Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control. RNA was2.2Solanum lycopersicum) cv. Moneymaker. Briefly, two to three fully developed lower leaves of tomato were dusted with Carborundum powder  and then inoculated with the extract of leaf inoculum prepared in a 0.02 M phosphate buffer (pH 7.4) containing 2% polyvinylpyrrolidone 10,000. Mechanical inoculation of test plants with water or nutrient solution samples was performed in such a way that a few drops (approximately 300-500 μl) of these samples were used as inoculum. Five to ten minutes after inoculation, the test plants were rinsed with tap water to remove the abrasive and kept in a quarantine greenhouse. Symptom development on test plants was monitored weekly, and infection was confirmed on newly developed leaves using a rapid one-step assay based on lateral flow immunochromatography  (hereafter: LFD) according to the manufacturer’s instructions or by testing extracted RNA using M&W RT-qPCR.Experiments were performed in a quarantine greenhouse with temperatures of 22 ± 2°C during the light period (16 h) and 19 ± 2 °C during the dark period (8 h). ToBRFV isolate (DSMZ PV-1236) was propagated on tomato  using RNeasy Plant mini kit , following the manufacturer recommendations, with minor modifications: specifically, without using 2-mercaptoethanol, and performing the final RNA elution with two consecutive washes with 50 µl  of RNase-free water pre-warmed to 65°C. To assess the quality of the RNA in the extractions, RNA samples were tested with RT-qPCR using  a probe , and onlPeriodic testing with M&W RT-qPCR of control plants grown in the same chamber of the quarantine greenhouse as the experimental plants were used to check the adventitious spread of ToBRFV during greenhouse manipulations. Due to limited space in the quarantine greenhouses, the tops of tomato plants were occasionally pruned to keep them at a maximum height up to one and half meter.2.3Three trays of three tomato plants each were placed in a greenhouse chamber: one with all three plants inoculated with ToBRFV, one with just one plant out of three inoculated with ToBRFV, and one with three healthy tomato plants. Plants were grown in Grodan rockwool cubes  floating on nutrient solution . The nut2.4Tomato leaves (1 g) infected with ToBRFV were macerated in tap water (10 ml) using extraction bags with synthetic intermediate layer for filtration  and tenfold dilutions were prepared using tap water as diluent. These dilutions were tested by LFD. RNA was extracted from the dilutions with QIAamp Viral RNA mini kit and tested using M&W RT-qPCR  filled with nutrient solution . Few weeSpecial care was taken to prevent any contact between the mechanically inoculated plants and the bait plants, and between the nutrient solution from the inoculated plants and the upper green parts of the bait plants. Styrofoam  was used keep the upper green parts separate from the root parts and the nutrient solution. During the first experimental period, infected plants and bait plants were grown in the same chamber of the greenhouse, and nutrient solution was pumped directly from the tanks containing inoculated plants to the root zones of the tanks containing bait plants using pumps and plastic tubes   using RNeasy Plant mini kit. All extracted RNA was analyzed using M&W RT-qPCR. In addition, the infectivity of ToBRFV in the nutrient solution was checked with mechanical inoculation of tomato plants as described above. Several control plants of tomato grown in substrate at the same time in the same greenhouse, and fresh nutrient solutions were also tested.2.7Ten-day-old seedlings  of tomato plants cv. Moneymaker were each planted in 18 cm diameter plastic pots with the substrate . For the first three weeks, 50 ml of infested water/nutrient solution was added to the substrate in each pot once a week using a syringe, and then 100–150 ml of infested water/nutrient solution was added per pot two to three times a week depending on the growth stage of plants. Precautionary measures were taken to prevent contact of green parts of the bait plants with the exterior of syringe and infested water/nutrient solution. The nutrient solution from the tank of inoculated plants from the second experiment described above was injected into the substrate of six pots  or without  previous ToBRFV outbreaks were chosen and drain water samples were gathered from a collection point before disinfection in the greenhouse water circulation system. RNA was extracted from the samples using the RNeasy Plus kit  and the samples were tested using ISF-ISHI-Veg RT-qPCR.To monitor the correlation of ToBRFV building up in water with the development of infection in plants, leaf samples from the young leaves at the top of the plant and from the sepals were also taken. RNA from collected samples was extracted and analyzed using the same procedure as for water samples.The infectivity of ToBRFV in drain water was assessed using bio-assay in a growth chamber. Five tomato plants of cv. Climbo of 10 days old were planted in styrofoam pots (480 ml) containing a soil mixture suitable for young plants (DCM potting mix for sowing and cutting). For the duration of the experiment, the plants were watered three times a week with 50 ml of ToBRFV-infested drain water collected from a grower with an active ToBRFV outbreak. Irrigation with ToBRFV-infested water was carried out for four weeks, and in the meantime the water was stored at 4°C. Before the treatment, RNA extracted from the infested water sample was tested with ISF-ISHI-Veg RT-qPCR. Additionally, five tomato plants of cv. Climbo of 10 days old were first dusted with Carborundum and then inoculated on the cotyledons with the same ToBRFV-infested drain water sample. Symptom development was monitored for up to four weeks after the start of the treatment and confirmed by ISF-ISHI-veg RT-qPCR analysis of extracted RNA from upper newly developed leaves. As a positive control, five plants were mechanically inoculated with ToBRFV-infected leaf material. The inoculum was prepared by homogenizing 1 g of frozen ToBRFV-infected leaf material (-20°C) in 3 ml phosphate buffer (pH 7.4) using metallic beads. As a negative control, five plants were inoculated with the phosphate buffer. The experiment was performed in a growth chamber under controlled conditions (25 ± 2°C during the light period (14 h) and 19 ± 2°C during the dark period (10 h)).In addition, genomic and coat protein degradation in drain water over time was assessed. A drain water sample of 2 L was collected from a grower with an active ToBRFV outbreak. Subsequently, 1 L of the sample was stored at 4°C and 1 L of the sample was stored at room temperature to monitor the integrity of the ToBRFV particles in drain water over time. Both samples were sub-sampled every two weeks. After RNA extraction using an RNeasy Plus kit, the water samples were tested with ISF-ISHI-Veg RT-qPCR and a near-full genome PCR (hereafter NFG-PCR) to determine the integrity of the RNA genome. The cDNA for the PCR was created using the qPCRBIO cDNA synthesis kit (PCR biosystems) with viral RNA as a template. The RT reaction was performed using the F-22 and F-6392 primers published by 33.1The first detection of ToBRFV RNA in Slovenia was done in a wastewater sample from 2017 using high-throughput sequencing . Later, 3.2Centricons were found to be efficient for concentrating ToBRFV from infested nutrient solution samples stored at 4°C for six months, as analysis of RNA from concentrated nutrient solution samples by RT-qPCR yielded values up to 8 Cq lower than analysis of RNA from nonconcentrated nutrient solution samples Table 2.The concentrated samples of nutrient solution with the estimated highest virus concentration (two samples with Cq 15 from the tray with three infected plants) were further examined by electron microscopy and one of these two also by mechanical inoculation of test plants. No virus particles were observed in these two samples under the electron microscope, and the virus did not replicate in test plants.3.3-8, Cq = 25), indicating that sporadic transmission to test plants from water samples with higher Cq is possible, although accidental transmission during the course of the experiment cannot be completely ruled out, although the results of controls were negative.The tests of dilution of ToBRFV infected plant material in water confirm that a higher viral load is required to detect the virus with LFD and by inoculation of test plants than when using RT-qPCR Table 1.3.4-2 and 10-4, and only one week in water spiked with ToBRFV at a dilution 10-6 . In addition, three separate experiments examined infection via the roots of tomato plants grown in other glass tanks in which only the roots were exposed to ToBRFV-infested water . In each experiment, six tomato plants were placed in the tanks with bait plants. Experiments 1 and 2 had two replicas. Five, seven, and eight weeks after the start of the experiment, the Cq values of M&W RT-qPCR of this nutrient solution were 19, 15, and 16, respectively, and the ToBRFV in the nutrient solution proved infectious at all these time points Similar to the experiments described above, ToBRFV was also detected in leaves of tomato plants grown in a substrate that had been irrigated with ToBRFV-infested nutrient solution and with ToBRFV-infested tap water Figure 33.6In Greenhouse A, eight rows were monitored weekly for a possible outbreak of ToBRFV, by randomly collecting pooled leaves of different tomato plants spread randomly across the row. In Week 5, a sudden drop in ToBRFV Cq value was measured in one row (143) Figure 4via mechanical inoculation of leaves ; however, this did not work via roots (none of the plants irrigated with this drain water for four weeks became infected)  Table 4.4In Slovenia, ToBRFV RNA was detected in a wastewater sample from 2017  and thenAs described in The ToBRFV concentration in Slovenian water samples was below the detection limit of serological testing (LFD) and mechanical inoculation of test plants, so it is not known whether the detected ToBRFV in Slovenian waters was present as infectious particles, as non-infectious particles, or only as RNA. However, the data presented here shows that in case of an active ToBRFV outbreak in commercial greenhouses, a much higher ToBRFV concentration can be reached in the drain water, and by mechanical inoculation of test plants it was confirmed that the ToBRFV particles detected in the water are infectious Table 4.-4 dilution, infectivity in water three weeks after water inoculation could not be confirmed, while four weeks after water inoculation, transmission to test plants by mechanical inoculation was successful  in many control plants, in water samples, and in swabs from walls that were never in direct contact with contaminated water or with infected plants, but were from the same chamber as heavily infected plants or heavily contaminated water (data not shown). In one case, ToBRFV was detected in collected control plants with a Cq value of 22, but a week later the analysis of the same plants resulted in a much higher Cq value. Since there was no significant drop in Cq values in these samples over periods from two to six months, and no symptoms were seen in the negative control plants, it is assumed that these were environmental contaminations that never led to real infection. However, this is very worrying as such low signals may also mean low virus titer, which theoretically could lead to infection in individual cases. Therefore, this should be investigated in further studies, because the situation found under experimental conditions is also likely to be the situation in commercial greenhouses during and after ToBRFV outbreaks as shown by The results of our studies have shown that water can be an important source of ToBRFV inoculum under the experimental conditions. Potentially, such a scenario in intensive production can lead to significant yield losses in tomato production. Therefore, the results of this study are very important for the introduction of new water monitoring systems that can be used for larger scale studies. An improved, more affordable water monitoring system can play an important role in controlling ToBRFV and other tobamoviruses.The original contributions presented in the study are included in the article/NM participated in the design of the study, drafting of the manuscript, and supervised with AV the conduct of the hydroponic transmission, soil transmission, and survival study experiments, conducted by MK, AV, IB, and JB. OMCF, IG-A, DK, JB, AV, and NM conceived and performed the experiments ToBRFV detection in various environmental waters. OMCF, AF, YL, IG-A, DK, KB, and NM planned and performed the experiments for testing the different approaches for ToBRFV RNA detection in water. EV and CV planned and performed the experiments related to commercial greenhouses. All authors contributed to writing and reviewing the manuscript and approved the submitted version.


Brainstem tumors represent a challenge. Their management and prognosis vary according to anatomopathological findings and genetic and bio-molecular fingerprints. We present our experience with pediatric brainstem tumors.All patients admitted for a brainstem tumor at the Pediatric Neurosurgical Unit at Hôpital Femme Mère Enfant hospital between January 1997 and December 2019 were considered. Patients data were obtained through a retrospective review of the medical records; follow-up was from the last outpatient consultation.One hundred and twelve patients were included. Eighty-five patients (75.9%) had open surgery or stereotactic biopsy. Thirty-five patients were treated for hydrocephalus. Sixty-six received an adjuvant treatment. Several protocols were adopted according to the SFOP and SIOP during this time period. The overall survival rate was 45% with a median follow-up of five years (range 1–18 year). However, the survival rate was very different between the diffuse intrinsic pontine gliomas (DIPG) and the others tumor types. If we exclude the DIPG (59 patients), of which only 1 was alive at 3 years, the survival rate was 90.6% (only 5 deaths over 53 patients) with a median follow up of 5 years.Our series confirms that benign tumors of the brainstem have a good survival when treated with surgical removal ± adjuvant therapy. Diffuse pontine gliomas continue to have a dismal prognosis. Individualized treatment based on molecular fingerprints may help to select the best adjuvant therapy and hence potentially improve survival. Brainstem tumors represent 15% of children brain tumors; 80% of children brainstem tumors are diffuse intrinsic pontine gliomas (DIPG) that have a dismal prognosis. Brainstem tumors were first described by Kummel in 1881 . The firIn 1989, a report of the French Speaking Society of Neurosurgery on Brainstem Tumors showed the progress achieved in term of radiological diagnosis using MRI, histological classification and genetic knowledge. The role of surgery to treat exophytic lesion of the mesencephalon, of the bulbo-medullary region or focal lesions anywhere in the brainstem was also recognized. However, the survival rate of diffuse intrinsic gliomas did not improve . The purAll patients who were admitted and diagnosed with a brainstem tumor in the Pediatric Neurosurgical Unit at the “Hôpital Femme Mère Enfant”  between January 1997 and December 2019 were included in this study. Patient data were obtained through a retrospective review of the medical records from our data base; follow-up data was from the last outpatient visit. All patients without a clinical history or radiological images and without a documented post-surgical follow-up were excluded from this study.The study protocol was approved by the local ethics committee.One hundred and twelve patients were included in this study. All patients were diagnosed with a cranio-medullary MRI with and without gadolinium. The male/female ratio was 0.94 and the age varied from 6 months to 18 years with a median age of 8.9 years. The symptomatology that led to the diagnosis was unknown in 44 patients (39.3%), progressive in 63 patients 56.2%), acute in 2 patients (1.8%) and incidental in 3 patients (2.7%). At diagnosis, 14% of patients presented a deficit of the mixed cranial nerves, 21% cerebellar troubles with balance disorders, 16% nystagmus and torticollis, 10% impaired consciousness while 38% presented with a mild intracranial hypertension. According to the classification of Choux  had open surgery or biopsy (open or stereotactic). For DIPGs, the biopsy was proposed to the family but in 27 patients the biopsy was refused so the diagnosis was based on neuroradiological criteria. th nerve palsy, 3 peripheral facial nerve palsy), 2 patients presented a Parinaud syndrome, 2 patients had worsening of the pre-operative cerebellar syndrome, one patient developed a left hemiparesis and 2 patients developed tetraparesis with respiratory troubles. In one case a small intratumoral hemorrhage was visualized without clinical symptoms, 2 patients developed an epidural hematoma that didn't need a surgical treatment, and two patients developed a pseudo-meningocele treated with furosemide.We tabulated the post-operative complications separately for patients who underwent biopsy and for those who underwent tumor removal. In the biopsy group, 29% of the patients (14/48) had postoperative complications. One patient died after a diffuse edematous reaction of the brainstem, 2 patients presented a reversible bradycardia, one patient had respiratory troubles that regressed after the administration of corticoids, 2 patients had worsening of the pre-operative cerebellar syndrome, 3 patients had worsening of the pre-operative motor deficit (complete deficit in one case), 5 patients presented new cranial nerves deficit . 29% (12/41) of patients who had surgery aimed at tumor removal had postoperative complications: 12 had a new cranial nerve deficit  was found in 18 cases. This mutation is characterized by the substitution of a lysine (K) with the methionin (M). It was absent in 10 cases. Amplification of PDGFRA was seen in two cases.Among 15 tested circumscribed benign gliomas, the presence of BRAF molecular alterations was recorded in 9 cases. BRAF alterations included KIAA1549-BRAF fusion that is more specific of pilocytic astrocytomas (5 cases) and the BRAF V600E mutation that is more frequent in ganglioglioma (4 cases).Two patients were diagnosed as AT/RT. One patient had an embryonal tumor with multi-layered rosettes (ETMR) with an ETANTR phenotype.We were able to define five groups of patients : diffuseSixty-six patients (59%), had postoperative chemotherapy according to the indications of our neuro oncological team and according to the protocol of the SFOP. Several types of protocol were used  following recommendations of the local neuro-oncological team. All patient with DIPGs received a complementary treatment.At last follow-up, the overall survival rate was 45% with a median follow-up of 7.5 years (range from 1 to 23 years). However, this survival rate was very different between DIPG and circumscribed/exophytic gliomas. For DIPG, the median survival is inferior to one year (10.7 months) with a range between 4 months and 36 months. The DIPG with a survival of 36 months was H3.1 mutated with the lost of PTEN. If we exclude the 59 DIPG , the survival rate of the non-DIPG tumors of our cohort was 90.6% (only 5 deaths over 53 patients) showing that benign tumors of the brainstem can have a good survival. The median survival for all patients with benign focalized or exophytic brainstem tumors was 13 years with a range varying from two to 23 years. Eleven patients had a survival superior to fifteen years with a range between 15 and 23 years.n = 2) for the tectal plate tumors (Grade IV astrocytoma and 1 patient not operated), 6.25% (n = 1) for the bulbo-medullary region tumors (ganglioglioma), 0% for the ponto-bulbar and ponto-mesencephalic region exophytic tumors, 9.1% (n = 1) for the latero-bulbar and bulbar region tumors and 25% (n = 2) for the ponto-cerebellar tumors (AT/RT). The mortality was 14.3%  classified brainstem lesion in 4 differents groups: exophytic lesions (group I), intrinsic lesions without enhancement (group II a), intrinsic lesions with lateral or ventral extension (group II b), intrinsic lesion with enhancement (group III); focal lesions (group IV) . We haveAll these classifications should be improved by including MRI and molecular analysis of the tumor in order to decide the best oncological treatment and assess their possible prognostic evolution. Indeed, to-date the main progress is represented by the bio-molecular and genetic knowledge of the Central Nervous System tumors. For brainstem tumors, two main oncogenic pathways have been individualized: the BRAF pathway that is usually associated with benign tumors and the histone pathway that characterizes malignant neoplasms. Diffuse midline glioma and DIPG are mostly characterized by the presence of the H3.3K27M or H3.1K27M mutation of the histone genes H3F3A and HIST1H3B respectively. The mutation inhibits the PCR2 complex and regulates gene expression via the global reduction in demethylation and trimethylation of lysine 27 residue of histone sub-unit , 14. ForCastel et al. in a biomolecular study concerning 62 patients, showed the loss of H3K27 trimethylation in 95% of the DIPG samples . Beside In our experience the treatment with targeted chemotherapy did not significantly change the rate of survival for DIPG because the median survival was inferior to one year (10.7 months) in patients with and without the mutation. Considering only patients treated with the BIOMEDE program and patients treated with the TRONC 98, there was no significant difference in the median survival with a survival of 13.7 months for the group (BIOMEDE) and 13.5 months for the TRONC 98 group. We have to stress that, in our cohort, biomolecular analysis was available only in a limited number of patients. This prevented us to establish significant statistical differences regarding the survival as these technologies were available only from 2015 onward.The necessity to include patients in targeted chemotherapy protocols was at the origin of trials encouraging to perform biopsies and we share this attitude.The importance of the biopsy for brainstem tumors has been highlighted by the progress on molecular targets for many general cancers  and by tIf a biopsy has to be done, different surgical techniques can be used: direct approach, stereotactic technique with a frame or frameless, stereotactic biopsy using image guidance. We have switched from a frame-based to a frameless biopsy technique (Medtronic) without losing precision and without increasing the rate of complications. We have not observed significant statistical differences concerning the rate of sequels with the different techniques used except for a slight increase with the direct approach because it was associated to a more extended surgical removal. In the literature, the rate of transient and permanent deficits following biopsy varies from 4% to 1% with a rate of 94.9% of diagnostic yield , 4.9% ofIn the future, it may be possible that the PET/MRI scans may help to establish a diagnosis of these tumors with the study of their own metabolism avoiding the need of a surgical biopsy.Our rate of post-surgical complications for biopsies for brain stem tumors was 22%. This higher rate of complications could be explained with the higher number of biopsies realized with a direct open approach with an attempt to remove a volume of tumor as large as possible avoiding clinical sequelae.The other reason of our high rate of complications may be due to the fact that we reported also transitory complications as cardiac rhythm troubles and respiratory failures during the procedures that were completely reversible; if we remove these short-lived complications our rate of post-biopsy complications goes down to 6.5% as in other series. Regarding the mortality rate, our 2.2% rate confirm that the location of this tumor exposes the patients to a real risk.In the future, liquid biopsy and liquid biome can take a great importance not only to establish the diagnosis but also to have an evaluation of treatments in the follow-up of the disease. Liquid biopsy is a developing technique in cancer studies using saliva, blood, urine, CSF to detect cells free tumors DNA (Ct DNA), circulating tumor cells (CTC) and tumor extracellular vesicles , 32. CSFThe first direct surgical approaches for a brainstem tumor were reported in the 1960s . Then, HProgresses of surgical techniques were useful to remove focalized brainstem tumors, bulbo-medullary and latero-bulbar tumors or exophytic mesencephalic lesions that could benefit from a large resection. The use of neurovavigation, intra-operative neurophysiology and ultrasonic aspirator have also permitted a better surgical outcome. Nevertheless, the surgical approach has to be chosen based on the anatomical localization of the tumor, its exophytic extension and the axis of the tumor’s growth. In our center, we generally use the sitting position except in cases of subtemporal approach to expose the lateral portion of the mesencephalic region or the lateral portion of the pontine region. The Mayfield headrest is used in patient older than three years old to avoid complication with the pins penetrating into the fragile bone of younger patients. The CUSA cavitron permits to remove tumors staying inside the tumor and with a weak power of aspiration and fragmentation it is possible to reduce the tumoral mass also on critical area as the floor of the fourth ventricle. We advocate an aggressive surgery for benign tumors such as pilocytic astrocytomas and ganglioglioma even if complete surgical removal was possible in only twenty patients of our cohort. A complete removal can be possible also at the level of the bulbo-medullary junction. A subtotal surgical resection was possible in 13 patients. In case of ganglioglioma of the latero-bulbar or latero bulbo-pontine region, a large removal can favor a quiescence of the tumor with a good evolution also for long time. For exophytic tectal plate tumors, we prefer the sub-occipital trans-tentorial approach, as for pineal tumors, that permits a very large exposure of the posterior mesencephalic region. We already reported our experience with tectal plate tumors . When tuOn the other hand, surgery has limited possibilities for DIPG. We believe that in the future new radiological tools could allow a diagnosis without surgical procedures and also that the individualization of tumoral targets with tailored chemotherapies and radiotherapy could help to improve the prognosis of these tumors. The bio-molecular studies and tailored chemotherapic treatment in our experience did not permit an improved survival rate of patients with diffuse brainstem gliomas.Our series confirms that histologically benign tumors have a good survival with surgical removal and adjuvant treatment. DIPG still have a dismal prognosis despite recent biomolecular characterizations and new chemotherapeutic regimens that have not been able to significantly improve their survival rate and the quality of life. Still, we believe that it is necessary to perform biopsies to obtain tissue for diagnosis and to perform molecular studies to improve the efficacy of adjuvant treatment. A better classification of brainstem tumors in children with the bio-molecular understanding of the oncogenic process will be in the future responsible for further improvement of more personalized treatment.The future depends mainly on the discovery of more effective drugs that could change the dismal evolution of infiltrating brainstem tumors. The better overall prognosis of exophytic lesions may be due to the fact that these lesions are less aggressive and consequently more accessible to an extensive surgical resection that needs to be balanced with morbidity prevention and with the ability to maintain z good quality of life.


To determine that early needle-knife sphincterotomy does not increase post-ERCP pancreatitis in patients with difficult biliary cannulation as compared to standard cannulation.This prospective single-centre cohort study was carried out at Pak Emirates Military Hospital from January 2021 to June 2021. Patients requiring ERCP were enrolled in the study (according to inclusion and exclusion criteria) and were subsequently allotted different groups according to the technique used for deep biliary cannulation. Qualitative data was analysed using frequencies and chi square statistics whereas, quantitative data was analysed using mean±SD and one way ANOVA test.The cohort included 114 patients with 52.6% male patients and predominance of relatively younger age group (31-45 years). The most common indication for ERCP was choledocholithiasis (36%) with an overall technical success rate of 96%. Deep cannulation was achieved either through standard cannulation (56%), double guidewire and/or pancreatic stent assisted (10.5%), use of early Needle-Knife Sphincterotomy (19%), NKS as a last resort (3.5%) or Transpancreatic Stenting and/or combined sphincterotomy (6%). Pancreatitis as a complication occurred in 4(3.5%) patients, bleeding in 2(1.8%), on-table desaturation in 2(1.8%) and perforation in 1(0.9%) patient. The occurrence of pancreatitis was only related significantly to inadvertent PD cannulation through univariate and logistic regression analysis whereas, multiple cannulations (>5), gender, age, classification of papilla and the use of early NKS had no impact on pancreatitis or the occurrence of other complications.NKS is an effective and safe modality for deep biliary cannulation and achieving technical success where cannulation is deemed difficult and does not increase the risk of PEP if done by experienced endoscopists in high volume centres. Although endoscopic retrograde cholangiopancreatography (ERCP) has become an important standard procedure for the management of pancreatic biliary diseases, the failure to achieve deep biliary cannulation still ranges from 5% to 18% with 2% to 10% risk of post-ERCP pancreatitis (PEP) as a substantial adverse event.2Needle-knife sphicterotomy (NKS) has long been considered a risk factor of PEP, perforation and bleeding in literature and has been historically tried only as a last resort.A thorough review of the literature revealed that only two studies have been conducted from Pakistan regarding the safety profile of NKS in terms of complications.This study was designed as a prospective single-centre cohort study carried out at Pak Emirates Military Hospital, Rawalpindi from January 2021 to June 2021 after obtaining ethical committee review  and patients’ consent.All patients, age 15 years and above with indication for ERCP and consenting to the study were enrolled. Exclusion criteria included age less than 15 years, non-consenting individuals, active shock and hemodynamic instability, coagulopathy, severe cardio-pulmonary disease and pregnancy.The study was performed in a dedicated tertiary care advance GI procedure suite by two high volume Consultant Endoscopists with 90% success rate of biliary cannulation and over 400 ERCPs per year. A strict cannulation protocol of a total of less than five cannulation attempts with no more than two inadvertent PD cannulations was followed. In some of the cases, the anatomy of the papilla compelled the endoscopists to resort to early NKS. If deep cannulation was not achieved through either of the methods; double guidewire and/or pancreatic stent assisted procedure or TPS was tried according to the indication of the procedure, the local anatomy, patient dynamics and operator’s discretion.Both the operators used the same technique for deep cannulation and the demographics, extensive cannulation data, laboratory and outcome measures were noted for every patient.All patients were observed for six hours post-ERCP and those with significant abdominal pain were retained for 24 hours.Cannulation attempt was defined as a continuous contact between the papilla and the sphincterotome for at least five seconds.early NKS. In some of the cases, NKS was tried earlier at the endoscopist’s discretion considering the local anatomy. In other cases, resortNKS as the last  was tried after five or more unsuccessful attempts with standard method or using guidewire assisted procedures. Difficult biliary cannulation was defined as more than five attempts at papillary cannulation, more than five minutes spent to cannulate after papilla was clearly visualized or more than one inadvertent PD cannulation.Technical success was defined as free and deep instrumentation of the biliary tree.Total time of the procedure was defined as the time from initial intubation to the procedure terminationPancreatitis was defined as abdominal pain and more than three times rise in serum amylase levels 24 hours post-procedure. Cholangitis was defined as fever of >38°C with abdominal pain for more than 24 hours.1Primary outcomes for our study included technical success and the occurrence of complications. Secondary outcomes included total procedure time, time to deep cannulation and goal attainment, inadvertent PD cannulation, PD stenting and number of attempts at cannulation.Sample size was calculated through OpenEpi sample size calculator with 80% power by using the postulation that PEP incidence is 5 to 20% with ERCP.A total of 114 patients were enrolled according to the inclusion criteria with 60 (52.6%) males and majority of the patients (42%) belonging to the age group 31 to 45 years. Fifty-three (46.5%) of the patients had no prior co-morbidities and 69 (60.5%) belonged to ASA-I class . The mosThe laboratory parameters are shown in Total time of the procedure varied among the groups significantly, owing to the fact that highly specialized cannulation techniques were used only after standard cannulation was tried and deemed unsuccessful. In this study, pancreatitis as a complication occurred in 4(3.5%) patients , bleeding in 2(1.8%) patients and on-table desaturation in two patients (requiring neck extension and high flow oxygen). Perforation occurred in one patient when NKS was tried as a last resort and the patient had an uneventful recovery through conservative management. The patients that underwent early NKS showed almost negligible complications with only a single patient presenting minor bleed that required adrenaline injection. The mortality rate was zero for our study with meticulous follow up of the cases with complications .The occurrence of pancreatitis was only related significantly to inadvertent PD cannulation through univariate and logistic regression analysis whereas, multiple cannulations (>5), gender, younger age (<60 years), classification of papilla and the use of early NKS had no impact on pancreatitis or the occurrence of other complications . An analPost ERCP pancreatitis (PEP) has been extensively studied over the recent years due to an increase in demand of pancreatico-biliary disease management. Sphincter of Oddi dysfunction (SOD), female gender, relatively young age, non-dilated CBD, inadvertent PD cannulation, multiple cannulation attempts in lieu of difficult biliary cannulation, contrast injection and a previous history of PEP have all been reported to be significant factors contributing towards PEP.12Fistulotomy and sphincterotomy have long been considered contributors to PEP and were only used as the last resort. However, this has been rigorously challenged and many researchers now believe that papillary and PD edema secondary to excessive manipulation is the reason for pancreatitis and NKS itself is a surrogate marker for difficult cannulation.13In our study NKS was not found to be responsible for PEP through univariate and logistic regression analysis. Although the risk of PEP was low for our study (3.5%) the only factor that did contribute to the occurrence of PEP was inadvertent PD cannulation. The rate of PEP for our study was comparable to many large-centre randomized studies6This raises the question whether pre-cut PEP is operator dependent and if yes, whom should be allowed to do biliary sphincterotomies? A study by Li et al concluded that on average, 13 sessions of NKS were required to achieve a success rate of 85% and 50 sessions were required to reduce the rate of complications below 5%.18The only other complication that was seen for our cohort undergoing early NKS was minor bleeding in a single case that needed intra-procedure adrenaline injection. Also the time required for deep cannulation was less when early NKS was applied as compared to standard cannulation. It is worthwhile to mention that younger age (<60 years) and female gender were not statistically significant for PEP in our study which might be due to non-matched nature of the sample. Another local cross-sectional study with 200 participants reported CBD perforation and hypoxia to be significantly related to NKSThis study also showed that the anatomy of papilla has no correlation with post-sphincterotomy complications nor with the success rate. Similar results have been reported by Park CS et al by studying 154 NKS cases with 33 cases presenting with peri-ampullary diverticulae.The study has its limitations including single-centre non-randomized design, non-consideration of SOD as a risk factor for PEP and a non-matched sample. The strengths of the study include extensive data collection, removal of operator bias and follow up of cases for complications that might appear late.This study concludes that NKS is an effective and safe modality for deep biliary cannulation and achieving technical success where cannulation is deemed difficult and does not increase the risk of PEP if done by experienced endoscopists in high volume centres.RSAK contributed to the idea, data collection, procedure, patient care and critical review.LA contributed to the study design, pro forma, statistical analysis and drafting of the manuscript. She is also responsible for the integrity and accuracy of the study.FS contributed to patient care and critical review.FS contributed to patient care and data collection.RZAK contributed to literature review and statistical analysis.


Cognitive impairment in multiple sclerosis (MS) can adversely impact participation in employment, activities of daily living, and wider society. It affects 40–70% of people living with MS (pwMS). There are few effective treatments for cognitive impairment in people with MS. Neuromodulation with intermittent theta-burst stimulation (iTBS) has potential for treating cognitive impairment in pwMS. This single-centre mixed-methods feasibility randomised controlled trial (NCT04931953) will assess feasibility, acceptability, and tolerability of procedures used for applying iTBS for improving cognitive performance in pwMS. Participants will be randomised into three intervention groups with varying lengths of iTBS treatment (from 1 to 4 weeks) and a sham-control group. Quantitative data will be collected at three time points . End of the intervention semi-structured interviews will explore the views and experiences of the participants receiving the intervention, analysed using framework analysis. Quantitative and qualitative data will be synthesised to explore the impact of the iTBS intervention. Ethical approval has been received from the Health Research Authority (21/LO/0506) and recruitment started in June 2022. The results will inform the design of an RCT of the efficacy of iTBS as a therapeutic intervention for cognitive impairment in pwMS. However, there has been evidence showing that high-frequency rTMS poses some side effects including seizure, headache, and neck pain. Intermittent theta-burst stimulation (iTBS) is a type of rTMS that uses short bursts of three high-frequency pulses administered with a short inter-burst interval that mimics the activity (firing rate) of specific types of neurons and reduces the administration time. iTBS potentiates plasticity of brain circuits in healthy individuals that lasts for a longer duration than the changes induced by standard rTMS protocols .A recent systematic review found reported improvements in working memory and Stroop task performance in predominantly young (mean age 26 years) healthy non-cognitively impaired volunteers receiving iTBS targeting the prefrontal cortex . FurtherThe neural circuit including the dorsolateral prefrontal cortex (DLPFC) is reported to have a role in regulating executive functions ,18,19. S22.1This study will test the feasibility and acceptability of iTBS as an intervention for improving cognitive performance in people with MS. Group allocation will allow comparison of feasibility and acceptability of iTBS administration schedules of different total durations  and assessment of the believability of the sham administration, which will inform the design of a phase III trial which will investigate the clinical- and cost-effectiveness of the treatment.2.2Group 1: iTBS intervention – daily sessions for 4 days over 1 week .Group 2: iTBS intervention – daily sessions for 4 days per week over 2 weeks .Group 3: iTBS intervention – daily sessions for 4 days per week over 4 weeks Control Group 4: sham iTBS intervention – daily sessions for 4 days per week over 2 weeks .A single-centre, mixed-methods feasibility randomised controlled trial will be conducted to compare four groups (target: 10 participants each) of iTBS administration. Participants will be randomised into one of four groups:Participants will complete outcome measures at three time points during the study: baseline, end of intervention (EOI) , and 8-week follow-up . Participants will undergo MRI scans at baseline and EOI. We will conduct post-intervention interviews to explore the participants’ experiences of the trial, including the tolerability of the protocol, acceptability of the visit schedule, and any perceived differences in cognition.2.2.1The primary objective is to assess the feasibility of the trial procedures, in terms of their acceptability and tolerability for people with MS.– Assess the feasibility of recruitment, including the proportion of eligible and consenting participants.– Assess the acceptability of overall trial participation for the 1-, 2-, and 4-week groups.– Assess the tolerability of the iTBS intervention (or sham), including any side effects.– Establish the believability of sham iTBS administration.To address this primary objective, we will:2.2.2– Measure changes in cognitive assessment scores by group between baseline and EOI assessments , to allow comparison of effect sizes between groups.– Measure changes in cognitive assessment scores by group between EOI and 8-week follow-up assessments, to allow comparison of effect sizes between groups.– Measure changes in participant-reported effects on cognition by group between baseline, EOI, and 8-week follow-up assessments, to allow the comparison of effect sizes between groups.– Explore (at the follow-up interview) participant views on meaningful change in cognition in relation to the demands of trial participation.– Measure changes in scores for anxiety, depression, and fatigue before and after intervention, to allow comparison of effect sizes between groups.– Measure changes in the effective connectivity between left DLPFC and left caudate nucleus, to allow correlation with changes in cognition.Secondary objectives are to:2.32.3.1– Clinical diagnosis of MS (any type of MS) according to the 2017 McDonald criteria  at leastper se, but to remove high-performing individuals [may have cognitive impairment, with a range of lower cognitive performance levels, for the purpose of this feasibility study.– Score of 55 or lower on the oral Symbol Digit Modality Test (SDMT). The SDMT threshold of 55 or lower has been chosen because it is 1 standard deviation (1sd) below the mean for young adults with high educational level, and this range includes the respective cut-offs for 1sd below mean values for other age and educational groups. This lenient cut-off value is not intended to select individuals with cognitive impairment ividuals . This al– Aged between 18 and 69 years.– Ability to give informed consent.– Able to commit to regular attendance in the clinic, for up to 4 times a week for 4 weeks and follow-up assessment 8 weeks after the end of trial procedures.2.3.2– Depressive symptoms present with a score of ≥15 (the threshold for moderate depression) on the Patient Health Questionnaire-9 (PHQ-9) .– Medical history of, or self-reported, seizures.– Co-morbid neurological conditions (in addition to MS), e.g., brain neoplasm, cerebrovascular events, epilepsy, prior brain injury, or brain surgery.– Contraindications to MRI scanning (identified by standard MRI safety screening questionnaire).– Contraindications to TMS, including hairstyles or piercings  that would impair magnetic pulse delivery.– Frequent panic attacks which are likely to prevent regular attendance or participation in MRI/TMS procedures.– Prior TMS intervention.– Pregnancy.– MS relapse within the preceding 6 weeks.– Significant mobility problems if they are likely to preclude regular attendance in the clinic, for up to 4 times a week for 4 weeks.– Involved with any other clinical trials involving medical procedures, interventions, or treatment.– Not fluent English speaker hampering the completion of questionnaires and cognitive assessments.2.4Participant flow through the trial is shown in ralloc.ado version 3.5.2 [Consenting procedures will be conducted by the research psychologist. Participants will be randomised after completing the consent form, and the research psychologist will be blinded to group allocation. Participants will be randomised to one of four groups in equal proportions (1:1:1:1 ratio) automatically using blocked stratified randomisation, stratified on: age group (≤40), gender (man or woman), and MS type (relapsing-remitting MS or secondary progressive or primary progressive MS). The randomisation schema was created on 9th November 2021 10:51 using on 3.5.2  in StataParticipants will be blinded as to whether their iTBS intervention is real  or sham (group 4) until the end of their study participation. Although only the 2-week intervention duration has a sham comparator , participants will not be made aware that the 1- and 4-week intervention durations do not have a sham comparator. Participants will be instructed not to tell the research psychologist the duration of their intervention, as this would reveal to the research psychologist that the patient received the real iTBS intervention if in the 1- or 4-week intervention groups. To standardise the assessment interval and maintain the blinded status of the research psychologist, the interval between baseline and EOI assessments will be fixed at 5 weeks, requiring commencement of the iTBS intervention to be staggered per group as per 2.5Depending on the group, participants will receive iTBS (or sham) intervention 4 times per week for 1, 2, or 4 weeks. Each intervention session will last around 30 min, including 20 min of iTBS. The iTBS (or sham) sessions will be delivered by two TMS operators that completed 15 h of training to deliver TMS. The sessions will be conducted in a private and quiet room located at the Clinical Research Facility of Nottingham University Hospitals. The first intervention session will be scheduled for weeks 2, 4, or 5 depending on the group. We designed this study to have, for all participants regardless of group allocation, a 5-week standardised gap between baseline assessment on week 1, administrated before any iTBS session and the EOI assessments on week 5, administrated immediately after the last iTBS session.2.5.1For participants allocated to one of the real iTBS groups, stimulation will be administered to the DLPFC target coordinates (see below for target identification) under StimGuide neuronavigation using a 70 mm Double Air Film Coil , connected to a Magstim Horizon Performance stimulator. The administration comprises bursts of 3 pulses at 50 Hz with a power of 80% of the resting motor threshold (RMT), at a burst frequency of 5 Hz  for 2 s, repeated every 10 s for a total of 190 s (600 pulses) . Blocks 2.5.2The sham iTBS administration will be performed under the same conditions and with an identical protocol including 50% RMT and equipment to the full administration, except that the 8-shaped iTBS coil is held orthogonally to the scalp surface, its side touching the scalp to provide a similar sensation of direct contact to the real intervention, with the axis of the coil parallel to the midsagittal plane. The centre of the coil is kept roughly 10 cm from the skull and does not deliver any stimulation in the direction of the cortical surface. The sound made by the iTBS coil is the same as the real intervention stimulation. The TMS operators display a sham coil position on the screen of the neuronavigation system, similar to that shown for the real intervention.2.6Study data will be collected and managed using REDCap electronic data capture tools ,30 hoste2.6.1Screening procedures will be conducted via telephone or videoconference by the research psychologist. The oral version of the SDMT and the PHQ-9 will be administered. Participants will be asked to complete a questionnaire regarding their demographics, MS diagnosis, and current treatments and to identify possible contraindications to MRI and TMS procedures.Patients fulfilling the eligibility criteria will be invited to participate in the trial. Those who do not meet eligibility criteria will be excluded and thanked for their time. Reasons for ineligibility will be recorded.2.6.2Following enrolment, missing information regarding the participants’ clinical characteristics  will be requested from the neurologist or usual MS care team, along with the participants’ most recent Expanded Disability Status Score . The use2.6.3All participants regardless of group allocation will complete the following procedures at baseline assessment in week 1.The Brief International Cognitive Assessment for MS (BICAMS): The BICAMS was developed by an international panel to support a cognitive assessment that is brief and universal, to include tests of mental processing speed and memory. It was developed to be optimised for small centres where staff may not have formal neuropsychological training . Tests iThe SDMT, assessing attention, working memory, and visual/spatial information processing speed .The California Verbal Learning Test-II, assessing verbal memory recall .The Brief Visuospatial Memory Test Revised, assessing visual memory .Digit Span (from WAIS-IV): this test comprises two conditions, namely Digit Span Forwards (DSF) and Backwards (DSB). DSF assesses attention, encoding, and auditory processing capacity while DSB assesses working memory and executive function .Patient Health Questionnaire (PHQ-9) is a module of the PHQ for recording severity of depressive symptoms  and has Generalised Anxiety Disorder (GAD-7) is a brief scale to screen for generalised anxiety disorder .The Perceived Deficits Questionnaire (PDQ) assesses subjective cognitive dysfunction in people with MS . The PDQThe Modified Fatigue Impact Scale (MFIS) is a self-report questionnaire exploring the effects of fatigue across physical, cognitive, and psychosocial modalities. It is a modified form of the Fatigue Impact Scale  and was The Edinburgh Handedness Inventory (EHI) assesses whether participants favour their left or right hand to inform the MRI analysis.Participants will undergo in-person baseline cognitive and mood assessment that includes:High-resolution T1-weighted volume, for image co-registration and segmentation;Resting-state functional MRI, for functional connectivity analysis;Fluid-attenuated inversion recovery, for MS T2 lesion quantification;Diffusion tensor imaging, for structural connectivity analysis;Participants will have a 3T MRI scan to allow target localisation, quantification of MS lesion burden, and characterisation of brain networks and connectivity. The MRI scan will include:The T1-weighted and resting state functional MRI will be used to estimate the iTBS intervention targets.2.6.4Two working days before the participants’ first visit, staff delivering the iTBS (or sham) intervention will ask participants if anything has changed since they were assessed which may impact their cognitive abilities, such as infection or MS relapse. If they have MS relapse or active/recent infection, they will be withdrawn from the study.The first iTBS (active or sham) session will begin with the estimation of the RMT as detected using electromyography (EMG) to record a motor response in the contralateral abductor pollicis brevis. This is achieved by administration of single pulses of TMS to the scalp over the approximate location of the primary hand motor area of the left hemisphere, identified using the StimGuide neuronavigation system to locate the C3 position on the scalp. Slight adjustments to the position of the coil and the angle relative to the scalp surface are made by the operator to optimise stimulation of the primary hand motor area. The TMS operator delivers single TMS pulses with varying intensities guided by the adaptive Parameter Estimation by Sequential Testing  to estimThe localisation of the left DLPFC target will be identified using a similar target-identification methodology to previous work ,44, usinThe participant then receives the intervention according to group allocation as described above in the ‘Intervention’ section.Following each administration, the participant is asked to report any sensations they may have experienced.In case of missed appointments, the research team will work with the participant to reschedule the appointment, providing that participants do not have more than four consecutive days without intervention (including weekends) and that the total afforded extra time does not exceed 50% of the initial intervention schedule.Thus, participants allocated to 1 week of intervention (group 1) will be allowed 3 extra days within which to complete the intervention schedule. Participants allocated to 2 weeks of intervention (groups 2 and 4) will have one extra week to complete it and participants allocated to 4 weeks of intervention (group 3) will be afforded up to 2 weeks during which to complete it. If the intervention cannot be administrated during the allowed time, this will be recorded as a protocol deviation.2.6.5Participants will undergo repeat cognitive assessments and all questionnaires (except for the EHI) as per the baseline assessment. Participants will have a 3 T MRI scan with an identical protocol to the baseline MRI. Participants will be asked to complete an in-house questionnaire exploring the experience and tolerability of the iTBS protocol and MRI scan.2.6.6At 8-week follow-up, participants will complete again the cognitive assessments and questionnaires (except for the EHI), followed by a semi-structured interview exploring the acceptability and tolerability of the intervention and any self-reported changes in cognitive ability experienced in daily life. The interview will also explore participants’ views on what a sufficiently meaningful and durable change in cognitive performance is to balance and justify the demands of the intervention  Participants will be asked for suggestions for improving the trial experience.2.6.7As this is a feasibility trial, participants withdrawing from the study before completing the protocol will be invited for an exit interview to explore the reasons for withdrawal.2.7As this is a feasibility study several endpoints will be collected to allow primary and secondary aims to be assessed, shown in 2.8We aim to randomise up to 40 participants (up to 10 participants per group), which should provide sufficient information to inform our feasibility objectives and to inform the design of a potential phase III RCT. Whitehead et al.  recommen2.9Data will be reported according to the CONSORT 2010 statement, extension for pilot and feasibility trials . The resQuantitative analysis will be used to evaluate the change in cognition, mood, and fatigue using the cognitive assessment and questionnaires. Where appropriate, parametric or non-parametric statistics will be used to estimate effect sizes and perform between-group comparisons to inform power and sample size calculations for a potential future phase III trial.Qualitative data (post-intervention interviews) will be analysed using framework analysis –51. Thes2.9.1Quantitative and qualitative data will be synthesised following the convergence coding matrix strategy ,54. The The research team will identify the key factors that provide insight regarding the impact and acceptability of the intervention. The research team will then look for agreement or disagreement between the findings from the different research methods.Convergence: Agreement between the data from both methodologies.Complementary: The findings from one methodology complement the rest of findings.Disagreement: The findings from each methodology contradict each other.Silence: Only one methodology offers information of a key finding.The convergence coding matrix follows four possible codes ,54:Conve2.10Patient and Public Involvement (PPI) representatives have been involved from the inception of the project. This project was presented and discussed at the Nottingham MS PPI group meeting in March 2019. Attendees were asked to rate the relevance of the study and methods used; the attendees believed that the study was highly relevant, and the planning was excellent.The research team also includes two PPI representatives who contributed to the development of the grant proposal and are involved in the review of participant-facing material, research team meetings to discuss progress, and commented on manuscripts for appropriateness of language.2.11The findings from this study will be disseminated to relevant stakeholders . A main manuscript reporting the feasibility of delivering the proposed intervention and outcomes will be submitted for publication. The findings will also be disseminated at national and international conferences to reach a wider academic audience. Finally, we will share the findings with organisations that work with people living with MS  by writing a lay summary report or presenting at conferences organised by and for people living with MS 2.12Recruitment commenced on 22 June 2022. At present, 32 potential participants have been screened, 24 have been recruited, 14 have completed the intervention (or sham), and 13 have completed the 8-week follow-up assessment.Ethical approval: The study received a favourable ethical opinion from the Health Research Authority (ref: 21/LO/0506) on 20 August 2021. The study is registered with ClinicalTrials.gov (NCT04931953).3This manuscript reports the study protocol for mixed-methods feasibility randomised controlled trial exploring the feasibility and acceptability of iTBS improving for cognitive performance in people with MS. Data regarding the feasibility and acceptability of the intervention will be collected using mixed methods research to allow for an in-depth understanding of the impact of the intervention and identify barriers for future implementation in a larger phase III RCT.


Whilst the treatment and prevention of overweight and obesity-related disease is managed by restricting daily energy intake, long-term adherence to dietary strategies appears unsustainable. Time-restricted eating (TRE) aims to position energy intake in an eating window under 12 h per day and offers an alternative behavioral intervention, which can aid weight management and improve cardiometabolic health. Adherence to previous TRE protocols is estimated at between 63 and 100%, although the accuracy of reporting is unclear. This study therefore aimed to provide an objective, subjective, and qualitative overview of adherence to a prescribed TRE protocol, and to identify any potential barriers affecting adherence. Adherence after 5 weeks of TRE was estimated at ~63% based on continuous glucose monitoring data when compared with time-stamped diet diaries. Subjective participant responses reported adherence at an average of ~61% per week. Barriers to adopting TRE, including work schedules, social events, and family life, were identified by participants during qualitative interviews. The findings of this study suggest that the development of personalized TRE protocols may help to navigate the barriers to adherence leading to improved health-related outcomes. There are three main versions of IER, including alternate day fasting (ADF), the 5:2 diet, and time-restricted eating (TRE). The primary aim of TRE is to restrict all daily energy intake into a timeframe lasting no more than 12 h, with less emphasis on calorie counting or adjusting diet quantity or quality. This dietary strategy focusing on the timing of energy consumption has been highlighted as a potential treatment demonstrated to reduce body weight, body mass index (BMI), total body fat percentage, waist circumference, and hip circumference in individuals with overweight and obesity ,9,10,11,There are numerous TRE strategies, which vary in the duration of their eating windows from ~4 to 12 h, distributed at different times throughout the day. What remains unclear are the mechanisms driving these health-promoting outcomes. TRE has been demonstrated to result in weight loss ranging from 1% to 4% of body weight in studies lasting 4 weeks , 8 weeksAttrition rates, however, are a consistent issue associated with weight loss-related dietary interventions . A revieEvidence for the biological, behavioral, psychosocial, and environmental factors affecting adherence to TRE is beginning to emerge within the literature ,29,30. QA questionnaire-based study analyzing the proof-of-concept of the current TRE protocol symmetrically restricting the daily eating window by 3 h found ~59% of participants with an eating window ≥12 h could feasibly incorporate the restricted eating pattern into their daily routine . The datThe studies described above were all conducted over a short timeframe between 4 and 24 weeks. Further, most studies have used self-reported diet diaries to monitor adherence, and these are subject to misreporting bias. Further research is needed to objectively assess adherence to TRE regimes, identify factors that may impact adherence, and explore preferences relating to the proposed TRE or other protocols.Therefore, the present TRE study incorporated a 3 h symmetrical reduction to habitual eating windows over 12 h by delaying and advancing first and last daily eating occasions by 1.5 h, respectively, with the aim and objective of: (i) exploring objective and subjective adherence to the protocol during 5 weeks of TRE; (ii) exploring the preference for the current TRE protocol and prospective use with a focus on the positives and negatives, and (iii) identifying the barriers to maintaining TRE.In this 7-week TRE study, participants were assigned to either the TRE (intervention) or control group using age, weight, and BMI to ensure that demographics were equivalent between groups. The study consisted of a 2-week baseline period followed by 5-weeks of control or TRE. The intervention length of 5-weeks was designed so that adherence could be reviewed and compared at three phases of the trial . The main purpose of this was to explore whether rates of adherence varied depending on the phase of the intervention. The intervention phase was deemed long enough to provide sufficient experience of following TRE under free-living conditions and to identify the barriers that impact on the ability to maintain adherence. An outline of the study and a timeline of procedures are provided in In total, there were 6 study visits to the Clinical Investigation Unit (CIU) at the University of Surrey, including screening, start of baseline, end of baseline, and after 2 weeks, 4 weeks and 5 weeks of intervention. Participants attended study visits at the end of baseline, and 2 weeks and 5 weeks of intervention in a fasted state (≥12 h). The study received a favorable ethical opinion from the University of Surrey ethics committee: UEC 2019 064 FHMS. This study was conducted in accordance with the ethical principles expressed in the Declaration of Helsinki. Recruitment began in January 2022, and participant enrolment started on 31 January 2022 and ended on 1 May 2022. The study ended on the 19 June 2022.At baseline, participants were instructed to maintain their habitual eating times, then either reduce the eating window by 3 h for the intervention phase (TRE group) or continue with routine eating times (control group). To reduce any potential bias towards early or evening eating, participants were instructed to delay and advance their first and last energy intake times by 1.5 h each in accordance with averaged eating time data recorded at baseline. Participants in both groups were allowed to eat ad libitum within their habitual or adjusted eating windows. Participants could consume water, tea and coffee without milk and sugar, sugar-free herbal teas, and sugar-free soft drinks during the fasting period. Under both conditions, participants were given a ±30 min timeframe in which they could adjust their eating window but were encouraged to maintain their routine as consistently as possible.2) males and females (aged 18–50 y) with normotensive blood pressure (<140/90 mm Hg) and an eating window ≥12 h were recruited to take part in this TRE trial. Potential participants were excluded if any of the following were reported: chronic illness, regular medication affecting appetite or weight, history of shift work, weight change of ±3 kg in the last 6 months, regular smoking, travel across ≥2 time zones one month prior to or during the trial, and current involvement in other clinical trials. After dropouts, n = 16 participants met the intervention (n = 8) and control (n = 8) conditions; n = 16 completed the exit questionnaire and n = 10 completed the exit interviews. An outline of the study consort is displayed in Non-obese  available for both iOS and Android smartphones and automatically uploaded to Nutritics software for analysis . Participants recorded 7 days of weighed food intake  in the lAdherence to the TRE and control group instructions was monitored by comparing CGM data to the time-stamped diet diaries. Freestyle Libre Pro iQ  continuous glucose monitoring (CGM) was used throughout the study to observe 24 h glucose responses in accordance with recorded dietary information. The sensor was inserted into the back of the upper non-dominant arm and scanned to confirm data were ready to record. Participants returned every two weeks to have a new CGM sensor attached. Due to sensor error or poor application, some glucose data are reported as missing.Objective scores for the rates of adherence to the TRE protocol were agreed by the research team. Adherence was evaluated using CGM data according to whether they met the following criteria: (i) the eating window after baseline was reduced by 3 h and was ≤12 h in total; (ii) first and last eating times were delayed and advanced by 1.5 h in accordance to baseline mean times; (iii) glucose increments began ±30 min within the self-reported first and last eating times. Controls were considered non-adherent if the reported eating window was <12 h. The first and last daily eating occasions were identified when glucose levels were elevated by ≥1 mmol/L for ≥1 h or longer.An example of how adherence and non-adherence was calculated is demonstrated in Adherence was coded as Yes/No for each 24 h period in an Excel spreadsheet. The percentage of adherence was calculated as:  × 100. When CGM data were missing, however, the number of available intervention days was recalculated to determine the number of days adherent.The exit questionnaire aimed to explore and quantify subjective rates of adherence to the protocol. For the TRE group, the questionnaire was designed to investigate perceived adherence to the 1.5 h morning delay and evening advance in eating times, and overall compliance with the 3 h reduction in habitual eating windows. For the control group, the exit questionnaire aimed to investigate their anticipated adherence if they had been selected to take part in the TRE group. An example of the questionnaires and the Likert scale responses used for both groups can be found below.Participants assigned to the TRE group responded to the following questions to mark their perception of adherence: (i) How did you find the TRE intervention? (Extremely difficult/Somewhat difficult/neither easy nor difficult/Somewhat easy/Extremely easy); (ii) On how many days of the week were you able to comply with the 1.5 h delay to the timing of first energy intake in the morning? ; (iii) On how many days of the week were you able to comply with the 1.5 h advance to the timing of last energy intake in the evening? ; (iv) Were you able to reduce your eating window by 3 h overall? .For the control group, participants were asked to estimate their level of adherence if they were assigned to follow the TRE intervention using the same response frames as the TRE group: (i) How do you think you would find the TRE intervention? (ii) On how many days of the week do you think you would be able to comply with the 1.5 h delay to the timing of first energy intake in the morning? (iii) On how many days of the week do you think you would be able to comply with the 1.5 h advance in the timing of last energy intake in the evening? (iv) Do you think you would be able to reduce your eating window by 3 h overall?In a semi-structured interview format, participants were asked to give feedback on their overall experience and attitudes towards following TRE. Participants in the TRE group were asked to provide an assessment of their adherence to the morning delay and evening advance in the timing of energy intake. Participants were encouraged to reflect on the factors that either enabled or restricted them from adhering to TRE, along with comments on their overall level of adherence to the protocol. Participants from the control group were asked these questions hypothetically to investigate their perceived likelihood of adherence to TRE. An overview of the exit interview questions can be found in Participant preference for TRE was assessed through the responses to the exit questionnaire.The exit questionnaire aimed to review the preference for TRE in participants from both groups. The questionnaire investigated post-study plans to follow TRE, how many days participants can realistically follow TRE, and the duration of restriction that could reasonably be adhered to. The questionnaires attributed to measuring the preferences towards TRE and the answers available on a Likert scale can be found below.TRE group participants were asked the following questions to explore preference for TRE: (i) Do you plan to continue with following TRE? ; (ii) Realistically, how many days a week do you think you could follow TRE on? (0/1/2/3/4/5/6/7); (iii) Please select the duration of time that you could consistently adhere to in terms of restriction to your daily eating window. (<0.5 h/0.5–1 h/1.1–2 h/2.1–3 h/3.1–4 h/>4 h).Control group participants were asked the following questions to explore preference for TRE: (i) How many days a week would you consider following TRE? ; (ii) Realistically, how many days a week do you think you could follow TRE on? (0/1/2/3/4/5/6/7); (iii) Please select the duration of time that you could consistently adhere to in terms of restriction to your daily eating window. (<0.5 h/0.5–1 h/1.1–2 h/2.1–3 h/3.1–4 h/>4 h).t-test to review any differences in subjective and perceived rates of adherence to and preferences for TRE between both groups. A chi-squared analysis was run to test the distribution of sex ratio between groups. A two-way repeated measures ANOVA was carried out to observe the effects of TRE by time, by group, and by time and group interaction on changes to first and last eating occasions and duration of the eating window. A Bonferroni test was carried out to identify in which group and between which different time points of the intervention did a significant change occur.Statistical analysis was undertaken using SPSS . Exit questionnaire responses were analyzed using an independent samples Interviews were recorded and transcribed in Microsoft Teams and were edited post-interview using Microsoft Word. The transcriptions were thematically analyzed to identify the key factors describing the experienced and anticipated positives and negatives of following TRE. In addition, participants from both groups identified some of the experienced and anticipated barriers to, and incentives towards, TRE.n = 11; 68%), Asian British , black, black British, Caribbean or African , or Middle Eastern . Half of the participants were aged between 19 and 23 years old . The remaining participants were aged between 25 and 46 years old .In total, 16 participants completed the intervention and were included in the analysis . An overview of the group characteristics can be found in p = 0.065). A post-hoc Bonferroni test revealed there was a significant delay to the timing of first energy intake in the morning between the end of baseline and after 2 weeks of intervention (p = 0.025) in the TRE group; however, the delay to morning eating time between baseline and week 5 of intervention was non-significant (p = 0.092). Overall, TRE in the intervention group had a significant effect on advancing the time of last energy intake in the evening (p < 0.001) and reducing the overall eating window between groups (p < 0.001). Specifically, post hoc analysis identified a significant difference to the timing of last energy intake of the day between the end of baseline and after 2 weeks of intervention (p < 0.001) and baseline and week 5 of intervention (p = 0.006) in the TRE group. The overall eating window in the TRE group was significantly reduced after the baseline phase at 2 weeks (p < 0.001) and 5 weeks of intervention (p < 0.001). A summary table with average eating times can be found in Adherence to the protocol was first analyzed using the self-reported time-stamped dietary assessment to estimate the average time of first and last energy intake of the day, and overall eating window at baseline and during the intervention.  evening B (p < 0.n groups C (p < 0.n = 1) and control group participants (n = 3).The use of CGM devices did not reveal any significant difference between groups in rates of adherence by time, group, or group × time effect, despite lower rates of adherence recorded at 2 weeks and 5 weeks in the TRE group. Based on the adherence rates listed in The data measuring adherence from the exit questionnaires are summarized in Although not a direct comparison, the results in n = 6, control n = 4) took part in the exit interviews expressing their views on TRE, adherence to the protocol, the positives and negatives of following TRE, their plans for future adherence, and the incentives or barriers to adopting TRE into their daily routines.A total of 10 participants (TRE n = 5). Factors that helped participants to adapt to the morning delay included taking breakfast to work (n = 1), working from home (n = 1), and not having the desire to eat in the morning (n = 1). Two participants found delaying morning energy intake challenging because they required energy to start their day (n = 1), and because of lectures in the morning (n = 1). Those who found it easier to advance in the evening stated this was because they cut out late-night snacking (n = 1), or they were able to eat an evening meal with their children (n = 1). Two of the participants in the control group said they would find the morning delay easy, but due to habitual snacking in the evening, they envisaged restricting this could be problematic. A control group participant indicated they would find it difficult to advance the last energy intake in the evening due to socializing or work commitments.The majority of TRE participants found the morning delay easier to adhere to  stated they had tried to continue with following TRE, although two did concede that family visiting and work schedules had impacted their ability to adhere. Most of the participants stated that they would prefer to follow TRE during the weekdays, and then have flexibility over the weekends (n = 7). Two participants concluded they would find it easier to implement TRE on the weekends because they have better flexibility then, as opposed to during the working week. If the participants were to follow TRE again, most participants said they would prefer to delay the first eating occasion so as to enable greater flexibility in eating in the evening (n = 7). Just two participants stated they would prefer to advance the last energy intake of the day. One participant reported they would not be inclined to ever follow TRE because they do not want to be restricted with when they eat, and would prefer to use their own self-awareness to determine when they eat.Encouragingly, a few participants (n = 4), with one participant benefiting from the structure of routine it provided. Improvements to sleep quality (n = 2), better digestion (n = 1), reduced bloating in the morning (n = 1), maintaining hydration (n = 1) and reports of having more energy in the morning were some of the positives of following TRE. Several participants from both groups described how taking part in the study made them consider their overall dietary quality, and influenced their eating habits by reducing their intake of highly palatable foods (n = 3).TRE was reported as being easy to follow (n = 5), work commitments (n = 3), evening leisure activities (n = 1), living arrangements (n = 1) and family life (n = 1). Another negative was the amount of planning that adhering to TRE required, as regards the consideration of when their routine enabled them to eat throughout the day, and whether this aligned with their desire to eat (n = 3). A lack of flexibility, modifications to eating behavior, fatigue, and unpredictable shift patterns resulting in skipped meals or snacking on highly palatable foods were highlighted as some of the negatives of following TRE.There were commonalities amongst the participants as regards the challenges with following TRE, including social events (n = 4) or the prevention of unwanted weight gain (n = 2). Participants from both groups described how the possibility of improved sleep would motivate them to take up TRE (n = 3). Glucose regulation (n = 1), benefits to gut health (n = 1) and mental health (n = 2), reduced alcohol intake in the evening (n = 1), and the avoidance of unhealthy foods (n = 2) also incentivized participants to follow TRE.The main incentive that participants shared was for induced weight loss (n = 5) and social activities (n = 5). One participant identified social media as a negative barrier that would prevent them from adhering to TRE. Specifically, they noted that food advertisements seen in the evening influence their desire to eat, and increase the likelihood of them eating. Living arrangements and, in particular, house sharing with friends were perceived as barriers to TRE, indicating that kitchen availability impacts when they could cook an evening meal (n = 2). Interestingly, one participant mentioned how an overly complicated TRE protocol may become a barrier itself if it is hard to follow. A lack of motivation or falling out of routine were also noted as potential barriers to adherence (n = 1). Stress and using food as a coping mechanism were also highlighted as potential barriers to maintaining TRE, especially in the evenings. Visiting family was noted as a factor that restricted one participant from continuing with adhering to TRE once the study was over.The factors acting as the greatest barriers to TRE uptake were work commitments  may be useful in identifying adherence to TRE. Our simple exit questionnaire has generated a wealth of quantitative data on self-reported adherence to the morning and evening constraints, and overall adherence to the 3 h reduction in the eating window. Our qualitative approach to exploring participant experiences helped us accumulate useful insights into the strengths and limitations of TRE, along with identifying some of the novel barriers and incentives that would motivate individuals to take up TRE.The limitations of the study included its symmetrical restriction of the eating window, which negates some of the preferences participants may have for morning or evening eating. Future research is required to ascertain to what extent preference towards early or delayed TRE are driven by chronotype. The small sample size and short study duration are noteworthy limitations of the study. Moreover, the small timeframes used to capture dietary intake over 7 days at baseline or 4 days during two separate intervention stages may not accurately reflect the dietary habits of the participants. Although CGM data provide a useful insight into glucose excursions in relation to dietary intake, some physiological responses may have gone undetected in response to low-carbohydrate meals or snacks. Furthermore, some other excursions may have been undertaken as a physiological response to waking . The numSelf-reported food diaries may provide a useful account of eating times, but accuracy in reporting is crucial for this to be successful. CGM devices used in cohesion with self-reported diaries may help with accurately reviewing adherence to TRE regimes with specified eating and fasting times. Future studies with larger sample sizes and longer intervention periods are required to investigate the effects of a self-selected TRE regime on rates of adherence.Despite being proposed as a simplified dietary approach, adherence to TRE is affected by lifestyle and behavioral factors. Overcoming these barriers to adopting TRE warrants a personalized approach in order to successfully incorporate TRE in relation to scheduling, social and family activities. The flexible scheduling of TRE is crucial to enabling individuals to self-select an eating window, along with a start and end time to eating occasions, which will maximize the likelihood of sustaining adherence.