from rCDI participants from the PUNCH CD2 trial investigating RBX2660 to reduce recurrent CDI . The institutional review board/research ethics board (IRB/REB) at each participating center approved the study protocol and clinical trial participants voluntarily provided fecal samples per consent requirements. The study design as well as clinical efficacy and safety outcome of this trial has been previously reported . BrieflyStool samples were collected by participants at home and shipped in a freezer pack via overnight courier to Rebiotix . Upon receipt at Rebiotix, samples were aliquoted and frozen at −80 °C with no added stabilizers until analysis. Time points were prior to receiving blinded RBX2660 or placebo study treatment  and 1, 4, and 8 weeks and 6, 12, and 24 months after blinded study treatment. A total of 113 participant stool samples from 27 participants who received RBX2660 and responded to the therapy were used in this study. An adequate number of placebo-treated samples were not available for metabolomic analysis, so they were excluded from this study.p values < 0.05.A mixed effect model was fit to determine if levels of each bile acid were statistically different after treatment at timepoints 1, 4 and 8 weeks when compared to baseline. The repeated measures factor was accounted for by treating the subject as the random effect in the model. The model fit was bile acid as a function of the time points for each of the 8 bile acids. It was observed for each of the models that the bile acid was significantly different after treatment when compared to baseline, with We sought to develop a multiplexed, high-throughput, and quantitative method to measure fecal BAs from clinical trial participants. Tandem liquid chromatography-mass spectrometry was used to detect abundant and rare BA species from wet fecal samples in a targeted manner using a BA standard library. Conventional primary and secondary BAs, including GCA, TCA, TCDCA, GCDCA, CA, CDCA, LCA, and DCA, were measured as well as typically rare secondary BA derivatives totaling 35 different BA species within each stool sample . Through2) typically being > 0.99 for all measured BAs in multiple experiments. Intra- and inter-day assay performance were measured for 19 BAs to assess accuracy and precision. Assay precision (%CV) largely ranged within 5–20% and accuracy (% accuracy) ranged within 75–125% across experiments performed on 3 separate days  as well as the lower limit of quantification (LoQ) and upper limit of quantification (ULoQ) of each BA were within workable range of expected amounts in human fecal matter A. The quate days B. These Since clinical samples are collected in different locations and kept frozen for storage, samples must be thawed at least once to allow for processing. Using the control stool sample, we performed freeze–thaw cycling and measured the stability of 16 representative BAs. After 2 rounds of freeze–thawing, the concentration of all measured BAs did not significantly change C. NotablTo test our approach, we measured BAs from available fecal samples of participants in the PUNCH CD2 trial of RBX2660 for rCDI. Total BA levels were elevated at baseline and moderately reduced after study treatment, remaining stable throughout the follow-up period (Week 1–Month 24) . PrimaryOur method allowed us to measure up to 35 BAs in a targeted manner including non-standard or typically rare secondary BA species that contain a 7-hydroxyl or 7-oxo group . Interestingly, this group of secondary BAs were moderately elevated in many baseline patient samples representing up to 50% of the total fecal BA pool . They apC. difficile lifecycle. TCA and its closely related species CA and GCA are well-studied germinants of C. difficile whereas LCA and DCA are associated with suppression of C. difficile outgrowth [p < 0.05). Taken together, the changes to these specific BA show that there are functional changes to the microbiome occurring post-baseline. An analysis was made to assess whether there are meaningful changes over time to key bile acids that have been shown to regulate the utgrowth ,20,21,22C. difficile lifecycle is complex, but in general it is thought that a fecal BA composition enriched for primary BAs is associated with dysbiosis and therefore is associated with an increased risk of CDI recurrence. In contrast, a fecal BA profile dominated by secondary BAs is normally seen in the healthy gut, which is resistant to C. difficile outbreaks [C. difficile colonization.The overall impact of BAs on the utbreaks . To quanHere, we describe the development and characterization of a bioanalytical method for targeted metabolomic assessment of BAs in human fecal samples. The assay involved quantification of up to 35 BAs in feces at varying dynamic ranges, designed according to the anticipated levels in disease and in health. With a focus on reproducibility and throughput, we optimized a protocol that is streamlined and well-controlled allowing for multiplexed assessment of BAs in hundreds of fecal samples across different experiments. Using this assay, we showed that large-scale fecal BA changes occurred in rCDI trial participants that received RBX2660 in the PUNCH CD2 Phase 2 clinical trial . The obsThe accuracy and precision of the assay were studied across 3 assay days and found to be highly reproducible. The assay also indicated that BAs are stable up to 2 freeze–thaw cycles , so samples from human trials should be analyzed within 2 freeze–thaw cycles. Room temperature stability of the fecal samples was not assessed, although a previous study has shown that temperature is not a confounding factor . In addiWater content in feces can vary from 60–90%, thus potentially complicating absolute fecal biomarker measurements ,31. HoweC. scindens [The unique longitudinal design of our clinical trial enabled us to track fecal BA profile changes at several time points following RBX2660 treatment. We found that there is an immediate deconjugation of primary bile acids as early as 1-week post-baseline, indicating that commensals expressing bile salt hydrolases (BSH) were restored at this early time point. Compared with baseline, the majority of week 1 samples had higher levels of 7-dehydroxylated BA including LCA and DCA, suggesting commensals harboring 7-dehydroxylation activity were either fully or in the process of being restored in these samples . Notablyscindens , were noC. difficile germinant TCA were among these showing a sustained drop in levels (p < 0.05). Reciprocally, secondary bile acids such as LCA and DCA, associated with C. difficile growth suppression, significantly increased (p < 0.05). By 8 weeks post-baseline, the fecal BA profile was dominated by the secondary BAs LCA and DCA. Notably, this shift in microbiome-dependent BA metabolism was sustained through at least 24 months. Acknowledging the low sample numbers and lack of head-to-head data, secondary BA appeared to restore more efficiently after RBX2660 administration in this trial than after antibiotics alone, including some in late-stage clinical development [When assessing specific BA abundance over the efficacy period of the trial (8 weeks post-RBX2660 treatment), specific BA species such as the elopment ,33.Clostridia class, which is known to harbor critical commensals that express 7-dehydroxylating genes [In addition to the conventional BAs that we and others have studied, our assay detected a wide range of secondary BAs that are expected to be uncommon in healthy human feces. Some of which have previously not been measured in a targeted manner in CDI patients. We found that a subset of baseline patients had elevated levels of these otherwise rare secondary BAs . These secondary BAs were abundant representing on average up to 20% 3.4–50% range) of the total fecal BA pool in a subset of baseline patients. The emergence of these BAs also appeared anti-correlated with conjugated primary BAs, suggesting that deconjugation of primary BAs occurs prior to their biochemical conversion to secondary BAs, as is the case for the synthesis of LCA and DCA . In lieu0% range In patient samples from the PUNCH CD2 trial, we demonstrated reduced fecal levels of primary BAs and restored secondary BAs in patients who responded to treatment with RBX2660 . A more comprehensive analysis, including comparing RBX2660 to placebo and responders to non-responders, will be conducted in a future study of a large phase 3 trial of RBX2660 to assess whether treatment is more efficient than placebo in mediating the shift towards a healthier BA profile. Combining traditional metagenomic sequencing with this robust bioanalytical method for targeted quantification of BAs will allow for a deeper analysis that not only examines restoration of microbiome diversity after treatment with RBX2660 but also assesses functional microbial activity that mechanistically contributes to the observed reduction in recurrent CDI.


Levilactobacillus brevis was identified in all six sourdoughs. Leuconostoc, Pediococcus, and Weissella were also dominant. Plate counts detected neither the presence of Pseudomonas nor members of the Enterobacteriaceae family, and LAB levels were, on average, barely 5.89 Log CFU/g for BL, and 7.33 Log CFU/g for HM sourdoughs. Data suggested that the microorganisms mainly imputable of BAs formation in sourdough are members of the LAB community.During a survey for isolating sourdough lactic acid bacteria (LAB), 20 dough samples produced at the bakery level (BL) or home-made (HM) were collected. An enzyme-based colorimetric method revealed a total biogenic amines (BAs) concentration in the range 41.4–251.8 ppm for six (three BL and three HM) sourdoughs characterised by unpleasant odours. Eight BAs generally investigated in foods were identified and quantified from these six samples by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). Only one HM sample contained almost all analysed BAs. Tryptamine was exclusively detected in HM sourdoughs (0.71–24.1 ppm). Putrescine, tryptamine, spermidine, and spermine were the only BAs detected in BL sourdoughs. MiSeq Illumina analysis was applied to study the total bacterial community of sourdoughs. LAB accounted from 67.89 to 92.17% of total bacterial diversity, and  One of the pathways of amino acid catabolism leads to their decarboxylation in the corresponding amines, namely biogenic amines (BAs), in a process catalysed by decarboxylases widely distributed in living organisms. In particular, bacteria are generally very rich in these enzymatic activities .Some BAs are involved in important and various physiological processes in humans, including brain activity, immune response, and cell growth and differentiation. For example, histamine, produced by histidine decarboxylation, is a powerful vasodilator that also stimulates gastric acid secretion; tyramine, produced by the decarboxylation of tyrosine, has a hypertensive action; serotonin, dopamine, and γ-aminobutyric acid (GABA), produced by decarboxylation of 5-hydroxytryptophan, 3,4-dihydroxyphenylalanine, and glutamic acid, respectively, are important neurotransmitters. On the other hand, other BAs, such as the diamines cadaverine and putrescine, produced by the decarboxylation of lysine and ornithine, respectively, are instead very toxic and responsible for the toxicosis caused by intestinal bacterial alterations ,3.Despite the physiological role of some BAs, the ingestion of foods containing high levels of these nitrogen compounds caused by an excessive microbial decarboxylation, determines several adverse effects on consumers’ health. Indeed, though a low amount of exogenous BAs can be rapidly detoxified by serum monoamine oxidase  and diamine oxidase  ,6,7. FurIt is noteworthy that, though BAs are not necessarily the casual agents of malodours in foods, their concentrations are well correlated with the development of spoilage off-flavours in some products ,12.Lactic acid bacteria (LAB) are primarily involved in the fermentation process of several alimentary raw materials , but theFurfurilactobacillus rossiae (formerly Lactobacillus rossiae), typically associated with Type I sourdoughs [On the other hand, in comparison with other fermented foods, sourdoughs and their derived products show low food safety risks . Howeverurdoughs ,20, as aurdoughs .Sourdough is a complex microbial environment originated from a mixture of flour and water where LAB and yeasts grow together, affecting the process of mass acidification (mainly LAB) and the increase of dough volume  . SourdouEven though sourdough fermented products undergo the baking process and fermenting cells are killed, BAs are quite resistant to heating, as confirmed by Kurt and Zorba , who estHere, with the aim of paying major attention to this issue, Type I sourdoughs produced at the bakery as well as house level, and characterised by undesirable odour profiles, were analysed for their BAs content, and the microbial communities were investigated by a culture-independent approach followed by plate counts.Scientific evidence suggests that BAs other than histamine should be considered for human health hazards, and the Food and Drugs Administration Agency (FDA) also proposed to use other BAs to evaluate fish freshness . HoweverIn this study, 10 dough samples from bakeries and 10 from local artisan producers were collected from throughout the Sicily region , and analysed for the total amounts of BAs by a modified enzyme-based colorimetric method . Only siThe content of BAs among the HM sourdoughs was higher in the BL ones, with average levels equal to 146.35 and 49.14 ppm, respectively. Moreover, whereas in HM sourdoughs, the variability between samples was considerable, limited variations were observed among BL sourdoughs. Although the three sourdoughs produced at the bakery level had a BA content around 50 ppm, among HM samples, 1HM sourdough was characterised by a BA content above 200 ppm.Based on these preliminary results, the quantitative and qualitative profile of the eight BAs  generally investigated in foods  was deteAccording to the results from the colorimetric assay, HM samples showed the highest content of BAs with values that were from three- to almost seven-fold higher than those detected in BL samples. Once again, 1HM sourdough contained the highest amount of BAs among HM samples.Regarding the qualitative amine profile of the investigated sourdoughs, sample 1HM was the only sample containing almost all studied BAs, except for cadaverine, which was not detected in any of the analysed samples. Moreover, 1HM was also the only sample to contain histamine and 2-phenylethylamine. In particular, the detected amount of histamine was about 4% of the total BAs content of 1HM sourdough. Tyramine and putrescine were found at the highest concentrations, representing over 80% of the total BAS content in 1HM and 3HM samples. On the contrary, a higher homogeneous distribution of putrescine, tyramine, spermidine, and spermine was observed for 2HM sourdough. Tryptamine was exclusively detected in HM sourdoughs, ranging between 0.711 and 24.023 ppm. Regarding BL sourdoughs, the average value of the sum of the eight BAs was around 25 ppm. For these samples, a reduced BAs variability indicated, as expected, that the preparation conditions of the sourdoughs from bakery were much less variable than those encountered at the home level. Putrescine, tryptamine, spermidine, and spermine were the only BAs detected in BL samples. In particular, putrescine and tryptamine accounted for over 75% of the total BAs content in the three samples. Tyramine content, which exceeded 60% of the total BAs in 1HM and 3HM, represented barely 2% of the total BAs content of BL sourdoughs. Among the different BAs, acute toxic effects are mainly caused by histamine and tyramine. Histamine intoxication causes a symptomatology known as “scombroid fish poisoning” with redness and skin rashes, burning in the mouth, severe headaches, heart palpitations, asthma attacks, and abdominal cramps . TyraminLeuconostoc, Pediococcus, Weissella, Erwinia, Acetobacter, and Rickettsiales were identified. It has to be noticed that besides Lactobacillus genus, the group of lactobacilli includes also the genera Companilactobacillus, Lacticaseibacillus and Levilactobacillus, recently reclassified by Zheng et al. [The DNA extracted from the six sourdoughs characterised by a detectable level of BAs was always successfully amplified in the bacterial V3–V4 16S rRNA gene region, and 441,902 paired-end sequences were obtained. Miseq Illumina analysis resulted in a very narrow taxonomy classification, since only lactobacilli, g et al. ). A smalg et al.  and cowpg et al. . The relg et al. , were coLactobacillus constituted barely 2.16% of total bacterial diversity of sample 2BL, but this genus accounted for 72.60% in sample 2HM, and was present in all sourdoughs analysed. Lactobacillus represents the main genus in Type I sourdoughs [Levilactobacillus brevis was identified from all six sourdoughs, and represented almost the half of total bacterial diversity (49.70%) in sample 1HM, and even more than half (62.09%) in sample 1BL; Companilactobacillus paralimentarius (3.47–35.38%) was detected in four samples, including all three bakery level sourdoughs; Lacticaseibacillus zeae was only hosted in sample 1HM at a very low relative abundance (0.81%). Lactobacilli are the typical LAB associated with sourdoughs [Lvb. brevis (formerly Lactobacillus brevis) and C. paralimentarius , as the main species representative of the obligate heterofermentative and facultative heterofermentative groups, respectively, identified from Italian Type I sourdoughs [Lcb. zeae (formerly Lactobacillus zeae) is less common in sourdough, but easily revealed when culture-independent methods are applied to this matrix [Leuconostoc, Pediococcus, and Weissella. Even though these genera mainly originate from raw materials, and both durum semolina and tender flour are used for bread-making in south Italy [Leuconostoc and Weissella species [Weissella represented 32.55 and 53.63% of the total bacterial community of the samples 3HM and 2BL, respectively, highlighting the important role of Weissellas in sourdough fermentation.LAB dominated all six sourdough samples with unpleasant odours. This bacterial group ranged from 67.89 (in sample 2BL) to 92.17% (in sample 3HM) of the OTUs identified. On average, bakery-level sourdoughs hosted LAB at a lower percentage (80.11%) than that revealed for home-made sourdoughs (86.67%). However, the LAB species composition was quite different among samples. urdoughs . Thus, turdoughs  with Lvburdoughs ,41,42. Ls matrix . The LABth Italy ,45, theyth Italy , and aretarius 3.–35.38% w species . HoweverAcetobacter (6.43%) were only detected in sample 2HM. However, the presence of Acetobacter spp. among sourdough microbial communities has been reported by several authors [Erwinia in sample 3BL. Also, the presence of this genus might be imputable to raw materials, since Erwinia species are reported as growth promoters for wheat [Regarding non-LAB OTUs, Rickettsiales were found in all samples (3.97–28.44%). Generally, their presence is imputable to environmental contaminations ; as, bei authors ,52,53. Aor wheat .The level of acidification of the six sourdough samples processed was estimated through pH measurement and TTA .The pH values of the samples collected from bakeries were  quite higher than those  of the sourdoughs produced at the home level. The last samples showed a pH comparable to those of sourdough produced in Sicily both at the artisanal as well as industrial level ,47,48. TThe microbiological investigations were exclusively performed for the six sourdoughs showing detectable BA levels. Cell densities of TMC  of sourdAlthough TMC levels detected for HM samples were lower than those generally reported for mature sourdoughs, which overcome 8.0 Log CFU/g , cell de7 CFU/g. Moreover, in this case, their levels were lower than those commonly registered for mature sourdoughs [7 CFU/g. Furthermore, mesophilic cocci counted on M17 were below the detection level. No big differences were observed among the four LAB groups (mesophilic rods (on mMRS) and cocci (on M17); typical sourdough LAB (on SDB) and presumptive Fructilactibacillus sanfranciscensis (on SFM)) for the samples 1HM and 3HM.LAB populations of HM sourdoughs , similarurdoughs ,57. AmonAs expected, the levels of LAB found for BL sourdoughs were particularly low. Except the 2BL sample, for which all four LAB groups were in the range 6.23–6.83 Log CFU/g, 1BL showed the presence of only two LAB groups at low levels , whereas 3BL was characterised by the absence of mesophilic cocci only, and the levels of the other LAB groups were very low (4.09–4.29 Log CFU/g). The absence of bacterial growth on M17 is imputable to the pH of this medium (>6.0), which is too high for the growth of typical sourdough LAB . HoweverDue to the undesirable odours emitted by the sourdoughs, all samples were investigated for the presence of members of the Enterobacteriaceae family, and pseudomonads that are generally involved in the process of generation of unpleasant off-odours . NeitherYeasts were also an object of enumeration . Only thIn all sourdough samples analysed, the levels of LAB were almost superimposable to TMC, clearly showing that the former group dominated the microbial communities.HCA classified the sourdoughs with unpleasant odours in accordance with their mutual dissimilarity and relationship  by usingHCA is a hierarchical ascendant analysis where the samples examined (sourdoughs) are graphically represented by a dendrogram from the closest one, i.e., the most similar, to the furthest apart, which is the most different sample . ConsidePseudomonas nor members of the Enterobacteriaceae family, which are generally involved in production of putrid and sulphuric odours. The levels of LAB were quite lower for the samples collected from bakeries, but Illumina analysis showed that the majority of OTUs were ascribed to LAB genera and species commonly associated to mature sourdoughs. Only Erwinia was identified among the Enterobacteriaceae family. However, all sourdoughs were positive for the presence of BAs, with those produced at the home level showing the highest concentrations. So far, BAs have been reported as spoilage off-flavours in fish and meat products, and this study provides evidence that sourdoughs should be better investigated for these aspects.Among the 20 sourdoughs of this survey, six samples, all characterized by unpleasant odours, showed detectable levels of BAs. The microbiological analyses conducted by classical plate counts detected neither the presence of Ten home-made (HM) sourdoughs and ten sourdoughs produced at the bakery level (BL) were collected throughout the Sicily region  . The samAll BL sourdoughs were propagated through mechanical fermenters applying the conditions reported in v/v) trichloroacetic acid was added to 5 g of the sample and homogenized for 10 min. Then, 10 mL of homogenate was diluted up to 100 mL using distilled water. After a clean-up step via centrifugation  and filtration, the supernatant was collected, and the pH was adjusted to 9 using 1 M KOH. Colour developing reagent was prepared mixing 1.5 M Tris buffer (pH 9.0), 400 mM 4-aminoantipyrine, and 40 mM phenol in a ratio 4:1:1 (v/v/v), respectively. Reaction mixture was prepared mixing 1 mL of each sample, 450 µL colour developing reagent, 500 µL of 300 mU/mL diamine oxidase, 50 µL of 175 U/mL horseradish peroxidase, and water up to 25 mL. After stirring and incubation at 50 °C in a water bath for 1 h, the absorbance of the reaction mixture was measured at 505 nm against a blank: a reaction mixture containing distilled water instead of the sample. The presence of BAs in all 20 sourdoughs was determined by a modified enzyme-based colorimetric method according to Yeh, Lin, and Hwang , with mix-axis of the calibration curve, whereas S is the slope of the regression curve.Quantification was performed using an external calibration curve of histamine dihydrochloride in the range of 1–80 ppm. The total amount of biogenic amines in sourdough samples was then expressed as ppm of histamine equivalents (HE) per grams of fresh weight using the following equation:escribed , accordiv/v) HCl and 1:5 (w/v) as the solvent ratio. Extraction was monitored by adding 50 ng of 2-PEA (phenylethylamine) to the solvent as an extraction standard. The solution containing the sample was vortexed for 2 min, sonic-bathed for 10 min, and, in order to separate solid from liquid components, centrifuged at 12,000× g for 20 min at 4 °C. In order to perform an exhaustive extraction, the process was repeated twice, and the aqueous layers combined. The resulting solution was diluted 1:10 (v/v) in 5 mM HFBA (heptafluorobutanoic acid) for the UHPLC run. HFBA was used as an ion pairing agent for the highly polar BAs. The analytical system chosen was a Shimadzu LCMS 8045  equipped with a NexeraXR UHPLC pump module . Elution was performed at 0.3 mL/min by a solvent gradient of A (5 mM HFBA in H2O) and B (5 mM HFBA in MeOH) on a Luna C18(2) reversed phase column  . The gradient was set as follows: in the first 2 min of the run, it was kept in isocratic conditions (20% B), then it reached 50% of B in 1 min; from 3 to 10 min, the gradient was readjusted to reach 100% of B and 100% B was kept for 5 min; finally, the column was allowed to recondition for 6 min.BAs were extracted only from the sourdough samples that showed detectable levels of BAs during the preliminary screening using 0.1% , 103.1 > 86.2 (cadaverine), 89.2 > 72.15 (putrescin), 122.1 > 105.1 (2-PEA), 138.2 >121.2 (tyramine), 161.1 > 144.1 (triptamine), 146.3 > 72.1 (spermidine), 203.3 >112.1 (spermine). All standards used were purchased from Merck .Total DNA from each sourdough was extracted using the Power Food™ Microbial DNA Isolation Kit  following the manufacturer’s instructions. The resulting DNA was then purified with the PowerClean DNA Cleanup Kit . DNA was quantified by Nanodrop8800 Fluorospectrometer . The DNA collected from the sourdoughs positive for the presence of BAs (considering both duplicate samples of the two distinct collection days) was pooled as two individual samples per pool, filling in one pool per sourdough. A proper balance of the DNA quantity was performed in each pool in order to obtain an equal representation for each individual sourdough.Escherichia coli positions 341 to 805) was amplified. Unique barcodes were attached before the forward primers to facilitate the pooling and subsequent differentiation of samples. To prevent preferential sequencing of the smaller amplicons, the amplicons were cleaned using the Agencourt AMPure kit  according to the manufacturer’s instructions; subsequently, DNA concentrations of the amplicons were determined using the Quant-iT PicoGreen dsDNA kit  following the manufacturer’s instructions. In order to ensure the absence of primer dimers, and to assay the purity, the generated amplicon libraries quality was evaluated by a Bioanalyzer 2100  using a High Sensitivity DNA Kit (Agilent). Following the quantitation, cleaned amplicons were mixed and combined in equimolar ratios.Amplicon library preparation, quality and quantification of pooled libraries, and pair-end sequencing using the Illumina MiSeq system  were performed at the Sequencing Platform, Fondazione Edmund Mach . Briefly, for each sample, a 464-nucleotide sequence of the V3–V4 region  of the 1https://github.com/yhwu/idemp/blob/master/idemp.cpp, accessed on 19 October 2021), and imported into Quantitative Insights Into Microbial Ecology . Sequences were quality-filtered, trimmed, de-noised, and merged using DADA2 [https://github.com/qiime2/q2-feature-classifier, accessed on 19 October 2021). A pre-trained Naive Bayes classifier based on the Greengenes 13_8 99% Operational Taxonomic Units (OTUs) database, which had been previously trimmed to the V4 region of 16S rDNA, bound by the 341F/805R primer pair, was applied to paired-end sequence reads to generate taxonomy tables. Data generated by MiSeq Illumina sequencing were deposited in the NCBI Sequence Read Archive (SRA), and are available under Acc. No. PRJNA772561.Raw paired-end FASTQ files were demultiplexed using idemp  for 2 min at maximum speed.The pH values were measured using the pH meter BASIC 20+ . Total titratable acidity (TTA) was determined using 10 g of each sample with 0.1 N NaOH (expressed in terms of mL of NaOH). All sourdoughs were previously homogenized in 90 mL distilled water with the stomacher BagMixerPseudomonas agar base (PAB) added with Cetrimide Fucidin Cephaloridine (CFC) supplement, incubated at 25 °C for 48 h; yeasts were investigated on yeast peptone dextrose (YPD) agar containing chloramphenicol (0.1 mg/mL) to inhibit bacterial growth, incubated at 25 °C for 48 h. All media and chemicals were purchased from Oxoid . Plate counts were performed in triplicate on each replicate sample to obtain a total of six data per sourdough.Ten grams of each sourdough were also homogenized in 90 mL of Ringer’s solution and subjected to the decimal serial dilution to perform microbiological analysis. Plate count agar (PCA) was used for total mesophilic count (TMC) after 72 h incubation at 30 °C under aerobiosis. mMRS and M17 were used for the development of rod- and coccus-shaped LAB, respectively, after 48 h incubation at 30 °C under anaerobic conditions by means of a hermetically sealed jar added with the AnaeroGen AN25 system. Sourdough LAB were specifically investigated on sour dough bacteria (SDB)  and San p < 0.05.Microbiological and chemical data were subjected to one-way analysis of variance (ANOVA) using XLStat software version 7.5.2 for Excel . The Tukey’s test was applied for pairwise comparison. Statistical significance was attributed to In addition, an explorative multivariate analysis was employed to investigate the relationships among sourdoughs. A hierarchical cluster analysis (HCA)  was carried out for grouping the sourdoughs according to their dissimilarities, measured by Euclidean distances, whereas cluster aggregation was based on Ward’s method . The inp


In Medicine, the term “scientific evidence” is no longer a designation used preferentially by doctors and researchers. Regulatory agencies and the judiciary apply it also, in order to support administrative and legal decisions.We believe, however, that some considerations need to be made about the interpretation of the statement “scientific evidence”, at the risk that generalizing the term would compromise its meaning.The qualification “scientifically proven” can be subjective and imprecise, when used without extensive critical analysis of the literature, because, at the limit of interpretation, it allows us to show that the conclusions of a single scientific article, chosen without selection criteria, are true. Each clinical research, in general, leads to the publication of a scientific article, which only “adds a brick to the wall of knowledge” on the subject. The “scientific truth” arises from the interpretation of this “wall”, that is, from the critical analysis of all articles published on the subject, which has been resulting from research with methodological accuracy. The type of study that best represents the “scientific truth” is the meta-analysis, which can also have its results contested due to technical limitations.In the meta-analysis, the evidence available in the literature is synthesized, statistically analyzed, and interpreted to answer a question, considering that there is often research indicating that treatment A is better than treatment B and vice versa.As there are rules and technical limitations for writing meta-analyses, it is not always possible to use them to answer all clinical questions. On the other hand, the selection, random or not, of a series of articles on a topic, can include research with methodological errors that compromise their results, even after they have gone through peer review and have been published in scientific journals. That selection of a series of articles can also be influenced by confirmation bias, in which the compiler tends to value studies that speak in favor of their belief.Another important aspect concerns the clinical significance of the research results. It is common for good scientific articles to point out that the effectiveness of one treatment, in relation to another, is statistically significant. However, this does not mean that it is necessarily clinically significant. The clinical relevance of a research result is initially analyzed, in a subjective way, by the author (who may be influenced by interpretation bias) and, later, by the medical community, according to their values and perceptions.It is also important to discuss the cost-effectiveness of treatments, as there are therapies with scientific evidence of being slightly more effective than others, at an extremely higher cost, which limits large-scale application. In some cases, there may even be pressure from the pharmaceutical industry to use the most expensive treatment, even if there are no significant clinical advantages.Thus, in the case of popularization of the terminology “scientific evidence”, it is necessary to establish standards to determine the scientific evidence of new treatments, as well as when establishing who will be responsible for the critical analysis of scientific data. It is better that the evaluators be medical researchers and not judges.The proposal to value decision-making in public health considering science is promising, especially when there is a rapid advance in the development of new therapies. However, even if the scientific evidence is confirmed, the establishment of treatment does not depend only on the validation of efficacy and safety through clinical trials, it also involves recognition by the medical community and a longer observation of its effects, the so-called proof of time. If only the idea of scientific evidence is accepted, in a generic and unrestricted way, it is likely that disputes of interpretation will occur and that important treatments will be postponed pending judicial decisions. If scientific results cause so much argument and controversy among the scientific community itself, imagine having lawyers added to the quarrel.Science and its results, in addition to being ephemeral, are too complex to serve as a single, inflexible reference for making administrative decisions. It is important to have a scientific background, but it must be part of a broader evaluation process. Therefore, we consider that, outside the scientific environment, criteria should be defined for the interpretation of what is scientifically proven.The authors declare no conflicts of interest.


METex14 skipping alterations. Analyses presented herein evaluated the relationship between tepotinib exposure, and efficacy and safety outcomes.Tepotinib is a highly selective MET inhibitor approved for treatment of non-small cell lung cancer (NSCLC) harboring METex14 skipping alterations. Efficacy endpoints included objective response, duration of response, and progression-free survival. Exposure–safety analyses included data from VISION, plus four completed studies in advanced solid tumors/hepatocellular carcinoma (30–1400 mg). Safety endpoints included edema, serum albumin, creatinine, amylase, lipase, alanine aminotransferase, aspartate aminotransferase, and QT interval corrected using Fridericia’s method (QTcF).Exposure–efficacy analyses included data from an ongoing phase 2 study (VISION) investigating 500 mg/day tepotinib in NSCLC harboring Tepotinib exhibited flat exposure–efficacy relationships for all endpoints within the exposure range observed with 500 mg/day. Tepotinib also exhibited flat exposure–safety relationships for all endpoints within the exposure range observed with 30–1400 mg doses. Edema is the most frequently reported adverse event and the most frequent cause of tepotinib dose reductions and interruptions; however, the effect plateaued at low exposures. Concentration-QTc analyses using data from 30 to 1400 mg tepotinib resulted in the upper bounds of the 90% confidence interval being less than 10 ms for the mean exposures at the therapeutic (500 mg) and supratherapeutic (1000 mg) doses.METex14 skipping alterations.These analyses provide important quantitative pharmacologic support for benefit/risk assessment of the 500 mg/day dosage of tepotinib as being appropriate for the treatment of NSCLC harboring NCT01014936, NCT01832506, NCT01988493, NCT02115373, NCT02864992.The online version contains supplementary material available at 10.1007/s00280-022-04441-3. MET exon 14 (METex14) skipping alterations. Recent guidelines for the treatment of NSCLC recommend tepotinib as a preferred first-line monotherapy option for patients with metastatic NSCLC and METex14 skipping alterations ..METex14 MET amplification-driven resistance to epidermal growth factor receptor (EGFR) inhibitors, the combination of tepotinib plus gefitinib showed anti-tumor activity in the INSIGHT study  v4.03 ), treatmAn exploratory graphical analysis was performed for each safety endpoint of interest to evaluate the potential association to tepotinib exposure quartile. The relationships between exposure quartile of tepotinib or MSC2571109A and grade ≥ 3 AE, treatment discontinuation due to an AE, and dose reduction due to an AE were also evaluated.KM plots, boxplots, or spaghetti plots of the safety endpoints, stratified by tepotinib, exposure quartiles were visually inspected to determine the feasibility of a model-based analysis.24h) on the hazard was modeled according to Eq. 0(t) is the base hazard. Covariate (risk factors) on the base hazard were evaluated as shown in Eq. drug is the drug effect and θi is the coefficient describing the impact of covariate (risk factor) covi.The occurrence of first edema event was described using a TTE model. Exponential (constant hazard), Weibull and Gompertz (hazard changes over time) distributions were tested, and the impact of drug exposure  score, metastatic status, number of prior systemic anticancer therapies in the locally advanced/metastatic setting, concomitant diuretic use, and creatinine clearance. The covariate effect was illustrated using Forest plots after 100 bootstraps.kin) of albumin. The structural model is described in Eqs. The time course of the changes in serum albumin was modeled using an indirect response model, assuming zero-order production and first-order degradation. The impact of tepotinib was assumed to affect the zero-order production rate constant , and steady-state AUC , AUC24h immediately preceding the event, the time-averaged AUC24h during the week prior to the event, or the time-averaged AUC24h during the 2 weeks prior to the event .The exposure metrics for tepotinib and MSC2571109A, used in the graphical analyses and in model-based analysis, were derived from a tepotinib population PK model , includiEmax (or Imax) models of drug effect (EFFdrug) were evaluated in the exposure–response models, as indicated by the exploratory graphical analysis  building procedure was performed with a forward inclusion phase and backward elimination phase . The forref is a reference covariate value for covariate m, to which the covariate model is normalized .where CovPK time-matched electrocardiograms (ECGs) collected in studies 001, 003, 004, and 005 (tepotinib multiple doses ranging from 30 to 1400 mg) contributed to an integrated concentration-QT interval corrected using Fridericia’s formula (QTcF) analysis . A seconMETex14 skipping alterations who received tepotinib 500 mg/day in the pivotal phase 2 VISION study were included in this analysis. The characteristics of patients from the VISION study, according to tepotinib exposure quartile, are shown in Supplementary Table S1. Covariates were generally balanced across tepotinib AUCτ,ss exposure quartiles, with the exception of minor trends towards a lower body weight  and a higher proportion of females  with increasing tepotinib exposure quartile. Mean tepotinib AUCτ,ss in the overall population was 25.3 µg·h/mL (range 4.7–51.1 µg·h/mL).A total of 146 patients with NSCLC harboring METex14 skipping alteration diagnosis [tumor vs liquid biopsy], histology [adenocarcinoma vs non-adenocarcinoma], body weight, number of non-target lesions at baseline or sum of longest diameters for target lesions at baseline).All 146 patients were included in the exposure–efficacy analyses for OR and PFS, and 66 patients who attained a response were included in exposure–efficacy analyses for DOR. Graphical analysis indicated that increasing tepotinib exposure was not associated with higher OR according to investigator- and independent-assessments, with OR 95% CIs that overlapped across exposure quartiles . Most patients were male (63.1%) and either Caucasian (57.1%) or Asian (29.9%) race. Median age was 66 years (range 19–93 years). NSCLC (40.3%) and HCC (27.5%) were the common tumor types, 68.9% had an ECOG PS of 1, and metastatic disease was present in 98.6% of patients.24h quartile (0.05–12.1 µg·h/mL) relative to tepotinib AUC24h > 12.1 μg·h/mL   was similar across all edema severity grades .Of the 499 patients in the pooled safety analysis set, 239 patients (47.9%) had at least one edema event. KM analysis of edema incidence indicates a longer time-to-first edema event within the lowest tepotinib AUC24h, did not have a discernible impact on the hazard model with a drop in objective function value (OFV) of less than –1.6 for all tested exposure–response models. A visual predictive check of the base model confirmed that it adequately described the probability of edema during tepotinib treatment . The final TTE model also revealed that advanced age was associated with an increased risk of edema, independent of tepotinib exposure. The median hazard ratio for risk of edema was estimated to be 1.3  for a 75-year-old patient relative to a typical reference 66-year-old patient  . A constant hazard  was found to provide the best description of the base hazard. Tepotinib exposure, expressed as time-varying AUCin in a Michaelis–Menten fashion). The visual predictive check plot of the indirect response model across the full population is shown in Fig. 50 associated with this effect was estimated to be 0.215 μg·h/mL (RSE 24.5%) (Supplementary Table S4). This represents 1% of the AUCτ,ss at clinical dose, suggesting that the time course of the effect on albumin was likely driven by the turnover rate of albumin, rather than an accumulation of tepotinib exposure.Median baseline serum albumin concentration was 38.4 g/L (range 16.0–72.0 g/L). There was a trend toward decreasing serum albumin concentrations with time in all studies. The time course of serum albumin concentration was described using an indirect response model, with tepotinib exposure-related inhibition of the formation of albumin  , or an MSC2571109A AUC24h of approximately 5 μg·h/mL   Fig. c. The tiCmax at the proposed clinical dose of 500 mg, and 7.54 ms at the geometric mean steady-state Cmax at the highest administered dose of 1400 mg (Supplementary Table S5). The impact of MCS2571109A was assessed in patients for whom matched tepotinib and MCS2571109A concentrations and ΔQTcF data were available using multivariate regression. At the proposed tepotinib clinical dose of 500 mg, the mean predicted ΔQTcF was 3.1 ms at a tepotinib Cmax of 1000.2 ng/mL and MSC2571109A Cmax of 319.3 ng/mL. At a tepotinib dose of 1000 mg, the mean predicted ΔQTcF was 5.2 ms at a tepotinib Cmax of 1199.4 ng/mL and MSC2571109A Cmax of 384.4 ng/mL. The upper bound of the 90% CIs of the predicted mean ΔQTcF at the observed geometric mean steady-state Cmax was 4.3 ms at the proposed clinical dose of 500 mg, and 6.8 ms at the highest administered dose of 1000 mg.Linear mixed effects modeling was used to quantitatively assess the effect of tepotinib concentration on QTcF. The model-based regression line of the population mean ΔQTcF and its two-sided 90% CIs, obtained by bootstrapping of 1000 datasets, is shown in Fig. N = 107 patients) showed that the upper bound of the 90% confidence interval of the estimated population mean ΔQTcF was 7.9 ms (Supplementary Table S5). Similar results were obtained in VISION cohort A with matched tepotinib and MCS2571109A concentrations; timepoint and categorical analyses for both the integrated population and the VISION cohort did not show any clinically significant changes (Supplementary Tables S6–9).Similarly, an additional QTc analysis of tepotinib at the clinical dose of 500 mg in cohort A of the VISION study .N = 255), treatment-related AEs led to dose reductions in 71 patients (27.8%), to temporary treatment discontinuations in 90 patients (35.3%), and to permanent treatment discontinuation in 27 patients (10.6%) [In updated safety analyses of the VISION study, comprising all patients in cohorts A and C who received tepotinib by July 1, 2020 ( (10.6%) . The medτ,ss and grade ≥ 3 AE or dose reduction, treatment interruption, or permanent treatment discontinuation due to an AE  falls within the observed tepotinib AUC range achieved in the VISION study, in which a flat exposure–efficacy relationship was observed. These data, therefore, indicate that efficacy would be maintained in patients who require a temporary dose reduction to 250 mg/day for the management of AEs. This is also supported by the observation that patients, with dose reductions in the VISION study, remained on treatment and continued to benefit from tepotinib for prolonged periods [Graphical and model-based analyses suggest a flat exposure–efficacy relationship for OR, DOR, and PFS in the VISION study. It is in agreement with our dose selection rationale that 500 mg/day regimen of tepotinib is expected to achieve close-to-complete (≥ 95%) intra-tumoral phospho-MET inhibition in the majority (> 90%) of treated patients , and ren periods . HoweverMETex14 skipping alterations [The most frequently reported AE for tepotinib is edema. While graphical analyses indicated that the risk of edema appeared to be lower in patients with tepotinib exposures within the lowest quartile, no readily apparent association between tepotinib exposure and edema grade was observed, and model-based analysis did not identify a discernible relationship between tepotinib exposure and the risk of edema. In summary, development of edema is clearly associated with the administration of tepotinib, but the effect seemed to plateau at low tepotinib exposures and the underlying exposure–response relationship therefore could not be fully quantified in the present analyses. Edema was also the most frequent cause of dose reductions and treatment interruptions in the VISION study, with a median time-to-first onset of 7.9 weeks (range 0.1–58.3) , 24. Theerations .Edema is a commonly reported AE among patients receiving MET inhibitors –36, suggTherefore, it is not surprising that both clinical efficacy and development of edema follow the same flat exposure–response relationship in this MET-driven tumor indication. This also suggests that temporary treatment interruptions, rather than dose reduction, may be a more effective approach to managing patients who develop edema.A trend towards decreasing serum albumin concentrations over time was noted in patients receiving tepotinib, with a decrease of 26% at steady state. Model-based analysis suggests that the inhibitory effect saturates at low exposure of tepotinib. Furthermore, both the risk and severity of edema were associated with serum albumin, with high baseline albumin likely providing some protection against early development of edema. There was also an apparent trend for positive association between magnitude of decrease in serum albumin and maximum severity of edema, with more severe edema seen in patients with the greatest reduction in serum albumin. This highlights an opportunity for further analyses to quantitatively evaluate the link between time course of changes in albumin and time course of edema. However, both the risk of edema and effect on serum albumin appeared to plateau at low exposure of tepotinib.The underlying mechanism(s) are poorly understood. Treatment-emergent hypoalbuminemia was also observed in other MET inhibitors , 40. OwiTepotinib treatment was associated with, on average, an approximately 30 μmol/L maximum increase in serum creatinine levels. This increase in serum creatinine plateaued with time and continued drug exposure, and was found to be reversible, based on data from healthy volunteers receiving single dose administration. No other clinical laboratory or clinical findings suggested a relation to kidney injury.50 of 3.6 μM and MATE2 with an IC50 of 1.1 μM, and MSC2571109A reaches a steady-state free peak concentration of 0.01 μM whilst inhibiting OCT2 with an IC50 of 0.04 μM.A potential explanation for the increase in serum creatinine is that tepotinib or MSC2571109A inhibit the elimination of creatinine through inhibition of the organic cation transporter 2 (OCT2) or the multidrug and toxin extrusion (MATE) transporters. At clinical doses, tepotinib reaches a steady-state free peak plasma concentration of 0.05 μM whilst inhibiting MATE1 with an ICThis hypothesis is supported by a recent report from Mathialagan and colleagues , who fou50 of 1.2 µM, which is 24-fold higher than the mean unbound steady-state Cmax of 0.05 µM achieved with the 500 mg clinical dose. There was also no meaningful effect of tepotinib on other key cardiac ion channels  up to the highest tested concentration of 10 µM. MSC2571109A had no effect on key cardiac ion channels, and no effect of tepotinib, or MSC2571109A was seen in dedicated cardiovascular safety pharmacology studies in rats and dogs.Concentration–QTc analyses for tepotinib and MSC2571109A showed no evidence of a clinically significant prolongation effect on QTcF interval. Based on linear mixed effects modeling, QTc prolongation did not exceed the threshold of 10 ms for either the proposed clinical dose of 500 mg, or for the highest administered dose of 1400 mg. In vivo and in vitro safety pharmacology data also suggest no anticipated risk for QT prolongation at the clinical dose of tepotinib (data on file). In these studies, tepotinib inhibited Kv11.1 (hERG) with an ICThe present exposure–safety analysis included a large number of patients pooled from different phase 1/2 trials, providing a robust assessment of the underlying exposure–response relationships. The flat exposure–efficacy relationship is consistent with the quantitative understanding of target modulation and, therefore, represents a classic case study of a model-informed dosing strategy confirmed by clinical data. A limitation of this analysis is that clinical situation with edema varies in anatomic location and intensity, as well as mixed and combined countermeasures, including overlapping dose reductions and temporary treatment interruptions plus other supportive therapies. The longitudinal profiles of edema, including onset/offset and severity of each episode, and its response to dose reduction/treatment interruption are still under investigation. The association between edema and serum albumin decrease has been observed in the exploratory graphical analysis, and further model-based characterization may provide some insight to support the hypothesis of its causal relationship. Such data will be necessary to inform the development of more comprehensive, pharmacometric models that may help elucidate the complex inter-relationships between the time course of tepotinib exposure, serum albumin, and the onset, severity and offset of edema.METex14 skipping alterations. The relationships between tepotinib exposure and edema, serum albumin, creatinine, lipase, amylase, AST, and ALT were also flat within the observed exposure range at a 500 mg daily dose. Concentration-QTc analyses indicate that tepotinib does not produce clinically relevant increases in the QTcF interval at the 500 mg daily dose. Taken together, these exposure–response analyses provide important quantitative pharmacologic support for benefit/risk assessment of the 500 mg once daily dosage of tepotinib, as being appropriate for the treatment of NSCLC harboring METex14 skipping alterations.In conclusion, a flat exposure–efficacy relationship was observed within the exposure range achieved after administration of tepotinib 500 mg/day in patients with advanced NSCLC harboring Supplementary file1 (DOCX 843 kb)Below is the link to the electronic supplementary material.


This study aimed to explore the longitudinal associations between eHealth literacy, health-promoting lifestyles, and health-related quality of life (HRQoL) among college students.From December 2019 (T1) to December 2020 (T2), we administered the eHealth literacy scale, Short-Form Health Survey (SF-12), and Short-Form Health-Promoting Lifestyle Profile Scale to 1,181 college students in Jinan, China. Participants were recruited for 12 months for the two-stage survey.P = 0.006); however, health-promoting lifestyles at T1 did not predict eHealth literacy at T2 . HRQoL at T1 predicted health-promoting lifestyles at T2 ; however, similar to the eHealth literacy finding, health-promoting lifestyles at T1 did not predict HRQoL at T2 . eHealth literacy was also bi-directionally associated with HRQoL, and the prediction effect of eHealth literacy at T1 to HRQoL at T2  was slightly higher than the prediction effect of HRQoL at T1 to eHealth literacy at T2 .Stable positive correlations were shown between eHealth literacy, health-promoting lifestyles, and HRQoL across time. The cross-lagged analysis showed that eHealth literacy at T1 predicted health-promoting lifestyles at T2 (β = 0.080, eHealth literacy and HRQoL may be antecedents for college students' health-promoting lifestyles. There may be significant bi-directional relationships between eHealth literacy and HRQoL. According to the China Internet Network Information Center, as of March 2020, there were 904 million Internet users in China, of whom student Internet users accounted for the highest proportion, at 26.9% . As actiAttending university is an important stage for young adults transitioning from school to society and is an important time for establishing positive health behaviors. However, college students often have unhealthy lifestyles, resulting in an increased risk of adverse health outcomes. A survey involving 2,422 Chinese college students find that the prevalence of lack of physical activity, sleep disorder, and bad eating habits were 62.0, 42.6, and 29.8%, respectively . It is, Health literacy is a recognized factor that influences individuals' health behavior and outcomes . As an eThe relationship between eHealth literacy and health status has recently received widespread attention. Studies show that low eHealth literacy can affect various health aspects, such as self-care for chronic diseases , depressPrevious cross-sectional studies confirm that health-promoting lifestyles are closely related to HRQoL , 30, 31;In view of the limitations and research gaps in previous studies, we conducted a 1-year follow-up study to analyze the longitudinal relationship between eHealth literacy, health-promoting lifestyles, and HRQoL among college students; to clarify the directions of effects among the three factors; and to provide high quality scientific evidence to help derive relevant interventions in the future.α/2 = 1.96, and δ = 0.05, resulting in a required sample size of 794. To control for invalid survey samples, we increased the sample size by 10%, resulting in 873 as the minimum sample size required for this study.This is a 1-year longitudinal study of 1,181 college students in China. In this study, we followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statemen . The reqliteracy . TherefoThe sample population included all college students in 30 classes of a university in Jinan City, Shandong Province. The inclusion criteria were smartphone ownership and absence of any clinical diagnosis of major physical and psychological diseases, such as cancer, COVID-19, depression, anxiety, and so forth, based on the participants' self-report. College students with a clinically diagnosed disease were not considered because this study focused on non-clinical samples in an attempt to provide scientific evidence for early prevention and intervention with respect to college students' eHealth literacy, health-promoting lifestyles, and HRQoL. Prior to initiating the study, we contacted the university's student administration to obtain their consent, after which we announced the recruitment to the class. During extracurricular time in December 2019 (T1), uniformly trained investigators administered a self-reported questionnaire to 1,235 participants. In December 2020 , we followed up with the same participants. Each survey took ~30 min. Preset codes were used to match participant responses for the T1 and T2 measurements, which safeguarded participants' identity. By matching the preset codes, we found that 54 people could not be followed up with, because they were absent from school during the T2's survey; these participants' data were excluded from the final sample. Finally, 1,181 participants who provided data twice were included in the study. All participants were gathered in a classroom to complete the survey, and all provided informed consent prior to the first survey and agreed to participate in the follow-up survey. The study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine (identification code: 2019-03-11). All research procedures adhered to the guidelines stipulated in the Declaration of Helsinki.Participants' eHealth literacy was measured using the Chinese version of the eHealth Literacy Scale (eHEALS), developed by Norman  and tranHealth-promoting lifestyles were measured using the short-form health-promoting lifestyle profile (HPLP), which was revised by Wei et al.  from theHRQoL was measured using the Chinese version of the Short-Form Health Survey (SF-12) . The scaCovariates consisted of participants' age, sex, Chinese ethnic group, place of residence, academic major, and self-reported family economic level.n (%) for categorical variables and mean  for numeric variables. Z-test was used to analyze the differences in the mean scores of eHealth literacy, health-promoting lifestyles, and HRQoL between the 54 participants with missing follow-up data and the 1,181 participants in the two surveys. Pearson correlation coefficients were used to test the association between eHealth literacy, health-promoting lifestyles, and HRQoL. IBM SPSS AMOS 23.0 was used to perform structural equation modeling with maximum likelihood to test a cross-lagged model. This model considers two or more variables at different time points, in which the estimated path coefficients of the pre-measured variables affect the post-measured variables. That is, the variables have time sequence relationship, which conforms to the principle of epidemiological causal inference, and can be used to explore the mutual predictive relationships or quasi-causal relationships between variables  to test the mutual predictive effects of the three variables. Model 1 was unadjusted, while Model 2 was adjusted for age, sex, Chinese ethnic group, residence, academic major, and self-reported family economic level. By comparing the magnitude and significance of these standardized regression coefficients, the strength and direction of the associations between variables were determined (2) divided by the degrees of freedom (df) of < 5,000, a comparative fit index (CFI) and Tucker-Lewis index (TLI) >0.900, and a root mean square error approximation (RMSEA) < 0.080  was used to conduct descriptive and correlation analyses. Data were presented as ariables . Specifitermined . The chi < 0.080 , indicatZ-test results showed that at the time of the first survey, there were no statistically significant differences in the scores of 54 participants with missing follow-up data and the 1,181 participants who completed two surveys on eHEALS score, HPLP score, and SF-12 score; this indicates that the missing samples during follow-up have no impact on the study.The Pearson's correlation analysis showed that eHealth literacy was related to health-promoting lifestyles and HRQoL at two time points, and that health-promoting lifestyles were also related to HRQoL at two time points .2/df = 2.680, CFI = 0.983, TLI = 0.943, RMSEA = 0.038). After adjusting for sociodemographic variables, Model 2 shows that eHealth literacy at T1 predicted health-promoting lifestyles at T2 ; however, health-promoting lifestyles at T1 did not predict eHealth literacy at T2 . HRQoL at T1 predicted health-promoting lifestyles at T2 ; however, similar to the eHealth literacy finding, health-promoting lifestyles at T1 did not predict HRQoL at T2 . In addition, eHealth literacy was bi-directionally associated with HRQoL, and the prediction effect of eHealth literacy at T1 to HRQoL at T2  was slightly higher than the prediction effect of HRQoL at T1 to eHealth literacy at T2 . Details are shown in Specifically, the autoregressive coefficients of the three variables from T1 to T2 were all statistically significant in both the unadjusted model and adjusted models, with an acceptable model fit  contacting the corresponding author via email.The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.SLi and GC designed, implemented the entire study, and wrote the first draft. SLi, GC, and YY conducted surveys. FZ, SLiu, and YG contributed to data disposal. YY and HX revised the first draft. All authors 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.


While the original stretch reflex threshold was velocity dependent in both muscles (p < 0.05), the latency correction rendered it velocity independent. Thus, the effects of latency correction on the stretch reflex threshold are substantial, especially at higher stretch velocities, and should be considered in future studies.Hyper-resistance is an increased resistance to passive muscle stretch, a common feature in neurological disorders. Stretch hyperreflexia, an exaggerated stretch reflex response, is the neural velocity-dependent component of hyper-resistance, and has been quantitatively measured using the stretch reflex threshold . In this study, we introduce a correction in how the stretch reflex threshold is calculated, by accounting for the stretch reflex latency . Furthermore, we evaluated how this correction affects the stretch reflex threshold in children and young adults with spastic cerebral palsy. A motor-driven ankle dynamometer induced passive ankle dorsiflexions at four incremental velocities in 13 children with cerebral palsy . The stretch reflex threshold for soleus and medial gastrocnemius muscles was calculated as 1) the joint angle corresponding to the stretch reflex electromyographic onset ; and as 2) the joint angle corresponding to the electromyographic onset minus the individual Hoffmann-reflex latency . The group linear regression slopes between stretch velocity and stretch reflex threshold differed in both muscles between methods ( Hyper-resistance is defined as an increased resistance to passive muscle stretch, commonly reported in people with the upper motor neuron syndrome. Three main contributors to hyper-resistance have been identified: non-neural tissue properties, neural velocity-dependent stretch hyperreflexia and neural non-velocity dependent involuntary background activation . CorrectR2) for the linear regression between the SRTs and stretch velocity  activity have been developed, allowing more accurate assessment of the joint angle at the SR EMG onset, also termed stretch reflex threshold (SRT). The SRT can be measured at different stretch velocities, and it is generally assumed that higher stretch velocities will result in earlier onset joint angles . Furthervelocity , which iR2 will change, and thus the adequacy of the TSRT method. Furthermore, we hypothesized that the TSRT angle will not significantly change as the latency correction will have only a small effect on the lower velocity SRTs, and consequently will not change the y-intercept of the regression line considerably.In the present study, we argue that the SRT and TSRT measures are influenced by a systematic error due to the lack of SR latency correction. SR latency is the duration between the SR being mechanically initiated at the muscle spindles  to its appearance in the EMG signal . For a given SR latency, the difference between the joint angle at the SR onset and at the SR EMG onset  will have a positive linear relationship with the stretch velocity. For example, if we consider a SR latency of 30 ms and two stretches performed at 50°/s and 300°/s, the errors of using the angle at the SR EMG onset are 1.5° and 9° respectively, simply because the EMG onset is delayed by the ∼30 ms monosynaptic SR latency. Thus, without the SR latency correction, the SRT is progressively overestimated to later angles as velocity increases. While the study by Levin & Feldman  acknowleThirteen children and young adults diagnosed with spastic cerebral palsy (CP), aged between 9 and 22 years participated in this study. None of the participants had lower limb surgery, serial casting, pharmacological treatments  or had participated in a resistance-training program for the lower limbs in the past 6 months. All participants were able to stand with both heels touching the floor . The present study is part of a larger nonconcurrent multiple-baseline research project called EXECP , prospecx-axis and the latter to the y-axis, which is the opposite of how this data has been presented in previous studies . Similarly, in MG the median regression slope in the original method [−0.021 (0.01)] was statistically different from the latency corrected method .p = 0.008) and MG (p < 0.001). Bonferroni post-hoc analysis revealed that SRTs in the two slowest stretch velocities occurred significantly earlier than SRTs at the fastest (291°/s) velocity for both Sol  and MG (55 and 110°/s: p < 0.001). With latency correction, no statistically significant differences across the stretch velocities were found for Sol (p = 0.552) or MG (p = 0.315). In the original method, SRTs occurred at statistically different joint angles for both Sol ] and latency corrected [0.27 (0.34)] methods were not statistically different (p = 0.301). In MG, R2 in the original method [0.91 (0.68)] was statistically higher than in the latency corrected method .corrected  was found. Likewise, MG TSRT [18° (2)] and MG TSRTcorrected  were not statistically different.No statistically significant difference between Sol TSRT [16° (11)] and Sol TSRTThe present work sought to verify the effects of the SR latency correction on the SRT and TSRT methods of stretch hyperreflexia assessment. The main findings were that latency correction significantly changed the SRT-velocity slopes and rendered the group-level SRT for both Sol and MG velocity independent. Thus, the lack of linear relationship between SRT and stretch velocity invalidates the use of a linear regression to find the TSRT.To group individual SRT-velocity slopes, we defined a ‘near zero slope’ as having a modulus value smaller than 0.01, which would result in a maximum 2.5° difference between the slowest and fastest stretch velocities used in this study. Since within participant and velocity SRT median range was 1.5° (min–max range: 0.1–16°), a slope smaller than 0.01 would cause changes in SRT that are indistinguishable from the subject’s natural variability. At the individual level, with SR latency correction, six participants had near zero slopes in Sol  while the other six had positive slopes . In MG, nine participants had near zero slopes and two had positive slopes. Interestingly, all participants that showed velocity independency, had an early SRT within the first 2° of the stretch, whereas positive slopes were present in participants with SRTs later in the range of motion. These individual differences suggest that stretch velocity has a negligible effect on participants with early SRTs. A possible explanation may be that the muscle-tendon unit is already under tension and/or the IA arc is highly excitable .The changes in the regression slopes caused by the SR latency correction towards positive values were expected since the correction shifts the SRTs of higher velocities to earlier angles. This means that when stretch velocity is increased, SRTs without the correction occurred progressively later in the range of motion, whereas the corrected SRTs occurred progressively earlier. Only the latter is an expected phenomenon of the velocity-dependent nature of hyperreflexia, and is also expected due to the viscoelastic behavior of the muscle-tendon unit .R2 was bidirectional among our participants: 1) negative slopes shifting towards near zero slopes reduced R2 (50% of participants in Sol and 82% in MG); 2) near zero slopes shifting towards positive slopes increased R2 (50% of participants in Sol and 18% in MG). This explains why the statistically significant effect of SR-latency correction on R2 was observed only in MG. The lower SRT variability in MG was probably due to the extended knee testing position, which placed the biarticular MG under considerably more tension than Sol. Overall, the low median latency corrected R2 values for both muscles  and high variability among participants, argues against the utilization of the linear regression to calculate the TSRT, at least in our sample. Nevertheless, previous studies have reported positive slopes without the SR latency correction for the same muscles that we studied  were found between methods in both examined muscles. Since latency correction had a minimal effect on the SRTs at slow velocities , even considerable changes in the regression slope had small effect on the TSRT.Although the regression slope changes with SR latency correction were unidirectional , its effect on  studied . These sSeveral important aspects of this study require further clarification: 1) a powerful motor-driven ankle-joint movement actuator was used to induce the stretches, whereas most of the aforementioned studies applied manual stretches. It took only 20–40 ms to achieve the target velocity in our actuator, which seems unlikely in manual stretches or even in the mechatronic device utilized by The present study demonstrated that it is vital to consider SR latency when assessing the SRT and consequently the TSRT. To the best of our knowledge, most if not all current research utilizing the SRT as a measure of hyperreflexia has incurred in this error, thus a careful re-examination of data is important to update our understanding of this promising assessment method. Future research should assess the SRT and concomitantly measure muscle fascicle velocity  and the joint angular acceleration profile of the stretch. This information would allow better comparison between studies and perhaps elucidate why some participants exhibit velocity dependent SRT while others do not.


Endoscopic Endonasal Surgery (EES) is an innovative surgical technique to remove brain tumors and lesions. Post-operative central nervous system (CNS) infections following EES are poorly described. The objective of this study was to define the epidemiology and characteristics of post-EES CNS infections.Adult patients who underwent EES between 1/2010 and 7/2021 were evaluated and included if microbiologically confirmed CNS infection occurred within 30 days of EES. Suspected contaminants, ventricular drain colonization, and pre-EES CNS infections were excluded.S. aureus, Enterobacterales, and P. aeruginosa. Among 20 patients with prior EES, pathogens included S. aureus (5/20), Enterobacterales (3/20), Enterococcus spp. (3/20) and polymicrobic infections (3/20). Overall, 35.1% (13/37) of patients developed CNS infection due to a pathogen susceptible to pre-EES prophylaxis. Among those colonized with MRSA at time of EES, 75% (3/4) developed MRSA CNS infection compared to 6.1% (2/33) of non-colonized MRSA patients (P=0.005). The overall 30-day mortality rate was 2.7% (1/37).Overall, 2005 patients underwent EES; 1.8% (37/2005) developed CNS infection. The median [IQR] age was 51 [42-60] years, 32.4% (12/37) were female, and 54% (20/37) had a prior EES. The most common indications for EES were tumor resection [67.6% (25/37)] and cerebrospinal fluid (CSF) leak repair [24.3% (9/37)]. Post-operative CSF leaks were documented in 70.3% (26/37) of patients and 24.3% (9/37) had an extra-ventricular drain or shunt in place for >48 hours at the time of infection. Ceftriaxone prophylaxis was prescribed in 64.9% (24/37) of cases and other regimens varied. The median [IQR] time from EES to diagnosis of CNS infection was 12 [6-19] days. The most common pathogens were A polymicrobic case was defined as >1 pathogen isolated from CSF (n=1) or from rhinocerebral tissue if CSF cultures were negative (n=11). Among polymicrobic cases (n=12), P. aeruginosa (n=5), Enterococcus spp. (n=4). and S. aureus (n=3) were predominant. Cases labeled as other consisted of Trichoderma spp, A. xylosoxidans, P. acnes, S. epidermidis, Peptostreptococcus spp.S. aureus, antimicrobial prophylaxis should ensure adequate coverage of this pathogen in addition to sinus flora, and programs may benefit from screening patients for MRSA colonization pre-EES. Our data also suggest that prophylaxis should target Gram-negative and other colonizing bacteria among patients with prior EES.CNS infection post-EES is rare and causative pathogens vary. Given the predominance of Ryan K. Shields, PharmD, MS, Infectious Disease Connect: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Roche: Grant/Research Support.