Ruminants’ milk is commonly used for supplying nutrients to infants when breast milk is unavailable or limited. Previous studies have highlighted the differences between ruminants’ milk composition, digestion, absorption, and fermentation. However, whether consuming different ruminants’ milk impact the appearance of the circulatory blood metabolites in the early postnatal life is not well understood. The analysis conducted here aimed to determine the effect of feeding exclusively whole milk from bovine, caprine or ovine species to pigs, approximately 7 days-old for 15 days, on circulatory blood plasma metabolites. Relative intensities of plasma metabolites were detected using a liquid chromatography-mass spectrometry based metabolomic approach. Seven polar and 83 non-polar (lipids) metabolites in plasma were significantly different  between milk treatments. These included polar metabolites involved in amino acid metabolism and lipids belonging to phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and triglycerides. Compared to the caprine or bovine milk group, the relative intensities of polar metabolites and unsaturated triglycerides were higher in the peripheral circulation of the ovine milk group. In contrast, relative intensities of saturated triglycerides and phosphatidylcholine were higher in the bovine milk group compared to the ovine or caprine milk group. In addition, correlations were identified between amino acid and lipid intake and their appearance in peripheral blood circulation. The results highlighted that consuming different ruminants’ milk influences the plasma appearance of metabolites, especially lipids, that may contribute to early postnatal life development in pigs. Human breast milk or infant formula is one of the factors influencing the developmental events during this period. Human milk is the best nutrient source for infant development. However, formula feeding plays a crucial role in satisfying the nutrient requirements in situations where breastfeeding is either limited or unavailable. Infant formulas are predominantly made with bovine milk. However, non-bovine milk  are also increasingly used due to their hypo-allergenic properties and easier digestion than bovine milk .Nutrient composition  and physMilk is largely digested in the stomach and small intestine. The released nutrients are absorbed in the small intestine and metabolised in host tissues. A pig study by Roy et al.  showed tMilk also contains nutrients like oligosaccharides known to escape digestion in the small intestine and used by the colonic gut microbiota . DigesteBased on the existing evidence, it was hypothesised that bovine, caprine or ovine milk with different nutrient compositions and gastric digestion rates would lead to a varied abundance of host and microbial metabolites appearing in the peripheral blood circulation. Therefore, the aim was to determine the effects of bovine, caprine, or ovine whole milk treatments on the circulatory blood plasma metabolome in pigs as a model of human infants. The samples were collected as part of a study that focused on structural gastric changes in bovine and non-bovine whole milk in early postnatal life . Liquid 2.2.1.® water for lipid analyses using LC-MS lipidomics.Three batches of milk samples collected during the study to feed the pigs were stored at −20°C before being freeze-dried and ground. The freeze-dried raw whole milk batches were analysed for standard amino acids using AOAC method 994.12, including HCl acid hydrolysis, followed by reversed-phase high-performance liquid chromatography (RP-HPLC). Cysteine and methionine were analysed using AOAC method 994.12, including performic acid oxidation. Tryptophan was analysed using alkaline hydrolysis, followed by RP-HPLC , 19. Fre2.2.The protocol used for the animal study was approved by the Massey University Animal Ethics Committee (MUAEC protocol 18/97) and described in detail elsewhere . Twenty-ad libitum sow’s milk. The experimental period comprises PND 7 to PND 21, where pigs were bottle-fed one of the milk treatments. From PND 7 to PND 18, pigs were bottle-fed either bovine, caprine, or ovine reconstituted whole milk powder diets , including vitamin and mineral supplements . From PND 19, the pigs were fed either bovine, caprine or ovine fresh whole milk obtained under chilled conditions from the Massey University No. 4 Dairy Farm , Dairy Goat Co-operative  and Phoenix Goats , and Neer Enterprises Limited , respectively. The fresh milk was provided for 3 days only due to a limited supply of fresh ovine and caprine milk.Upon arrival, pigs were randomly assigned to three diet groups  such that there were 8 pigs per milk treatment. From birth to PND 6 , these pigs consumed The pigs received iso-caloric and iso-volumetric amounts of each diet on a BW basis  from PND 7 to PND 13, which was considered the acclimatisation period for the pigs to learn to drink from the bottle with a rubber teat. The diets were balanced for protein content from PND 14 to PND 21 (2 g per kg BW). From PND 14 to PND 18 (reconstituted powder), the pigs received equal amounts of protein  and equal volumes of diet . From PND 19 (fresh milk), the pigs received their respective milk volumes based on equal amounts of protein per kg of BW. Balancing the protein intake between groups allowed the investigation of the effects of structural changes in ruminants’ milk on raw whole milk gastric digestion, which was the study’s primary objective . On the 2.3.®; Franklin Lakes, NJ, United States). Immediately after the blood collection, the blood sample was centrifuged at 4,500 rpm for 10 min at 4°C, and the plasma was removed and stored at −80°C until required.The pigs were anaesthetised using Zoletil 100  reconstituted with 2.5 mL each of ketamine and xylazine (both 100 mg/mL). Blood samples were drawn from the left ventricle and were collected in evacuated blood collection tubes containing EDTA  using the Pico-Tag method  as descr2.5.2.5.1.® ultrapure water was purchased from Merck Millipore . Ammonium formate (HPLC grade) and internal standards  used in extraction solvent were purchased from Sigma-Aldrich . SPLASH® lipidomix® mass spectrometry standard was obtained from Avanti® , which included all of the major lipid classes .All the chemicals and solvents used were LC-MS grade unless specified. Chloroform , methanol, acetonitrile, isopropanol, and formic acid were purchased from Thermo Fisher Scientific . Milli-Q2.5.2.5-tryptophan, d10-leucine, d2-tyrosine, and d7-alanine), precooled at −20°C was added to each 2 mL microcentrifuge tubes containing 100 μL of plasma. The mixture was vortexed for 30 s and then incubated for 60 min at −20°C. Then 400 μL of Milli-Q® water was added to each sample, vortexed for 30 s and centrifuged for 10 min at 11,000 rpm at 4°C. Subsequently, 200 μL aliquots of the supernatant and 250 μL of the bottom layer were transferred into new 2 mL microcentrifuge tubes for polar and non-polar metabolites analyses. The pooled polar quality control samples were prepared by combining 100 μL of the supernatant from each sample into a new tube, vortexed for 30 s and then aliquoted into multiple microcentrifuge tubes. Similar procedures were followed for non-polar quality control samples, except the aliquots of 80 μL were taken from the bottom layer. Blank samples were prepared using the above procedures, except the samples were replaced with 100 μL Milli-Q® water. All samples and blanks were dried under a stream of nitrogen at room temperature and stored at −80°C.Plasma samples were thawed overnight at 4°C and vortexed. Extraction solvent of 800 μL containing chloroform: methanol  containing formic acid (0.1%) for polar metabolites and 800 μL of chloroform: methanol (2:1 v/v) for non-polar metabolites. The reconstituted polar metabolite mixture was vortexed for 15 s, then centrifuged for 10 min at 11,000 rpm at 4°C. Aliquots of 100 μL of polar extract and 200 μL of lipid extracts were transferred into a vial containing a volume insert. Then, 7 μL of SPLASH2.5.3.™ HILIC column, 2.1 mm × 100 mm, 2.6 μm particle size , and 2 μL of samples onto the CSH-C18 column, 2.1 × 100 mm, 1.7 μm particle size , respectively. The mobile phases used for chromatographic separations were 10 mM ammonium formate in water (solvent A) and 0.1% of formic acid in acetonitrile (solvent B) for polar metabolites and 10 mM of ammonium formate in water/acetonitrile/isopropanol (5:3:2 v/v) (solvent A) and 10 mM of ammonium formate in water/acetonitrile/isopropanol (1:9:90 v/v) (solvent B) for non-polar metabolites. The mass spectral detection for polar metabolites was performed in positive and negative ionisation modes. In contrast, for non-polar metabolites, mass spectral detection was only performed in positive ionisation mode, as it captures most lipids.Metabolites were analysed using a LC-MS-9030 mass spectrometer coupled with a Nexera-x2 ultra-performance liquid chromatography system  as described by Abshirini et al. . Briefly2.5.4.in silico generated MS/MS lipid fragmentation spectra were used for polar and non-polar metabolite feature identification, respectively. Afterwards, data normalisation was conducted using the quality control samples  and the features with a quality control coefficient of variation >30% were removed. Identified missing values were treated with the k-nearest neighbour method using Metaboanalyst (version 5.0) (Raw data files were converted to centroid mzML format using the Shimadzu file converter and were uploaded to MS-DIAL software (version 4.48)  for subsion 5.0) . The humion 5.0)  and Metlion 5.0)  were useSimilar to the procedures applied to plasma non-polar sample preparations, LC-MS run and data processing were followed for milk lipidomic analysis except for samples volume, i.e., 200 μL each of extracted sample was used for milk lipidomic analysis.2.6.post hoc analysis on FDR-adjusted ANOVA, performed using the agricolae package for R.The concentration of amino acids in plasma samples and three batches of each milk treatment were analysed using a one-way analysis of variance (ANOVA) of the rstatix R (version 4.02) package. Amino acid concentrations with a false discovery rate (FDR) < 0.05 between milk treatments were considered significantly different. The Fisher’s least significant difference test was used for Milk lipids’ relative intensities data were expressed as relative percentage (%) for calculating the lipid intake in the last meal. The percentage of each lipid was calculated within each lipid class. Lipids within the same class, e.g., TGs, ionise at a similar rate, whereas lipids belonging to different classes ionise differently in the electrospray source; hence, the lipids were not expressed as a percentage of total lipids but as a percentage within the lipid class.2) and analysis of variance of cross-validated residuals (CV-ANOVA), i.e., Q2 value approximately >0.5 and CV-ANOVA p-value < 0.05, were considered as a good model for multivariate data. Permutation tests involving 100 permutations were used to check the robustness of the model. Features with a variable important for projection (VIP) score > 1 identified using the PLS-DA model were used for metabolites selection. One-way ANOVA was conducted on selected metabolites using the Metaboanalyst platform (version 5.0)  and FDR < 0.05 (identified using t-tests) was considered significant. Metaboanalyst (version 5.0) was used for heatmap visualisation and hierarchical clustering was performed using the Ward’s method.Multivariate statistical analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were conducted to investigate differences in polar and non-polar metabolite profiles in plasma samples in response to different milk treatments using SIMCA (version 16). Validation of the PLS-DA model was performed using the predictive ability of the model (Qion 5.0) . Metabolp < 0.05 and rho > ±0.5 were considered significant and visualised using the corrplot package in R. Last meal intake was used for correlation analysis as the nutrient appearance in the peripheral blood is expected to be due to the last meal after 18 h of fasting.The association between milk nutrient intake (amino acids and lipids) in the last meal and plasma metabolites were assessed using Spearman correlations. Correlations with 3.3.1.Ovine milk had significantly higher (FDR < 0.05) concentrations of all the amino acids and total amino acid (TAA), essential amino acid (EAA), branched chain amino acid (BCAA), and large neutral amino acid (LNAA) compared to bovine milk and caprine milk . Bovine For the last feed, protein contents (2 g of protein per kg BW)  were mat3.2.Four hundred and ninety-five features were detected during the initial lipidomics data analysis. After filtration and removal of background noise, 88 features were identified and used for subsequent statistical analyses.2FC > ±1 and FDR < 0.05) than in bovine milk  with a good fit showed a clear and robust separation of polar plasma metabolites between milk treatments  were used for multivariate analyses. The PCA model could not resolve any separation of polar plasma metabolites between milk treatments . Howevereatments ,C.2FC > ± 1 & FDR < 0.05) than in the bovine milk group (The relative intensity of seven plasma polar metabolites was significantly different (FDR < 0.05) between milk treatments and contributed the most to the separation between milk treatments (VIP > 1) . Out of lk group . No signlk group . Overalllk group .Most plasma polar metabolites identified using LC-MS metabolomics were amino acids. Their concentrations were then quantified using the HPLC method. The plasma concentration of five amino acids, namely asparagine, leucine, lysine, proline, and tyrosine were significantly higher (FDR < 0.05) in the ovine milk treatment group compared to bovine or caprine milk treatment groups . In addi3.4.A total of 438 features were obtained during the initial lipidomic data analysis process. After filtration and removal of background noise, 324 features were identified and used for subsequent statistical analyses.p-value = <0.001) with a good fit showed clear and robust separation of plasma lipids between milk treatments  between milk treatments and contributed the most to the separation between milk treatments (VIP > 1) . Therefo2FC > ±1 and FDR < 0.05) than in the caprine milk group (The relative intensity FC of 11 lipids (TG) in the ovine milk group was significantly higher than in the bovine milk group . In contlk group . Converslk group .Overall, the relative intensity pattern of plasma unsaturated TG obtained with the ovine milk treatment was distinct from caprine and bovine milk treatments, whereas the intensity pattern of PC obtained with the bovine milk treatment was distinct from those from caprine and ovine milk treatments .3.5.As pigs were fed different volumes of milk in their last meal to match protein intake  between milk amino acid intakes of the last meal  metabolites using an LC-MS-based metabolomics approach in young pigs as a model of the human infant. The result showed plasma polar and lipid metabolite changes in response to ruminant milk treatments. In total, 90 polar and lipid metabolites with differential relative intensities in the plasma samples discriminated bovine, caprine, and ovine milk treatments, and some of these metabolites were correlated with milk nutrient intakes in the last meal.4.1.Differences in relative intensities of polar plasma metabolites, predominantly those involved in amino acid metabolism, were observed between milk treatments. Similar results were observed when plasma amino acid concentrations were quantified. The intake of ovine milk led to higher relative intensities of proline, tyrosine, and isoleucine and amino acid derivatives  and higher concentrations of asparagine, leucine, lysine, proline, and tyrosine in plasma than bovine or caprine milk treatments even though protein intakes were similar. A similar observation has been reported in a study in women aged 20–40 years, showing ovine milk consumption also resulted in higher postprandial plasma concentrations of leucine, lysine, and proline compared to bovine milk . HoweverIn the present study, pigs received similar amounts of amino acids in their last meal due to balancing the protein intake based on body weight. Correlation analysis between amino acid intake and plasma amino acid appearance showed that only tyrosine intake was positively correlated to its plasma appearance. Hence, elevated levels of plasma amino acids in response to consuming ovine milk may be due to increased and/or more rapid amino acid absorption. This hypothesis is supported by a faster rate of gastric protein emptying in pigs consuming whole raw ovine milk than in pigs fed whole raw caprine or bovine milk . AdditioThe higher levels of some plasma amino acids observed in response to the ovine milk treatment could influence processes such as protein synthesis and degradation, hormone synthesis, and neurotransmission. For instance, proline is involved in glutamate neurotransmitter synthesis, whereas tyrosine is involved in dopamine and norepinephrine synthesis . Hence, de novo synthesis, and the gut microbiome  relative intensities were observed between milk treatments. Pigs fed bovine milk had higher relative intensities of plasma lipids, except for unsaturated TG than those fed ovine or caprine milk. A study involving four-week-old pigs fed bovine or caprine milk showed that their plasma lipid profiles had predominantly TG, PC, SM, DG, and ceramides . HoweverIn the present study, plasma saturated and unsaturated TG relative intensities differed between milk treatments. Pigs fed ovine milk showed higher relative intensities of plasma unsaturated TG than that of caprine or bovine milk, possibly due to faster gastric lipid emptying . In addiNegative correlations were observed between specific TG intake and its appearance in circulation in the current study. The complex digestion and absorption patterns of lipids might help to explain this phenomenon. For example, TG undergo hydrolysis of fatty acids and monoacylglycerol in the small intestinal lumen. These metabolites then enter enterocytes and are incorporated into TG, which are packaged into chylomicrons that are transported via the lymph to the systemic circulation . TherefoThe changes in relative intensities of lipids in the peripheral circulation could influence lipid synthesis, cell signalling, and the structural components of cell membranes. For example, phospholipids are precursors of the neurotransmitter acetylcholine , 53 whic4.3.The main strength of this study was the application of metabolomics for analysing plasma metabolites. This approach provided a comprehensive analysis of measurable metabolites in a biological sample, which otherwise would have been missed with the targeted approach that detects only specific metabolites. Furthermore, using the LC-MS analytical platform is another strength of this study which offered high sensitivity and detection of many metabolites compared to other analytical platforms like nuclear magnetic resonance.Another advantage of this study is using pigs as a model for human infants to understand the plasma metabolome changes in response to different milk treatments. Furthermore, considering the higher similarity between pigs and humans in terms of gut and brain physiology, anatomy, and development than in other non-primate models like rodents , 60, theThere are also some limitations which should be considered in interpreting findings. The use of metabolomics helped the detection of many plasma metabolites that were significant between milk treatments. However, most features remain unidentified. Additionally, LC-MS based metabolomics approach does not provide absolute quantification of metabolites, unlike nuclear magnetic resonance. No control groups of pigs fed with sow’s milk showing basal expected plasma metabolic profile were compared with the pigs fed with other species’ milk. This choice was because biological samples were collected from a study aimed to compare structural changes in ruminants’ whole milk on pigs’ digestion in early postnatal life, and using pigs fed with mother’s milk was not required for the experiment.Another limitation of this study was the use of a single time-point for evaluating the peripheral blood plasma metabolome profile in response to milk treatments, making it difficult to ascertain whether the observed changes were an immediate diet effect or an early postnatal developmental effect. Furthermore, only male pigs were used to understand the changes in plasma metabolite relative intensities. However, a study has shown that the plasma metabolome of children with autism spectrum disorder was sex-specific . Hence, 5.In conclusion, this study demonstrated that the consumption of whole milk from various ruminant species  affects the metabolite profiles in the circulatory plasma of young pigs, serving as a model for human infants. The analysis of plasma metabolites using LC-MS-metabolomics revealed significant alterations, particularly in lipid profiles, between milk treatments. Specifically, the study found that the relative levels of unsaturated TG were higher in response to ovine milk consumption, whereas saturated TG levels were higher with bovine milk consumption. Furthermore, the correlation analysis highlighted a relationship between some nutrients’ intake and their appearance in the peripheral blood circulation, indicating the influence of milk nutrients on their distribution in the bloodstream. These findings contribute valuable insights to our understanding of the metabolic effects of different ruminant milk consumption and lay the groundwork for future research investigating the potential consequences of changes in circulating metabolites during early life development.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by Massey University Animal Ethics Committee (MUAEC protocol 18/97). The study was conducted in accordance with the local legislation and institutional requirements.The animal experiment was codesigned by DR, CM, and WM. DR and CM conducted the pig experiment. AJ assisted with collecting biological samples, conducted the laboratory experiments and data analysis, and wrote the original draft. CM and NR helped in structuring the paper and critically reviewed the paper. KF helped with the metabolomics data analyses. CG prepared the samples for plasma amino acid analysis by HPLC. CM, NR, KF, WY, JM, WM, RD, and CG advised and critically reviewed the paper. WM and NR sourced the funding for the study. All authors contributed to the article and approved the submitted version.AJ was supported by a PhD Fellowship from the Riddet Institute through funding provided by the NZ Ministry of Business, Innovation and Employment (MBIE), Smarter Lives: New opportunities for dairy products across the lifespan Grant (C10X1706). Funding for this study was provided by MBIE through two programmes: the New Zealand Milk Means More (NZ3M) programme and the Smarter Lives Grant programme. CM, WY, KF, and WM are also supported by both MBIE grants. NR was supported by the Smarter Lives grant from 2018 to 2019 and the NZ3M grant.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer CB declared a past co-authorship with the author RD to the handling editor.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.


Comparing diagnostic performance between RFR and FFR, RFR demonstrated diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 85.0%, 80.0%, 86.7%, 67.1%, and 92.7%, respectively. We analyzed the RFR value in the hyperemia zone (0.86–0.93) according to positive (RFR: 0.86–0.89) and negative (RFR: 0.90–0.93) areas. PPV in positive area is 47.8%  and NPV in negative area is 87.7% (95% CI: 80.3% to 93.1%). Excellent correlation exists between RFR and FFR and the diagnostic value of RFR without hyperemia compared with FFR in establishing the accurate functional significance of coronary artery stenosis was shown. RFR alone could evaluate the functional significance of coronary artery stenosis without unnecessary hyperemia, except in the positive area.Resting full-cycle ratio (RFR), an alternative to fractional flow reserve (FFR) for evaluating intermediate coronary artery stenosis, helps reduce patients’ time, cost, and discomfort. However, the validation data for RFR and FFR are lacking. We aimed to assess the diagnostic accuracy of RFR and FFR and evaluate effective decision-making for revascularization using their values. Patients subjected to an invasive physiological study for intermediate coronary artery stenosis in Yongin Severance hospital between October 2020 and April 2022 were prospectively and consecutively recruited. We evaluated the correlation between RFR and FFR measurements and the diagnostic performance of RFR (≤ 0.89) versus FFR (≤ 0.80). In all, 474 intermediate coronary stenosis lesions from 400 patients were evaluated using RFR and FFR values. There was a strong linear relationship between RFR and FFR (r = 0.75, 95% CI 0.70–0.78, Trial registration: URL: http://trialsearch.who.int; Unique identifier: KCT0005255. Therefore, current guidelines recommend evaluating coronary pressure-derived FFR to evaluate the hemodynamic significance of intermediate coronary lesions in patients with symptomatic angina and undocumented ischemia5. However, FFR has several disadvantages, including the need to induce maximal hyperemia, which requires additional time and cost and to minimize the effect of coronary microcirculation by administering pharmacological agents such as intravenous or intracoronary adenosine, which causes discomfort for patients6. Therefore, the hurdle of hyperemia in clinical practice could reduce FFR use. Recent studies have reported that the rate of FFR-guided PCI for intermediate lesions was only 6.1% in the United States, 8.2% in Japan, and 5.1% in South Korea9.Fractional flow reserve (FFR) is a reliable index of the functional severity of coronary artery stenosis, which is determined using coronary pressure measurement during cardiac catheterization. Notably, several studies have shown that invasive coronary physiology assessment could improve clinical outcomes by appropriately selecting patients who may benefit from percutaneous coronary interventions (PCI) with stent implantation13. Notably, several non-hyperemic pressure ratios (NHPR) have been developed, including the entire cardiac cycle, and one of them is the resting full-cycle ratio (RFR). RFR evaluates the hemodynamic significance of coronary stenosis by identifying the lowest distal arterial pressure (Pd)/arterial pressure (Pa) ratio within the entire cardiac cycle. The validation of a novel non-hyperemic index of coronary artery stenosis severity i.e., the RFR (VALIDATE RFR) study reported that RFR is diagnostically equivalent to iFR14. However, data regarding the comparison between RFR and FFR in patients with intermediate coronary artery disease are limited17. Therefore, the present study aimed to assess the diagnostic value of RFR and FFR in real-world practice.Instantaneous wave-free ratio (iFR) has been developed to overcome the limitation of FFR requiring maximal hyperemia. With accumulating evidence, iFR, which is similar to FFR, is also recommended for evaluating the hemodynamic significance of intermediate coronary stenosis in the current guidelineshttps://trialsearch.who.int/Trial2.aspx?TrialID=KCT0005255) and adhered to the ethical guidelines of the Declaration of Helsinki. The funding sources did not participate in the design or conduct of the study, analysis or interpretation of the data, or the decision to submit the manuscript for publication.The Invasive physiologic assessment of coronary artery stenosis by RFR and FFR (ICE-FLOWER) study was a prospective, single-center observational study conducted at Yongin Severance Hospital in South Korea. Patients aged > 19 years with suspected ischemic heart disease (IHD) between October 2020 and April 2022 were enrolled in this study if diagnosed with de novo stenosis. We excluded patients with single lesion in acute coronary syndromes, left main coronary artery disease, stenosis in a coronary artery bypass graft, and a life expectancy of < 1 year. For non-culprit lesions in patients with acute coronary syndrome, we performed RFR/FFR immediately if the patient was stable, and staged RFR/FFR measurement was done within 7 days if the patient was unstable. This study protocol was approved by the Institutional Review Board of Yongin Severance Hospital , and all participants provided written informed consent before participating in the study. The study protocol was registered in the International Clinical Trial Registry Platform (KCT0005255) on July 24th, 2020  using CASS workstation 7.4 . All QCA images were retrospectively re-analyzed by two independent observers with > 5 years of experience in cardiac catheterization. All diameters were determined as the average of the diameter values obtained independently. The reference diameter was determined using the proximal and distal reference diameters.Coronary angiography (CAG) was performed by five interventional cardiologists with extensive experience in CAG and PCI according to current guidelines and standard technique using a femoral, proximal, or distal radial approach19. After confirming that there was no pressure drift, the pressure wire was re-advanced to the distal portion of the target vessel to evaluate the FFR value. In all lesions, FFR values were measured with hyperemia, achieved using an intracoronary (IC) bolus injection of 2 mg nicorandil . Based on previous studies regarding IC bolus injection of nicorandil for hyperemia, FFR measurement was obtained during maximal hyperemia, defined as a period between 20 and 50 s after IC bolus injection of nicorandil20. The pressure drift was checked after FFR measurement. In patients with multivessel disease, there were sufficient time intervals between measurements of each vessel21.Physiological assessments of the intermediate coronary lesions  were performed as follows. All coronary physiologic measurements were performed using a 0.014″ intracoronary wireless pressure wire  and automatically calculated using the QUANTIEN™ system (Abbott Vascular Inc.) and OPTIS™ Mobile System (Abbott Vascular Inc.). The pressure wire was equalized to the aortic pressure, and equalization was then performed after placing the pressure wire on the tip of the guiding catheter and removing contrast media using saline flushing. First, the pressure wire was advanced distally to a target vessel to evaluate the RFR value. The instantaneous Pd/Pa was continuously measured throughout five cardiac cycles to calculate RFR, defined as the point where the Pd/Pa ratio was the lowest during the entire cardiac cycle. After the RFR measurement, the pressure wire was pulled back into the tip of the guiding catheter to check the presence of pressure drift. A final Pd/Pa between 0.97 and 1.03 is considered acceptable22. The diagnostic accuracy, positive predictive value (PPV), and negative predictive value (NPV) of RFR in the hyperemia zone were re-evaluated. We also evaluated the relationship between RFR and Pd/Pa.We evaluated the correlation between RFR and FFR measurements and the diagnostic performance of RFR to identify FFR-positive coronary stenosis. Hemodynamically significant stenosis was defined as FFR ≤ 0.80 and RFR ≤ 0.89. Based on the actual RFR cut-off of 0.89, the gray zone with an RFR value between 0.86 and 0.93, called the “hyperemia zone,” is further subdivided into the positive (RFR 0.86–0.89) and negative (RFR 0.90–0.93) areas14. Statistical significance was set at P < 0.05 (two-sided). All statistical analyses were performed with SPSS statistical software  and R software .All data were expressed as mean ± standard deviation (SD) or numbers (%) of patients. The 95% confidence intervals (CIs) of the means of continuous variables and percentages of categorical variables were calculated with t-tests and Clopper–Pearson (exact) approaches, respectively. Pearson’s correlation coefficient (r) between RFR and FFR was computed with 95% CIs. The receiver operating characteristic (ROC) curve was used to represent the overall prediction performance of the RFR for FFR ≤ 0.80 and Pd/Pa ≤ 0.92 with the area under the ROC curve (AUC). Youden’s index was used to determine the optimal RFR cut-off against FFR ≤ 0.80 and the prediction of Pd/Pa ≤ 0.92. Prediction performance measures, such as sensitivity, specificity, PPV, NPV, and accuracy were calculated. The cut-off value of RFR calculated using Youden’s index was determined to be 0.89, consistent with the value in previous studies23 Participants’ baseline clinical and angiographic characteristics are shown in Tables A total of 474 stenoses from 400 patients were investigated and included in this study. A STAndards for the Reporting of Diagnostic Accuracy Studies (STARD)-type flow chart depicts this process as shown in Fig. p < 0.01). Figure p < 0.01).Figure p < 0.01)  of 0.90 . Using Pd/Pa ≤ 0.92 as a reference, the sensitivity, specificity, PPV, NPV, and overall diagnostic accuracy of RFR were determined as 86.8%, 92.6%, 82.5%, 94.6%, and 90.9%, respectively.  of 0.96 , the value was divided into positive (RFR: 0.86 to 0.89) and negative (RFR: 0.90 to 0.93) areas to evaluate the accuracy of RFR with FFR, which was 47.8% (95% CI 33.8%–62.0%) in the positive area and 87.7% (95% CI 80.3%–93.1%) in the negative area. Outside the hyperemia zone, the accuracy of RFR < 0.86 with FFR ≤0.80, and RFR >0.93 with FFR >0.80 were 84.2%  and 96.8% , respectively ; 1.80, 95% confidence interval (CI); 1.07–3.07; This prospective observational study (ICE-FLOWER) analyzed the validation of a novel non-hyperemic resting physiologic index using RFR for coronary artery intermediate stenosis in patients with IHD. Herein, we reported several clinically important findings to support the effective application of RFR with FFR in daily clinical practice. First, RFR value showed a good correlation with FFR value in the present study as compared to that in the previous study that used iFR, which is the gold standard of NHPR. Second, compared with the FFR (≤ 0.80) value, RFR (≤ 0.89) value in the present study showed a favorable accuracy rate of 85.0%, which was comparable to that of iFR or FFR in other studies. Third, RFR value demonstrated low accuracy (47.8%) in the positive area of the hyperemia zone (0.86–0.89); therefore, additional FFR would be required to evaluate functional coronary stenosis accurately in the positive area.24. However, the rate of FFR-guided PCI for intermediate coronary lesions in real-world practice is very low because of time, cost, and risk of adenosine- or nicorandil-induced maximal hyperemia9. NHPRs have emerged recently, and among them, iFR demonstrated non-inferiority compared with FFR in two randomized control trials; further it is recommended in the guidelines for class 1A11. RFR is one of the NHPR indices, and it has been introduced for obtaining the absolute Pd/Pa value of the entire cardiac cycle14. The present study demonstrated a good correlation between RFR and FFR and excellent diagnostic accuracy of RFR (> 85%) as compared to FFR in real-world practice. A few recent studies showed that RFR was diagnostically equivalent to iFR and had a comparable value with FFR25. In two recently published studies comparing RFR and FFR, the accuracy rate was 78% in a study from Germany that analyzed 712 lesions and 79% in a study from Spain that analyzed 380 lesions17. These accuracy rates were similar to those in previous studies comparing iFR and FFR27. Regarding the validation of RFR, the present study showed a higher accuracy rate than previous studies.FFR-guided PCI has shown superior clinical outcomes in randomized trials, and it is recommended as class 1A in current guidelines for intermediate coronary artery stenosis28, was found to be a significantly influencing factor. Further, there is an ongoing debate with growing interest whether discordant lesions should be revascularized29. To overcome such disadvantages and limitations, few studies have suggested a hybrid approach using FFR and NHPR simultaneously during decision-making for revascularization22. However, the accuracy of iFR or RFR in the hyperemia-free zone is reported to be > 90% in some studies, except for the gray zone with a value of 0.86–0.9330. When the hyperemia zone was excluded, the accuracy of the correlation of RFR with FFR increased in the present study (93.1%) compared to that in other studies. In addition, we further analyzed the hyperemia zone by dividing it into positive (RFR: 0.86–0.89) and negative (RFR: 0.90–0.93) areas. Notably, the accuracy rate in the negative area was 87.7%, similar to that in the hyperemia-free zone, whereas the accuracy rate in the positive area was only 47.8%. Previous iFR studies suggested the need for a hybrid approach in the entire hyperemia zone because of the low accuracy rate22, and a recent RFR-FFR study also reported a low accuracy rate of 68% in the hyperemia zone (RFR: 0.86–0.93)16. However, because the present study showed that the accuracy rate in the negative area was similar to that in the hyperemia-free zone, we cautiously suggest that RFR alone could predict the functional significance of the intermediate lesion, except in the positive area of the hyperemia zone (13.8% of participants in the present study). The strategy of additionally implementing FFR in the positive area of the hyperemia zone might improve the diagnostic accuracy of RFR and can avoid unnecessary adenosine- or nicorandil-induced hyperemia to ensure patients’ comfort and time and cost savings. In the future, a large-scale randomized trial should be conducted using FFR and NHPR, including iFR with RFR, which can overcome the limitations of iFR and RFR to evaluate intermediate coronary artery stenosis accurately.Discordant findings of NHPRs and FFR have shown worse prognosis compared to concordant negative indices in previous trials. In our study, only diabetes mellitus known as microvascular dysfunction, a discordant factor also identified in previous studiesThis study has several limitations. First, it was a prospective observational study conducted in a single center. Second, among NHPR, iFR, which is the gold standard, could not be compared directly with RFR in this study. However, since RFR showed a strong correlation with Pd/Pa, it might be replaced by the iFR value. Third, the percentage of stenosis > 70%  was limited, which reduced the validity in reference to angiographically significant lesions  in real-world clinical practice for intermediate coronary lesions. In addition, for further analysis of the hyperemia zone, RFR alone could evaluate the functional significance of coronary artery stenosis without unnecessary hyperemia, except in the positive area (RFR: 0.86–0.89) of the hyperemia zone.Supplementary Information.


Frontline mental health, emergency, law enforcement, and social workers have faced unprecedented psychological distress in responding to the COVID-19 pandemic. The purpose of the RCT  study was to investigate the effectiveness of a Group EMDR (Eye Movement Desensitization and Reprocessing) therapy (Group Traumatic Episode Protocol—GTEP) in the treatment of Post-Traumatic Stress Disorder (PTSD) and Moral Injury. The treatment focus is an early intervention, group trauma treatment, delivered remotely as video-conference psychotherapy (VCP). This early intervention used an intensive treatment delivery of 4x2h sessions over 1-week. Additionally, the group EMDR intervention utilized therapist rotation in treatment delivery.N = 85 completed the study.The study’s design comprised a delayed (1-month) treatment intervention (control) versus an active group. Measurements included the International Trauma Questionnaire (ITQ), Generalized Anxiety Disorder Assessment (GAD-7), Patient Health Questionnaire (PHQ-9), Moral Injury Events Scale (MIES), and a Quality-of-Life psychometric (EQ-5D), tested at T0, T1: pre—treatment, T2: post-treatment, T3: 1-month follow-up (FU), T4: 3-month FU, and T5: 6-month FU. The Adverse Childhood Experiences – International version (ACEs), Benevolent Childhood Experience (BCEs) was ascertained at pre-treatment only. Post Hoc comparisons of the ITQ demonstrated a significant difference between T1 pre  and T2 post  (t11.58) = 15.68, p < 0.001). Further changes were also seen related to co-morbid factors. Post Hoc comparisons of the GAD-7 demonstrated significant difference between T1 pre  and T2 post  (t = 6.22) = 4.41, p < 0.001; with significant difference also with the PHQ-9 between T1 pre  and T2 post  (t = 6.30) = 3.95, p < 0.001, d = 0.71. The treatment effect occurred irrespective of either ACEs/BCEs during childhood. However, regarding Moral Injury, the MIES demonstrated no treatment effect between T1 pre and T5 6-month FU. The study’s findings discuss the impact of Group EMDR therapy delivered remotely as video-conference psychotherapy (VCP) and the benefits of including a therapist/rotation model as a means of treatment delivery. However, despite promising results suggesting a large treatment effect in the treatment of trauma and adverse memories, including co-morbid symptoms, research results yielded no treatment effect in frontline/emergency workers in addressing moral injury related to the COVID-19 pandemic.Results highlight a significant treatment effect within both active and control groups. The NICE (2018) guidance on PTSD highlighted the paucity of EMDR therapy research used as an early intervention. The primary rationale for this study was to address this critical issue. In summary, treatment results for group EMDR, delivered virtually, intensively, using therapist rotation are tentatively promising, however, the moral dimensions of trauma need consideration for future research, intervention development, and potential for further scalability. The data contributes to the emerging literature on early trauma interventions.Clinical Trial Registration:Clinicaltrials.gov, ISRCTN16933691. Frontline mental health, emergency, law enforcement, and social workers have faced unprecedented psychological distress in responding to the COVID-19 pandemic, with around 22% meeting the criteria for Post-Traumatic Stress Disorder [PTSD]–a mental health condition caused by a traumatic experience . ContinuThe dynamic nature of PTSD in the immediate aftermath of trauma presents both opportunities and challenges concerning early trauma interventions . FurtherReduce trauma stress reactions.Provide authentic support.Enhance coping.Empower resilience.Minimize the risk of burnout and vicarious traumatization.Diminish the risk of developing other mental health and psychological difficulties with the potential to impact psychological well-being.EMDR therapy is an empirically supported treatment for psychological trauma, endorsed by the World Health Organisation (2013), United Nations High Commission for Refugees 2022) and the International Society for Traumatic Stress Studies (2019). It is a psychological treatment for pathogenic (trauma) memories and their associated stress symptoms using a model of pathogenesis and change known as adaptive information processing . Shapiro and the A humanitarian crisis emerges when an event threatens a population’s health, safety, and well-being. COVID-19 creates a familiar narrative frequently witnessed in humanitarian emergencies where demand for mental health provision outstrips the supply available . As BryaThe Group Trauma Episode Protocol [GTEP], developed by For frontline and emergency workers, stigma remains a substantial barrier to seeking psychological support . Diop etvice-versa  a safe, and efficient treatment intervention for Frontline/Emergency/Keyworkers, who experience psychological trauma in response to COVID-19 with regards to trauma sequalae and co-morbid symptoms.Is Early Intervention EMDR Video Group Therapy (VGTEP) a relevant and effective treatment intervention for Frontline/Emergency/Keyworkers, who experience psychological trauma in response to COVID-19 with regards to both recruitment and retention to the study?via a social networking and advertising strategy coordinated by TRN Ireland, which involved media outlets and radio stations in Ireland, Northern Ireland, and Great Britain. Interest in participation into the study was such that recruitment, although from the Island of Ireland, also included international participants from United States, Canada, Australia, New Zealand, Greece, and Turkey. The treatment intervention used for the clinical trial was the EMDR Group Traumatic Episode Protocol – version 9  used as a video-conference psychotherapy (VGTEP).The University of Worcester (United Kingdom) granted ethical approval for the study [CBPS19200030]. The research study strictly adhered to the approval granted. Additionally, the study adhered to the British Psychological Society (BPS) Guidelines on ‘Conducting Research with Human Participants during COVID-19 (2020). The study registered as a clinical trial ID ISRCTN16933691 (2020) and hosted by the Trauma Response Network (TRN) – Ireland, an NGO organization offering early trauma interventions. Research participants recruited A Randomized Control Trial (RCT) Delayed Start Design used for the clinical study compared an active treatment intervention versus a control group (delayed treatment – 1 month) using the same intervention. The clinical trial conducted from July 2020 – March 2022 during the primary lockdown periods. The RCT consisted of two cohorts:o Active Cohort 1: EMDR therapy VGTEP Treatment  implemented as an intensive intervention for 1 week.o Control Cohort 2: 4 weeks Delayed intervention of EMDR therapy VGTEP. Treatment involved an intensive intervention within 1 week.The study incorporated six data points including T0 (control group), T1 (pre), T2 (post), T3 (1-month FU), T4 (3-month FU), and T5 (6-month FU) . All incThe RCT generates six hypotheses to determine that the Virtual EMDR Group Intervention was safe, relevant, effective, and efficient:Hypothesis 1: There is no difference between active and control (delayed) groups regarding trauma sequelae—measured with the International Trauma Questionnaire (ITQ) (re (ITQ)  when comHypothesis 2: There is no difference between active and control (delayed) groups regarding co-morbid sequelae – measured with the Patient Health Questionnaire (PHQ-9) ( (PHQ-9)  and the  (PHQ-9)  over theHypothesis 3: Treating the trauma sequelae will reduce the level of moral injury as demonstrated with the Moral Injury Events Scale (MIES) (( (MIES)  and Benevolent Childhood Experiences (BCEs) (s (BCEs)  regardinHypothesis 6: There is no difference between active and control groups regarding quality of life using the EQ-5D – generic measure of an individual’s health status when comparing between T1 and T5.The clients used for this clinical trial were Frontline Health and Social Care workers or Emergency/First Responders directly working on the frontline of the COVID-19 pandemic utilizing the following inclusion/exclusion criteria:Inclusion criteria:o Adults (18 years and above).o Front Line Health, Emergency and Social Care Workers experiencing Psychological Distress and Trauma in response to frontline working addressing the COVID-19 virus.o Currently in active employment.o Symptoms indicative of psychological distress and impact of psychological well-being and functioning measured using the Impact of Events Scale Revised Score of 24 and above.Exclusion criteria:o Non-trauma exposure.o Impact of Events Scale Revised score of 23 or below.o Undergoing current treatment .o Present evidence of psychosis.As mentioned earlier as this was a Republic of Ireland Research initiative, research participants were recruited either from the Island of Ireland or internationally. A recruitment initiative was carried out utilizing media organizations within the Republic of Ireland. Recruitment to participate in the clinical trial used self-selection access through the Trauma Response Network (TRN) Ireland website through a secure platform. Each participant was also assessed by TRN Ireland administrative staff, who provided further information about the study outlining what participation involved. Those wishing to participate were provided with a detailed, research participant information sheet and research consent form.The Impact of Events Scale–Revised IES-R;  was the Mirabilis Health Institute, Belfast, Northern Ireland, provided medical supervision for the clinical trial and was responsible for clinical risk assessment and triage.N = 72 with N = 36 in each arm of the study. This figure allowed for a 10% drop-out rate. Randomization done through sequence generation using a computer algorithm allocating each research participant a unique client number (UCN) to ensure anonymity to the clinical team. N = 192 clients assessed for eligibility and N = 97 individuals excluded from the study as their IES-R scores were < 24, leaving a total of N = 95 subject to randomization.In determining the sample sizeThe clinical trial used the following measures:o International Trauma Questionnaire (ITQ) – an 18 question, self-reporting measure focused on both PTSD and Complex PTSD, consistent with ICD-11 .o Generalized Anxiety Disorder Assessment (GAD-7) which objectifies and assesses the degree of anxiety severity .o Patient Health Questionnaire (PHQ-9) which objectifies and assesses the degree of depression severity .o Subjective Unit of Disturbance/Distress (SUD) – a scale of 0 to 10, measuring the subjective intensity of disturbance or distress currently experienced by an individual .o Moral Injury Event Scale (MIES) – 9-point self-reporting scale exploring perceived transgressions, betrayals, or violations of an individual’s moral code .o Adverse Childhood Experience International Questionnaire (ACE-IQ) – tested at Pre point only with the intention to measure exposure to adverse childhood experiences (< 18 years old).o Benevolent Childhood Experiences Score (BCEs)  – testedo EQ-5D – generic measure of an individual’s health status. The EQ-5D is a preference based HRQL across five dimensions; mobility, self-care, usual activities, pain/discomfort, and anxiety/depression.Measure evaluated after each VGTEP session: SUD.Measures evaluated at T1 only: ACE-IQ and BCEs.Measures evaluated at T0, T1, T2, T3, T4, and T5: ITQ, GAD-7, PHQ-9, EQ-5D.Measures evaluated at T1 and T5 only: MIES.via the Trauma Response Network Ireland Administrators and was collected independent to the research team.All psychometrics were sent out electronically to all research participants Once research participants met the study’s inclusion criteria the TRN administrative team randomly assigned participants using a random allocation software package to either the active group Immediate Treatment Group) or a 1-month Delayed Treatment Group. Each group intervention involved a 1-week block of intensive treatment consisting of four, two hours sessions of Group EMDR therapy, delivered as a remote treatment intervention. The platform used for the VGTEP sessions was ZOOM Enterprise version 5.9.1. The TRN Ireland administrative team emailed a ZOOM link for each of the treatment sessions – evenings of Monday, Wednesday, and Thursday, and Saturday morning. At the commencement of each treatment session research participants were instructed to only use the unique client number as a means of identification, and not their name. Clinical members of the research teams had no means of identifying any of the research participants. Participants were required to attend all four treatment sessions. Each session lasted approximately 2 h. Each group consisted of six participants who remained as a group for the duration of the treatment intervention. In addition, the VGTEP session lead, and the emotional protection worker (EPW), were also randomly allocated to the treatment sessions. As per the research protocol, session leads and EPWs introduced themselves at the start of each VGTEP treatment session, and importantly, however, the research participants revealed no information about themselves. Additionally, it was emphasized to the research participants that the VGTEP intervention required no disclosure of anything about the trauma memory – consistent with the EMDR Blind 2 therapist protocol. Clinical Leads and Emotional Protection workers were also randomly allocated to the VGTEP treatment intervention ensuring therapist rotation by the TRN Administrative team.The EMDR Group Treatment Intervention delivered as video-conference psychotherapy (VGTEP) consisted of seven steps: 1. Trauma regulation exercise, 2. Trauma target selection, 3. Anchor to a past resource, 4. Identification of a future belief, 5. Trauma confrontation of the target trauma memory, 6. Enhancement of resilience and post-trauma growth, and 7. Closure grounding exercise. Dual attention, and bilateral stimulation, a distinct element of EMDR therapy, are used in steps 2 and 5. Step 5 addresses three of the significant points of disturbance relating to the target trauma memory. Trauma processing within the confrontation stage of VGTEP involves 27 occasions of focus on the trauma target material.Each VGTEP session was digitally recorded and subjected to treatment fidelity checks carried out by an independent EMDR Europe Accredited Consultant/Clinical Supervisor. When necessary, the Emotional Protection worker utilized the breakout room within ZOOM to manage any participant if they needed I:I support or guidance. Before the commencement of the trial, training was provided for both the VGTEP clinical leads and Emotional Protection workers, this ensured familiarity with the clinical trial, treatment fidelity, triage and risk assessment procedures, welfare checks, team support, and clinical and research supervision. Mirabilis Health Institute, Belfast, Northern Ireland, provided onward referral and Consultant Medical Supervision for the clinical trial.Regarding treatment fidelity and integrity a treatment protocol manual was created for the clinical trial, based on the Elan Shapiro GTEP Training Manual – version 9 . Each treatment session was video recorded and was independently fidelity checked by an international expert in the Group Traumatic Episode Protocol (GTEP). Additionally, all psychometrics were acquired independently to the research team, with the entire data set only handed over to the team for data analysis once all the 6-month follow-up data had been obtained.p value of <0.05 considered significant. Using RM-ANOVA models Eta squared η2 is used as an indication of effect size  statistical software package. Descriptive data analyzed used either independent sample t-tests or fischer exact test. For further descriptive statistics, mean ±, standard deviation (±SD) for numerical variables and percentage (%) for categorical data. Tests for normality and homogeneity of variance determined no violations, therefore repeated measures RM-ANOVA analyzed each variable at T0, T1, T2, T3, T4, and T5 time points. The rationale for utilizing RM-ANOVAS is that they have been frequently used in various RCT studies testing EMDR therapy . Skewnesect size .N = 46) and control (N = 39) groups. Per the therapist rotation model, N = 11 EMDR therapists carried out the 64 treatment interventions, and N = 26 Emotional Protection Team members were utilized. VGTEP Therapists, Emotional Protection Workers, and research participants were blinded in the intervention.Within the research trial, 64 VGTEP treatment sessions were carried out during two periods of government lockdown during the COVID-19 pandemic. This equated to approximately 128 h of treatment intervention, including both active , and then T1 (pre-treatment), T2 (post—treatment), T3 (1-mth FU), T4 (3-mth FU), and T5 (6-mth FU). N = 85 for periods T1, T2, T3, T4, and T5. A repeated measures RM-ANOVA conducted to compare the impact of the Virtual GTEP intervention on trauma symptoms using the International Trauma Questionnaire (ITQ) evaluated at T0 (control group), T1 – pre, T2 – post, T3–1-mth FU, T4–3-mth FU, and T5–6-mth FU. For the control (delayed) group there was no statistical difference between T0 and T1. Results, highlighted in F(4–332) = 106.84, p < 0.001. Post Hoc comparisons of the ITQ demonstrated a significant difference between T1 pre  and T2 post  (t11.58) = 15.68, p < 0.001). This was also seen between T1 pre and T3 1-mth FU  (t13.77) = 20.06, p < 0.001; T1 pre and T4 3-mth FU  (t14.02) = 21.98, p < 0.001; and T1 pre and T5 6-mth FU  (t15.11) = 24.12, p < 0.001. Finally, there was a significant difference between T2 post and T4 3-mth FU d = 0.41, and T5 6-mth FU d. In testing Hypothesis 1: There is no difference between active and control (delayed) groups regarding trauma sequelae—measured with the International Trauma Questionnaire (ITQ) when comparing T1, T2, T3, T4, and T5. As mentioned earlier results revealed no statistical change between T0 and T1 for the control group, therefore, post hoc comparison determined no statistically significant difference between the active and the controlled (delayed) treatment interventions. In determining the overall effect size of the treatment intervention regarding the ITQ, as a repeated measures ANOVA used 2 as an indication of effect size: η2 = 0.01 , η2 = 0.06 (medium), and η2 = 0.14 (large). Results of the ITQ treatment effect size calculated at η2 = 0.420 indicating a large treatment effect.Concerning the ITQ, N = 340 participant episodes of VGTEP. The SUD treatment effect size calculated at η2 = 0.295, p < 0.001, indicating a large treatment effect.During the VGTEP session there are stages where the participants subjective unit of disturbance is assessed. This subjective measure ascertained in steps 1, 2, 5, and 6 of the VGTEP protocol, however, per the research protocol SUD measures obtained pre and post step 1, and at points step 2 and step 6. The rationale for this is to consider changes in subjective distress during both the trauma regulation (step 1) and trauma confrontation (steps 2–6) parts of the VGTEP protocol. 2 = 0.189, p < 0.001), and therefore is of clinical relevance and benefit.p < 0.001) decrease  sessions results demonstrated a significant reduction in both groups over time for both trauma regulation (Step 1) and trauma confrontation (Step 2 and 6) with no statistical difference between the active and control (delayed) groups. The overall effect size for the trauma regulation piece @ p < 0.001, with an effect size calculated at η2 = 0.189, and trauma confrontation η2 = 0.295.For the VGTEP SUD scores for the individual  and the Patient Health Questionnaire (PHQ-9), evaluated at T0 (control group), T1 – pre, T2 – post, T3–1-mth FU, T4–3-mth FU, and T5–6-mth FU. Results determined no statistical difference between the two treatment group interventions for either the GAD-7 or the PHQ-9.F(5–190) = 42.3, p < 0.001. There was no difference between T0 and T1. However, Post Hoc comparisons of the GAD-7 demonstrated significant difference between T1 pre  and T2 post  (t = 6.22) = 4.41, p < 0.001; T1 and T3 1-mth FU  (t = 8.67) = 5.78, p < 0.001; T1 and T4 3-mth  (t = 7.78) = 7.78, p < 0.001; T1 and T5 6-mth FU  (t = 8.93) = 6.72, p < 0.001. In determining the overall effect size of the treatment intervention regarding the GAD-7, the overall effect size calculated as η2 = 0.202 indicating a large treatment effect.For the GAD-7 results demonstrated a significant reduction in both groups over time F(5–190) = 43.3, p < 0.001. Furthermore, like the ITQ and GAD-7, there was no statistical difference between T0 and T1. However, post hoc comparison of the PHQ-9 demonstrated significant differences between T1 pre  and T2 post  (t = 6.30) = 3.95, p < 0.001, d = 0.71; T1 and T3 1-mth FU  (t = 6.22) = 4.95, p < 0.001, d = 0.82; T1 and T4 3-mth FU  (t = 6.39) = 5.49, p < 0.001; and T1 and T5 6-mth FU  (t = 8.89) = 7.15, p < 0.001. In determining the overall effect size of the treatment intervention regarding the GAD-7, the overall effect size calculated as η2 = 0.136 which is on the threshold of a large treatment effect.For the Patient Health Questionnaire (PHQ-9) results also demonstrated a significant reduction in both groups over time Regarding hypothesis 2 the null hypothesis is accepted – no difference was observed between the active and control groups demonstrating that the VGTEP intervention was influential in the treatment of co-morbid features of anxiety and depression.Hypothesis 3: Treating the trauma sequelae will reduce the level of moral injury in active and control groups.The Moral Injury Events Scale (MIES) was selected for this study as it has been adapted and applied within civilian research, has the advantage of measuring both events and symptoms, and, as a psychometric, is brief and straightforward in wording . AlthougRegarding hypothesis 3, the results do not support this hypothesis. Although the VGTEP intervention effectively treated trauma, anxiety, and depression symptoms, results demonstrate no discernible treatment effect on moral injury.Hypothesis 4: Using Trauma-Focused Therapist rotation will demonstrate no change in clinical diagnosis between T1 and T5, for both active and control groups.N = 85). At T1 N = 24 had sub-clinical PTSD, N = 28 PTSD, and N = 33 Complex PTSD. Of the research participants with sub-clinical PTSD at the start of the treatment intervention results indicated that 95.83% remained sub-clinical at T5 6-month FU, with 4.17% meeting the criterion for PTSD. Those meeting the criterion for Complex PTSD results suggest a 72.73% improvement to sub-clinical, however, 21.21% showed limited improvement from the VGTEP treatment intervention.Regarding the diagnoses of PTSD and Complex PTSD, using the ITQ as a diagnostic indicator, Despite using a therapist rotation model within the clinical trial results highlight significant alterations in diagnosis at T5 6-mth FU. The VGTEP interventions had marked efficacy with both the sub-clinical and the PTSD research participants. In the sub-category, only one research participant was referred for additional help and support. With a dropout rate of 10.53% from the clinical trial, and 11.76% finding the treatment intervention insufficient, the overall recovery rate from the VGTEP intervention is estimated at 77.71%. Results confirm that hypothesis 4 is not supported – a favorable recovery rate was achieved using a therapist rotation model.Hypothesis 5: There is no difference between active and control groups regarding quality of life using the EQ-5D – a generic measure of an individual’s health status and quality of life.F(1–83) = 48.8, p < 0.001. Post Hoc comparisons of the EQ-5D demonstrated a significant difference between T1 pre  and T5  t(−6.99) = −14.3, p < 0.001. In determining the overall effect size of the treatment intervention regarding the EQ-5D, calculated as η2 = 0.159 indicating a large treatment effect. There was not statistical difference between each of the groups. A more in-depth review of the health economic data will follow in a later paper.Hypothesis 6: A correlation exists, within the target population, between Adverse Childhood Experiences (ACEs) and Benevolent Childhood Experiences (BCEs) regarding evidence supporting resilience and posttraumatic growth.p = <0.01 as highlighted in A Pearson correlation coefficient was computed to assess the linear relationship between Adverse Childhood Experiences (ACEs) and Benevolent Childhood Experiences (BCEs). Results revealed a moderate, negative correlation between the two r = −0.312, **At T5 6-month FU, every research participant contacted to provide brief feedback about their lived experience of undergoing the VGTEP intervention. A further research paper will explore this in more detail. However, Undertaking any large-scale, early intervention clinical trial is always difficult and present significant logistical challenges. Doing so during periods of Government-Imposed lockdown was also immensely demanding. The rationale for the study was to carry out a multi-component research project which focused on an important population at the frontline of the COVID-19 response. The realities of the COVID-19 pandemic, and carrying out clinical research, meant that everyone involved – the research participants, research team, and clinicians were all exposed to the ‘lived experience’ realities of COVID-19. Therefore, a reasonable conclusion is that the universality level for this whole study was extraordinarily high. As highlighted within the literature review, defining early intervention is not straight forward. Considering the ongoing nature of the pandemic for the entire duration of this RCT there is an argument cogent argument for this qualifying as an early intervention study.An important reality of the current time was that lack of availability of trauma-informed early intervention for frontline emergency workers paradoxically assisted recruitment for our study. The consequence of this resulted in most research participants highly motivated, and appreciative to being included. Despite the eventual recruitment, a larger sample would have further enhanced the study’s findings. However, as this was an unfunded study, it was essential to make the limited resources we had stretch as far as possible.The headline results from this RCT suggest that EMDR therapy, delivered remotely through a virtual platform was effective in the treatment of both PTSD and complex PTSD. This is an important finding considering the critical elements of group treatment, remote intervention, intensive delivery, and therapist rotation. When demand outstrips supply regarding access to trauma interventions research supporting the benefits of group treatment suggest significant resource benefits. Further research is needed to explore the potential benefits of this in relation to task-shifting.However, based upon this data set, results do not support its efficacy as a treatment for Moral Injury. Although results are favorable regarding the ITQ, GAD-7, PHQ-9, SUD, and EQ-5D, demonstrating that the VGTEP intervention appeared relevant to the research population under investigation, results also suggest that the intervention was well tolerated, potentially safe, and effective as a treatment intervention. Intriguingly, there was no demonstrable difference between active and control (delayed) groups. Despite promising results, caution also needs to be exercised. The absence of another trauma-focused treatment intervention as a realistic comparator suggests that more research is required to ascertain a more reliable efficiency indicator. For example, future early intervention should consider comparing VGTEP with another Trauma-focused CBT Group Intervention.Overall, the dropout rate for the study was just below 11%. There are two ways of looking at this; firstly, this level is particularly favorable to the context in which the research study operated – during a global pandemic. A contrary perspective relates to the tolerability of any psychological treatment intervention. There will always be dropouts related to factors unrelated to the study intervention – this was certainly the case for some but by no means all. However, following up on those that did drop out and relate to the treatment intervention highlighted that for some, it was too intense, too powerful, and too overwhelming, bearing in mind everything else going on in people’s lives at the time.There are four other factors to consider. Firstly, treatment was delivered as video-conference psychotherapy. Overall results suggest that this was generally well tolerated. Although there were occasional technical glitches, fortunately, these were relatively infrequent. Indeed, the ZOOM platform performed robustly for much of the study—Secondly, the intense delivery of the treatment within 1 week. Again, results suggest that this was well tolerated, although admittedly, it does require a significant commitment from research participants. Further research needs to explore the potential health economic benefits of delivering trauma treatment in this manner. Thirdly, the VGTEP intervention involves non-disclosure of any trauma material. This aspect has advantages and disadvantages. Advantages include a greater willingness to work on traumatic material without the need for disclosure. This maximizes both power and control for the research participants. If trauma memories involve shame, fear of retribution, or a shattering of assumptive networks then there are clear benefits to non-disclosure. Disadvantages include a difficulty in demonstrating cause and effect. Although research participants were invited to work with recent material there was no means to accurately monitor this. This both a strength and limitation to this study. That said, previous research demonstrates qqclear benefits from working with non-disclosed trauma material , 2022. NThe study’s results shed interesting light on the therapist rotation model. Instinctively, therapist rotation seems counterintuitive, considering the therapist relationship critical to outcome. Results from this study run counter to this narrative. Why is therapist rotation effective? There are several reasons to consider. The VGTEP intervention is highly structured – there are distinct benefits from this, reassurance, familiarity, containment, etc., however, the intervention is relatively passive. This argument may provide a rationale for why VGTEP was effective for PTSD/C-PTSD, but not Moral Injury (MI), is that MI requires more active engagement. This would require further investigation and study, and if proven correct, would necessitate further adaptation in EMDR group interventions.The GTEP protocol involves 27 episodes of trauma confrontation and requires further research and investigation, particularly into its distinctive parts – which are essential, which not? Results from this study demonstrate an overall treatment effect; however, further dismantling studies need to explore whether there might be more effective or efficient means of achieving similar or better results.Results from Another critical point of note relates to Adverse and Benevolent Childhood Experiences. Results from this study parallel an earlier study  demonstrWe recognize that follow periods reported within this RCT study are short , howeverA further, distinct limitation of the study relates to the issue of moral injury. Further research is needed to adapt EMDR group interventions more specifically more sufficiently for this important issue. Secondly, with the benefit of hindsight, incorporating more qualitative data surrounding the participants experience would have been useful. Presently, there is no absolute clarity surrounding agents of change other than from the psychometric data. Thirdly, it would have been useful to ascertain better insight into the target memories worked on by the research participants. This was always going to be challenging based the critical aspect of non-disclosure. A further aspect related to this would have been to determine if research participants may indeed have been willing to disclose their trauma targets. Fourthly, would be to consider if more detailed debriefing sessions after the VGTEP intervention may have further enhanced the study’s findings. Fifthly, as the research participants were ‘self-selecting’ this leads to the possibility of bias and ambiguity. By not capturing or clarifying motivation for being part of the study there is a fair degree of uncertainty about this aspect. Furthermore, a distinct limitation was not being able to ascertain which trauma targets were moral injury, or not. A final limitation relates to the sub-clinical PTSD participants. The data set suggests that many from this group remained ‘sub-clinical’ post-intervention, however, there is not means to determine cause and effect, that the intervention prevented the onset of PTSD. To ascertain this with more clarity further research is needed.A contemporary debate within the EMDR therapy literature relates to the value of trauma regulation. The data presented in The COVID-19 pandemic has presented significant challenges individually, collectively, and geopolitically. Ascertaining treatment interventions that can be delivered remotely safely, relevant to trauma populations, that are both effective and efficient, is paramount. In addition, the advantage of treatment given through video-conference platforms potentially increases availability and access. Critical to this relates to technical components, including availability, access, and functionality in making such treatment interventions viable on a fundamental level of scalability.This research suggests the potential efficacy of the Group EMDR intervention; incorporating a therapist rotation model adds a contemporary facet that seems counterintuitive to the existing academic literature. In addressing the global burden of psychological trauma and the necessity for task-shifting, this study offers interesting findings to this debate.The primary objective and rationale for this study were Frontline and Emergency colleagues working on the frontline of COVID-19 traumatized by their endeavors. This study contributes to the emerging knowledge base that Group EMDR therapy has something significant to contribute.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number (s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by University of Worcester. The patients/participants provided their written informed consent to participate in this study.DF conceived the study, was the chief investigator for the entire project, the principal data analyst, and the primary author of this research manuscript. JM was responsible for project management. MK was responsible for secondary data analysis, senior leadership, and support to the project. LK, PM, IM, DM, TP, PP, CM, and IB were vital research team members and treatment interventions. ZZ was responsible for treatment fidelity. DF, LK, JM, PM, IM, DM, TP, and PP were all clinical leads regarding the VGTEP treatment intervention. All authors contributed to the article and approved the submitted version.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.


It has been postulated that flumazenil (FMZ) is efficaciousin the management of anxiety disorders via the removal of αTo assess the safety and feasibility of continuous low-dose FMZ infusions forthe management of generalised anxiety disorder (GAD) and collect preliminaryefficacy data.Uncontrolled, open-label pilot study.Participants had a primary diagnosis of generalised anxiety disorder (GAD)and received two consecutive subcutaneous continuous low-dose FMZ infusions.Each infusion contained 16 mg of FMZ and was delivered over 96 ± 19.2 h. Thetotal dose of FMZ delivered was 32 mg over approximately 8 days. Sodiumvalproate was given to participants at risk of seizure. The primary outcomewas the change in stress and anxiety subscale scores on the DepressionAnxiety Stress Scale–21 between baseline, day 8, and day 28.Nine participants with a primary diagnosis of GAD were treated withsubcutaneous continuous low-dose FMZ infusions; seven participants met thecriteria for treatment resistance. There was a significant decrease inanxiety and stress between baseline and day 8 and baseline and day 28. Therewas also a significant improvement in subjective sleep quality from baselineto day 28 measured by the Jenkins Sleep Scale. No serious adverse eventsoccurred.This study presents preliminary results for subcutaneous continuous low-doseFMZ’s effectiveness and safety in GAD. The findings suggest that it is asafe, well-tolerated, and feasible treatment option in this group ofpatients. Future randomised control trials are needed in this field todetermine the efficacy of this treatment. GAD is further complicated by highcomorbid rates of major depressive disorder (MDD), present in 52.6% of lifetime GADcases, and any comorbid anxiety disorder occurring in 51.7% of lifetime GADcases.4Anxiety disorders are a group of highly prevalent, chronic, and comorbid disordersthat are ranked as the ninth most health-related cause of disabilityglobally.912 depending on a number offactors,13 pharmacological interventions are often employed and typicallyinvolve treatment with selective serotonin reuptake inhibitors (SSRIs) or serotoninnoradrenaline reuptake inhibitors (SNRIs).1114 However, otherpharmacological approaches have been used, including tricyclics, benzodiazepines(BZDs), pregabalin, quetiapine, buspirone, moclobemide, and more recently,agomelatine and vortioxetine.1419While non-pharmacological interventions are the first-line management forGAD,20 and may lead todiscontinuation in as many as 22%.21 Compounding this,discontinuation of these drugs can result in a withdrawal syndrome, which isestimated to occur in 55.7% of patients.22 Symptoms of the withdrawalsyndrome include anxiety, insomnia, irritability, shock-like sensations, dizziness,nausea, fatigue, and headaches.23 Given the commonality betweenthe symptoms of SSRI/SNRI withdrawal and anxiety disorders, clinicians mayincorrectly reinstate SSRI/SNRI treatment resulting in unnecessarycontinuation.24 In addition, the less commonly used treatments are associatedwith other limitations, such as abuse potential for BZDs and pregabalin25 and metabolicside effects for quetiapine.26 While there is an array ofpharmaceuticals used in the management of GAD, each comes with its own limitations,including a significant number of patients not responding to existingpharmacotherapy and remaining treatment-resistant. As such, there is always a needto search for novel treatments that are efficacious, particularly for the estimated30% of treatment-resistant patients.28While SSRIs and SNRIs show efficacy in GAD, they are associated with side effects,including sexual dysfunction, nausea, and worsening of anxiety at the start oftreatment, which can be bothersome for patientsA)receptors result in anxiolysis and negative modulators produce an anxiogeniceffect.29 Typically, GABAA receptors containing theα2 subunit (e.g. α2βγ2) are responsible for theanxiolytic effects of BZDs and are expressed in the hippocampus, cortex, striatum,and nucleus accumbens.30 Recently, it was theorised that flumazenil (FMZ), anantagonist at the allosteric BZD binding site on the GABAAreceptor,31 could be useful in the management of anxiety disorders . The theory postulates that chronic stress resultsin paradoxical reactions to the endogenous neurosteroid allopregnanolone throughalterations in the expression of certain GABAA receptor subtypes anddecreased GABA-mediated inhibition in the presence of allopregnanolone.3234 FMZ has been shown to causeinternalisation of these receptors, which may result in an anxiolysis independent ofα2 subunit-containing GABAA receptors.35 As chronicstress may be present in and related to GAD,36 theoretically FMZ may showefficacy in reducing GAD symptoms. However, administration of FMZ comes with severalbarriers: low bioavailability (16%), extensive first-pass metabolism, and shorthalf-life (0.7–1.3 h).37 To overcome these barriers, FMZ has been delivered via acontinuous infusion both intravenously and subcutaneously, primarily in themanagement of BZD withdrawal;38 however, this is the firststudy to investigate the theory of an anxiolytic action of FMZ in anxiety disorders,more specifically, in GAD. To test this theory, a small cohort of treatment- andnon-treatment-resistant participants with a primary diagnosis of GAD receivedsubcutaneous FMZ infusions. Treatment resistance was defined as having received orcurrently receiving a therapeutic dose of any pharmacotherapy for GAD for anadequate period (at least 6 weeks) and still experiencing clinically significantsymptoms as assessed by the treating psychiatrist.Dysfunction of the gamma-aminobutyric acid (GABA) system has been associated withanxiety disorders, and modulation of the GABA system can result in anxiolysis oranxiogenesis, whereby positive modulators of GABA type A  criteria.A small pilot naturalistic open-label observational study of participants beingtreated with subcutaneous FMZ infusions for GAD meeting the The study was conducted at an outpatient class B day hospital . The study was approved by Southcity Medical Centre Human ResearchEthics Committee (001//2019) and recognised by the University of WesternAustralia Human Research Ethics Committee (2019/RA/4/20/5926). All participantsgave written informed consent. Data were collected between March 2021 and June2022.DSM-5for GAD.39 Inclusion criteria were: (1) met the DSM-5diagnostic criteria for a primary diagnosis of GAD, (2) adult aged 18 years andabove, and (3) willing and able to give informed consent for data collection.Exclusion criteria were: (1) had initiated or changed the dose of anypsychotropic medication that could be used in the management of GAD (e.g. SSRIsand SNRIs) in the last 6 weeks, (2) had previously received low-dose continuousor implant FMZ for any indication, (3) currently pregnant or breastfeeding, (4)untreated hyperthyroidism, which may be a differential diagnosis for GAD, and(5) using BZDs daily as FMZ has been shown to reduce BZD use in high-dose users.40 The choice to excludeparticipants using BZDs daily was made to reduce the confounding effect thatdecreasing or ceasing BZDs may have on anxiety levels . Participants taking BZDs on an as needed bases (i.e. not daily) were notexcluded as FMZ is less likely to affect low-dose BZD users.40 Inaddition, it may have been difficult to find participants withtreatment-resistant GAD who were not using any BZDs. Conversely, alcohol use wasnot an exclusion criterion as it has not been shown to be anxiolytic oranxiogenic in alcohol use disorders.41Participants were patients referred to the outpatient clinic for assessment andtreatment for GAD symptoms using FMZ, with or without a history of treatmentresistance. All participants underwent an assessment by the treatingpsychiatrist (S.A. or S.H.) and met the criteria in the 42Laboratory tests  were taken prior to theinfusion as a routine procedure. TFT was measured only at baseline to excludehyperthyroidism. Follow-up blood tests and FMZ blood levels were takenopportunistically as part of standard safety monitoring. FMZ blood levels weretaken at least 6.5 h  after theinfusion start to allow for distribution and steady state to be achieved. Thequantification of free FMZ was done by liquid chromatography–massspectrometry/mass spectrometry (LC-MS/MS); the procedure is accredited by theNational Association of Testing Authorities .43 Thesyringe was then inserted into the SpringFusor® pump manufactured byGo Medical Industries Pty Ltd , which allowedparticipants to be ambulatory for the duration of the infusions. Allparticipants needed to be released into the care of a nominated person for thefirst 24 h following the insertion of the FMZ infusion and were encouraged onlydo activities they felt comfortable completing while carrying the syringe. Thesubcutaneous route was chosen (instead of the intravenous route) as thisprocedure was shown to be comfortable in a cohort of 13 participants receivingFMZ for BZD withdrawal.43 Participants were told to return to the clinic tochange the infusion syringe, tubing, and needle after 4 days; however, if thiswas not possible, participants were given a syringe to take home and changethemselves, which they were instructed to store in the fridge until needed.Participants were trained at the appointment on how to change the syringe wherenecessary. Therefore, all participants received 32 mg of FMZ at an approximaterate of 4 mg/24 h for approximately 8 days.All participants received two consecutive subcutaneous continuous low-dose FMZinfusions inserted by nursing staff trained in the procedure. A subcutaneousbutterfly needle was inserted into the anterior abdominal wall, lateral to theumbilicus, connected to flow control tubing (flow rate: 0.31 ml/h) and asyringe, which contained the FMZ solution (16 mg/30 ml/96± 19.2 h).44 Asalcohol acts on the GABAA receptor similarly to BZDs, sodiumvalproate 500 mg twice a day was given to participants with a history of alcoholmisuse for seizure prophylaxis for the duration of the infusions and thenceased. To our knowledge, there are no known drug–drug interactions between FMZand sodium valproate and it has been used as seizure prophylaxis in BZDwithdrawal studies; however, interactions have not been explicitly investigatedand may be possible due to the enzyme inhibition caused by sodiumvalproate.45 Notwithstanding, given the duration of sodium valproatetreatment , a clinically significant interaction is unlikely.The risk of seizures using low-dose FMZ in BZD withdrawal has been previouslydocumented and sodium valproate has been used for seizure prophylaxis.46 The minimum clinically important difference (MCID) forthe primary outcomes has been previously reported.47 The MCID for the stressand anxiety subscales were 3.18 and 4.04 based on a move from the inpatient tooutpatient category described by Ronk et al.47 for themean stress and anxiety values at baseline, day 8, and day 28. It is importantto note that the values reported by Ronk et al.47 weremultiplied by two to make scores comparable with the DASS-42. As such, the MCIDvalues are half of those reported by Ronk et al.47Participants completed questionnaires for the efficacy analysis at baseline anddays 4, 8, 14, and 28 (±1 day). The primary efficacy outcome measure was thechange in the Depression Anxiety Stress Scale–21 (DASS-21) score for the anxietyand stress subscales.46 thesix-item short form of the Spielberger State Anxiety Inventory (SSAI-6)score,48 and the Jenkins Sleep Scale (JSS).49 The JSSwas only assessed at baseline and day 28 as the scale measures sleep-relatedissues over the past 30 days.Secondary outcome measures included the DASS-21 score for depression,50 The DASS-21 has beenvalidated in a three-factor structure, utilised by a diverse range of clinicaland non-clinical, cultural, and ethnic groups.5154 Higher scores indicate ahigher frequency of experiencing negative emotional states.46 Ofinterest to this study, the stress subscale is most highly correlated withGAD.55The DASS-21 is a validated and commonly utilised tool for assessing the negativeemotional states of depression, anxiety, and stress.48The SSAI-6 produces similar scores to the full 20-item Spielberger State AnxietyInventory offering a briefer scale for participants and therefore, was chosen toreduce response errors and unanswered items.49 The JSS was originallydesigned for clinical research; the scale has internal reliability and isvalidated in different patient cohorts.49The JSS addresses four different sleep difficulties: initiating sleep,maintaining sleep, frequent waking across the night, and daytime sleepinessafter normal sleep duration.Adverse events were self-reported by participants meeting the inclusion criteriathat commenced low-dose FMZ treatment.p value of 0.017 for DASS-21 and SSAI-6outcomes. JSS scores were compared at baseline and day 28 using a paired-samplest test where assumptions of normality were met for scoresand score differences.Data were included for analysis if the participant had received at least one16 mg FMZ infusion (approximately 4 days), provided a baseline, and met theinclusion criteria with no exclusions. Descriptive statistics were reported forall efficacy outcome measures. Differences between mean depression, anxiety, andstress scores (DASS-21) and SSAI-6 scores from baseline, day 8, and day 28 weremeasured using a repeated measures analysis of variance (ANOVA) whereassumptions of normality, homogeneity of variance, and sphericity were met. Theα value was set at 0.05. Pairwise comparisons were made with aBonferroni-adjusted One participant did not complete the SSAI-6 scale on day 28. As such, thismissing value was imputed using the worst observation carried forward, which wasthe participant’s baseline value. Sensitivity analysis was completed using thebest possible outcome for the SSAI-6, which is a score of 6.Eleven participants met the inclusion criteria and were recruited. Twoparticipants were excluded from the efficacy analysis due to withdrawal fromtreatment. One participant withdrew before treatment commenced and the otherwithdrew during the first infusion due to a seized syringe with an estimateddose of 13 mg of FMZ delivered . As such, this participantdid not receive the anticipated therapeutic dose of FMZ and was excluded fromthe efficacy analysis; however, their safety outcomes were still included .Nine participants were included in the efficacy analysis. The sample comprisedfive males and four females . The mean = 9) varied significantly across the three timepoints,F  = 11.08, p < 0.001, partialη2 = 0.58. Pairwise comparisons further revealed that stresslevels at day 8  and day 28 were significantly lower thanbaseline . Mean stress scores on days 4 and 14 were 8.00(SD = 5.03) and 6.56 (SD = 4.50), respectively (The ANOVA results showed that stress scores on the DASS-21(ectively . The redn = 9) varied significantly across the three timepoints,F = 15.06, p < 0.001, partialη2 = 0.65. Pairwise comparisons further revealed that anxietylevels at day 8  and day 28 were significantly lower thanbaseline . Mean anxiety scores on days 4 and 14 were 4.89(SD = 3.59) and 5.67 (SD = 4.03), respectively (The ANOVA results showed that anxiety scores on the DASS-21(ectively . The redn = 9) did not varysignificantly across baseline , day 8 , and day 28 , F  = 2.88, p = 0.086. Sensitivity analysis using the bestoutcome score did not change the statistical significance of this outcome. MeanSSAI-6 scores on days 4 and 14 were 12.89 (SD = 4.14) and 12.33 (SD = 5.77),respectively.The ANOVA showed SSAI-6 scores (n = 9) varied significantly across the three timepoints, F  = 4.65,p = 0.026, partial η2 = 0.37. However, pairwisecomparisons did not reveal any significant differences between any of thetimepoints (p > 0.05).The mean baseline depression score was 11.33 (SD = 4.24) and decreased to 6.67(SD = 5.36) on day 4, 6.00 (SD = 3.91) on day 8, 5.44 (SD = 4.42) on day 14, andslightly increased to 7.00 (SD = 6.95) on day 28 . The ANOt test was used to compare mean JSS scores(n = 9) between baseline  and day 28. On average, the participant’s scores were 3.78 pointslower [95% confidence interval (CI) = 0.45–7.10] after treatment with FMZ. Thisdifference was statistically significant, t(8) = 2.62,p = 0.031, Hedges’ g = 0.79.A paired-samples Eight participants provided FMZ blood levels during the infusion . Oneparn = 5).Overall, 14 adverse events were reported by eight participants during theinfusion period . The mos55 In addition, neither theDASS-21 nor the SSAI-6 measures sleep disturbances, which is a symptom listed in theDSM-5 for GAD and, therefore, the improvement in the JSS scoreis consistent with an improvement in GAD symptoms.39Despite a small cohort of participants, a significant and clinically importantreduction in anxiety and stress levels, measured using the DASS-21, and significantimprovements in subjective sleep quality, measured using the JSS, were observed.While the inherent limitations of an open-label, uncontrolled study prevent thesynthesis of any conclusions about the efficacy of treatment, this pilot studyprovides a feasible study design to evaluate the efficacy of treatment if applied ina randomised and controlled setting. Of high importance in future study designs isthe significant difference in the stress subscale from the DASS-21, as this is mosteffective at evaluating symptoms corresponding to GAD.28 Accordingly, anxietydisorders have a serious impact on health, both mental and physical, and represent asignificant cost burden to healthcare systems. This is explained by multiple medicalevaluations and the treatment of physical manifestations  coupled with a decreased quality of life and productivity.56 Thishighlights the pertinence of further evaluating pharmacological options for thetreatment of these resistant disorders, while minimising the common side effectsassociated with other commonly prescribed drugs to reduce these impacts.Importantly, in this cohort of participants, there were no reports of a withdrawalsyndrome, and the troublesome side effect of sexual dysfunction was also notobserved, which is commonly seen with SSRIs and SNRIs.22 The most commonly experiencedadverse event was fatigue, which may be indicative of increased GABAergic tone.Although FMZ is typically an antagonist at the BZD binding site of theGABAA receptor, there are data that demonstrate FMZ acts as apositive allosteric modulator at α4 containing GABAAreceptors, which may account for the fatigue experienced during theinfusions.5759Alternatively, fatigue is a symptom of MDD and may represent a symptom of thisdisorder; however, only one patient had this diagnosis at baseline.Treatment-resistant anxiety disorder patients have been shown to have a very poorquality of life and a high rate of suicide attempts.4 While the ANOVA wassignificant, pairwise comparisons with a Bonferroni adjustment did not reveal anydifferences between the mean depression scores from baseline to days 8 and 28. Thechanges in the DASS-21 depression score may be explained by the high degree ofoverlap between the DSM-5 diagnostic criteria for MDD andGAD.60DASS-21 was used to monitor changes in depression symptoms as depression is highlycomorbid with anxiety disorders.61 and decreases in response to chronic stress.6265 Consequently, theGABAA receptor subunit conformation has been demonstrated to changeafter chronic exposure to and withdrawal from allopregnanolone. This results inincreased expression of α4β2δ GABAA receptors, which are lesssensitive to GABA-induced hyperpolarisation and may contribute to anxiety symptomsdue to decreased inhibition.32 Since FMZ has been shown todecrease cell surface expression of α4β2δ GABAAreceptors,35 it was hypothesised that treatment with FMZ could result inanxiolytic effects that last beyond the duration of treatment.32 Results fromthis study support this theory; however, future randomised control studies areneeded to determine the efficacy of FMZ infusions in the management of GAD.FMZ’s efficacy in the management of anxiety disorders has been postulated to berelated to the release of the neurosteroid, allopregnanolone, which increases inresponse to acute stressThe main limitations of this study are the small sample size and the open-labeldesign limiting the interpretation of FMZ’s effect; however, as a pilot study, ithas provided the information required to assess the feasibility of future clinicaltrials. The participants represented in this small cohort had several comorbidpsychiatric conditions. While this could be seen as a limitation as a specifictreatment population is not defined, this is also a strength as it provides data onthe use of FMZ in a more common presentation of GAD, which will often involvecomorbid psychiatric disorders. The efficacy, safety, and tolerability profilecannot be generalised until randomised control trials with large sample sizes of GADparticipants are conducted. Finally, the use of sodium valproate may have confoundedthe anxiety scores at day 8 due to its mood stabilising effects; however, it isimportant to highlight that only two participants received sodium valproate up today 8, and results at day 28 were still significant. Nevertheless, despite the smallsample size, there was still a significant difference in anxiety measures frombaseline in a predominately difficult to treat population with treatment-resistantanxiety. These changes also occurred in participants who were currently receivingpharmacotherapy. No participants needed to discontinue their current pharmacotherapyto receive FMZ, which prevented confounding the results with potential withdrawalsyndromes.38 differences between theintravenous route should be explored (e.g. bioavailability andCmax), particularly in BZD users where bolus doseshave been shown to precipitate withdrawal,6671 suggesting an anxiogeniceffect of FMZ at certain concentrations. While the infusion procedure is moreinvasive than current oral first-line treatment options, such as SSRIs, any activecomparator (pharmacotherapy) study designs should also assess the acceptability ofthis procedure and the side effects compared with standard oral daily treatments.Since seizures have been reported in trials assessing FMZ for BZDwithdrawal,44 a larger cohort of patients that would not havepharmacodynamic GABAA receptor changes from chronic BZD use needs to beassessed. In addition, as treatment-resistant GAD patients are often treated withBZDs, FMZ should also be investigated in this cohort to determine whether BZD usecan be decreased or ceased while still observing an improvement in anxiety levels.While most participants did not receive sodium valproate for seizure prophylaxis,the precipitation of a seizure from FMZ cannot be ruled out in non-BZD usingpatients, which should be considered when designing larger clinical trials.While these results indicate that FMZ may be efficacious in the management of GAD,randomised control trials are required to make a conclusion on the efficacy oftreatment. Although the subcutaneous route of administration has been favoured inthe literature more recently,Significant reductions in anxiety symptoms in participants with a primary diagnosisof GAD, most of whom were treatment-resistant to one or more pharmacotherapies, wereobserved on the anxiety and stress subscales of the DASS-21 in an open-labeluncontrolled study design. These pilot data suggest that FMZ is safe in themanagement of GAD with or without treatment resistance and, as such, furtherresearch should be directed to confirm these results and determine the efficacy in arandomised and controlled setting.