Background: Magnetic resonance-guided high-intensity focused ultrasound (MRgHiFUS) has evolved into a viable ablative treatment option for functional neurosurgery. However, it is not clear yet, how this new technology should be integrated into current and established clinical practice and a consensus should be found about recommended indications, stereotactic targets, patient selection, and outcome measurements.Objective: To sum up and unify current knowledge and clinical experience of Swiss neurological and neurosurgical communities regarding MRgHiFUS interventions for brain disorders to be published as a national consensus paper.Methods: Eighteen experienced neurosurgeons and neurologists practicing in Switzerland in the field of movement disorders and one health physicist representing 15 departments of 12 Swiss clinical centers and 5 medical societies participated in the workshop and contributed to the consensus paper. All experts have experience with current treatment modalities or with MRgHiFUS. They were invited to participate in two workshops and consensus meetings and one online meeting. As part of workshop preparations, a thorough literature review was undertaken and distributed among participants together with a list of relevant discussion topics. Special emphasis was put on current experience and practice, and areas of controversy regarding clinical application of MRgHiFUS for functional neurosurgery.Results: The recommendations addressed lesioning for treatment of brain disorders in general, and with respect to MRgHiFUS indications, stereotactic targets, treatment alternatives, patient selection and management, standardization of reporting and follow-up, and initialization of a national registry for interventional therapies of movement disorders. Good clinical evidence is presently only available for unilateral thalamic lesioning in treating essential tremor or tremor-dominant Parkinson's disease and, to a minor extent, for unilateral subthalamotomy for Parkinson's disease motor features. However, the workgroup unequivocally recommends further exploration and adaptation of MRgHiFUS-based functional lesioning interventions and confirms the need for outcome-based evaluation of these approaches based on a unified registry. MRgHiFUS and DBS should be evaluated by experts familiar with both methods, as they are mutually complementing therapy options to be appreciated for their distinct advantages and potential.Conclusion: This multidisciplinary consensus paper is a representative current recommendation for safe implementation and standardized practice of MRgHiFUS treatments for functional neurosurgery in Switzerland. Magnetic resonance-guided high-intensity focused ultrasound (MRgHiFUS) provides a novel ablative treatment option currently applied mainly for essential tremor (ET) and to a lesser extent for tremor-dominant Parkinson's disease (PD) , 2. HoweFunctional neurosurgical treatment of brain disorders requires multidisciplinary collaboration among various clinical specialties. Apparent elegance of MRgHiFUS technology and simplicity of intervention processes make it easy to forget about the complexity of the pathophysiology of tremor. With the expected sharp rise in MRgHiFUS intervention numbers and given that MRgHiFUS is only at the beginning of its technological maturation curve, it is important that all specialists involved in care and management of patients suffering from neurological and psychiatric disorders have access to up-to-date information about the relevant technical and medical peculiarities of MRgHiFUS-based interventional therapy.Many issues concerning application of MRgHiFUS in functional neurosurgery remain controversial, whereas currently no clear guidelines regarding MRgHiFUS treatment exist. In order to aggregate and harmonize current clinical knowledge and experience regarding state-of-the art therapy of various movement and pain disorders with a special focus on MRgHiFUS, two complementary workshops and consensus meetings in Zurich as well as one additional online meeting were held, gathering participants from most of the specialized centers for the treatment of movement disorders in Switzerland.Here we present the consensus statement on clinical application of MRgHiFUS for functional neurosurgery as formulated by the newly established Swiss MRgHiFUS Working Group.Although focused ultrasound has been applied as a neurosurgical tool for more than 70 years , only thThe only commercially available transcranial MRgHiFUS system  consists of a phased array piezoceramic helmet with 1,024 elements that can precisely focus ultrasound waves into circumscribed brain locations. A 1.5 or 3 Tesla MRI scanner  for target verification, real-time thermal feedback and treatment planning, guidance and lesion confirmation complete the MRgHiFUS equipment.MRgHiFUS has emerged as a neurosurgical option mainly for the therapy of movement disorders , 2 but aIn Switzerland, three centers, the University Hospitals Zurich and Geneva, and the SoniModul AG Solothurn, are offering MRgHiFUS interventions in a clinical setting. In Zurich, clinical applications of MRgHiFUS in the brain have been studied since 2006 and, as early as 2009, the first focused ultrasound interventions in the central lateral nucleus of the thalamus for chronic neuropathic pain as a pilot application were reported .Motivated by the installation of a new clinical treatment system for MRgHiFUS at the University Hospital Zurich and the imminent addition of MRgHiFUS functional neurosurgery interventions into the reimbursement catalog of the Swiss public health system, the Swiss Movement Disorders Society (SMDS) board decided to develop a set of guidelines for the reintroduction of basal ganglia ablative functional neurosurgery in Switzerland with MRgHiFUS technology. The guidelines should build on the experience of movement disorders specialists from classical radiofrequency lesioning, deep brain stimulation (DBS) and MRgHiFUS and accordingly, a workgroup was formed (Swiss MRgHiFUS Working Group) involving neurologists, neurosurgeons and medical physicists from all over Switzerland.Invitations to participate in the meetings were sent to all involved experts in Switzerland (1) all chairmen of neurological departments of Swiss university hospitals and cantonal hospitals, (2) all heads of movement disorders units in Switzerland, (3) all neurologists and neurosurgeons already performing MRgHiFUS treatments in Switzerland, and (4) all members of the SMDS (see Participants for more details about the invitation process). For workshop preparation, participants received a booklet containing a list of all participants and their affiliations, a comprehensive literature research and overview over the pertinent publications on MRgHiFUS and basic information about MRgHiFUS technology (see Selection of literature for literature selection). The literature was selected by the organizing committee, LS, GK and AK, and comprised the literature cited in this manuscript. Furthermore, they were invited to provide suggestions for key questions and topics to be discussed. The objectives of the meetings and the consensus statement were to provide a specialist framework and guidance to those embarking on MRgHiFUS therapy including patient selection, indication, procedure, aftercare, alternatives, and educational opportunities, to discuss and highlight the potential, challenges, benefits and distinctive risks of MRgHiFUS, and to promote scientific activities and participation into clinical trials and registries.All known experts in the field of movement disorders in Switzerland were contacted via direct e-mail by the first author (LS). In total 19 experts from 15 centers and members of 5 societies across Switzerland participated in the two workshops and consensus meetings and the online meeting, comprising 14 neurologists, 4 neurosurgeons and 1 medical physicist. Nine of the participants had personal experience with MRgHiFUS treatment. Summarizing the Hôpital Fribourgeois (HFR), Hôpitaux Universitaires de Genève (HUG), Center Hospitalier Universitaire Vaudois (CHUV), Kantonsspital Aarau (KSA), Universitätsspital Zürich (USZ), Luzerner Kantonsspital (LUKS), Kantonsspital St. Gallen (KSSG), Neurocenter of Southern Switzerland EOC Inselspital Bern, Zentrum für Neurorehabilitation Zihlschlacht, Kliniken Valens, and Kinderspital Zürich, all regions of Switzerland are represented .The selection of the literature followed PRISMA guidelines and is illustrated in The workshops and consensus meetings took place in Zurich on 2.12.2019 and 17.01.2020, respectively, in the form of moderated, interdisciplinary roundtable discussions. A third meeting was held online on 26.07.2021 to comply with the hospital regulations of the Covid-19 pandemic. Meeting minutes were recorded by the first author (LS) and reviewed by all participants.The first meeting was moderated by a Swiss journalist, former redactor and director of a large Swiss media company and advisor for several Swiss cantonal health directors, who was booked for this occasion by the SMDS board to guarantee a fair and balanced discussion.The process of the consensus meetings is illustrated in During October 2019 and January 2020, the organizers drafted a meeting agenda including a list of relevant topics and questions to be discussed by the new Swiss MRgHiFUS Working Group. Based on a comprehensive literature review, special emphasis was put on current experience and practice, and areas of controversy regarding clinical application of MRgHiFUS for functional neurosurgery. All participants were asked to provide additional topics for the conference which were disseminated among the group together with the relevant literature in advance.Should lesioning  be considered a viable treatment option for movement disorders despite the success of reversible DBS?After the introduction of DBS, Professor Alim-Louis Benabid, one of the pioneers of DBS, claimed he would never perform a lesion again in a diseased brain . However, initial clinical success and technological potential of MRgHiFUS warrant reopening a therapy field that was considered closed.Technically, the innovations distinguishing MRgHiFUS from radio-frequency ablation (RFA) is (i) incisionless delivery of therapy, (ii) intraoperative image guidance, (iii) intraoperative ablation control, and (iv) progressive and better-controlled ablation. Together, they enable a more precise ablation of small functional targets deep in the brain which, owing to improved anatomical and pathophysiological understanding, are much clearer defined than in the initial days of lesioning. Compared with radiosurgery , thermal lesions created by MRgHiFUS become immediately effective and allow both intra-operative image-based and neurological assessments of induced lesions.While physiologically thermal lesions created by RFA and MRgHiFUS are similar, they differ in spatial geometry with MRgHiFUS lesions being much sharper delineated than RFA. Therefore, it will be challenging to translate previous clinical experience and literature from RFA interventions toward new MRgHiFUS lesioning. Most importantly, modern image guidance will help reduce adverse effects from mistargeting and will require an unbiased reevaluation of stereotactic targets both in terms of therapeutic benefit and in terms of associated risks.The effects of MRgHiFUS on brain tissue are well-known and it could be histologically demonstrated, that an ablative effect is reached beyond a specific threshold over 50°C . De VlooThe workgroup unequivocally recommends further exploration and adaptation of MRgHiFUS-based functional lesioning interventions as a complement and possible alternative to existing treatment approaches such as DBS and confirms the need for outcome-based standardized evaluation of these approaches based on a unified registry.For which indications should MRgHiFUS be included into standard of care; which indications should require further evaluation in clinical trials and for which indications should application of MRgHiFUS be discouraged?Technically, MRgHiFUS can create small thermal lesions in the thalamus and subthalamic area. Larger targets, or targets that are located deeper or more peripherally, could potentially also be accessed, but might need prolonged treatment times or adapted patient positioning. Obvious candidates for MRgHiFUS treatments are therefore patients with most indications that are currently treated interventionally, especially with DBS:tremor (ET and PD tremor) by ventral intermediate thalamic nucleus (VIM) lesioningPD with fluctuations and dyskinesias by unilateral subthalamic nucleus (STN) lesioningdystonia by bilateral globus pallidus internus (GPi) lesioningchronic neuropathic pain by centromedian thalamus (CMT) and central lateral thalamus (CLT) lesioningpsychiatric disorders such as OCD with anterior limb of internal capsule (ALIC) lesioningRecent randomized controlled studies (RCTs) have shown that MRgHiFUS treatment works well for ET , 12, 13.Because of its proven effectivity and low complication rates, MRgHiFUS could develop from a second-choice treatment option for patients with contraindication to DBS into a preferred option for a very specific sub-group of patients . F.i. patients with only mild tremor severity  might be a good choice as well as patients of advanced age (too old for DBS), or patients with very asymmetric tremor involving the dominant hand.With ET being mainly an isolated disease with typically only low neurological comorbidity, MRgHiFUS could be offered by specialized multidisciplinary interventional treatment centers in an easier way.Unilateral MRgHiFUS lesioning of VIM for tremor-dominant PD has received FDA clearance in 2018 and seems to be a viable treatment option . Involvement of a specialized neurological department, offering alternative treatments such as conservative therapy, drug delivery pumps or DBS, is mandatory. Beside neurological, also neuropsychological and neuropsychiatric expertise needs to be integrated, typically in the form of multidisciplinary boards.For the treatment of dystonia, only a single case of MRgHiFUS ablation in the ventral oral anterior thalamic nucleus (VOA) has been reported so far. Although it might be a potential indication, further studies are needed despite the technical and medical challenges involved in conducting the intervention . Most prAblative treatment of dystonia has an interesting potential. The key issue will be the safety of the procedure. Currently, only very little experience exists with MRgHiFUS pallidotomies, therefore the risk/benefit ratio may not be estimated properly. Thus, treatments should only be performed within randomized controlled trials. Based on the paucity of experience so far , MRgHiFUS treatment for dystonia is currently not recommended for clinical application.Therapy-resistant pain was one of the first indications treated with MRgHiFUS . The treLesioning of the VPM and VPL could be associated with sensory deficits and therefore, the CMT should be preferred. Alternatively, the lateral portion of the cuneocerebellar tract (CCT) could be used.No general recommendation can be made for patients suffering from chronic pain. Indications for treatment including MRgHiFUS should be restricted to the confines of RCTs and should be made within the framework of an interdisciplinary pain board.Regarding epilepsy therapy and targeting of e.g., the anterior thalamic nucleus (ANT) with MRgHiFUS, no relevant data exists, no experience was reported, and this therapy therefore currently remains widely hypothetical and experimental. Lesioning the ANT might be a promising option in therapy-resistant epilepsy. Still, this would be a completely novel treatment and should only be performed within the confines of a study protocol.Beside ET and tremor-dominant PD, MRgHiFUS treatments for movement disorders and other brain disorders in general are still under clinical evaluation or even considered experimental.For cases of intractable OCD, DBS as well as ablative neurosurgery have been tried as escalation therapies . Among tTreatment of intractable OCD with ventral MRgHiFUS capsulotomy might be efficacious. So far, only very small studies have been published and a sufficiently long follow-up seems to be crucial to correctly evaluate the efficiency of the treatment. Furthermore, ventral capsulotomy must be evaluated against DBS, for which more experience is published from randomized controlled trials. At the given time, treatment of intractable OCD with MRgHiFUS should be restricted to the confines of RCTs.In which clinical settings should MRgHiFUS be performed?There is presently no evidence available regarding required clinical settings for MRgHiFUS.Therapeutically, MRgHiFUS is an incisionless, ablative procedure involving low but real risks of neurological complications and adverse effects due to unintentional effects to non-targeted functional units, unexpected side effects of network modulation and, most significantly, microscopic and macroscopic bleeding. It is therefore considered essential that MRgHiFUS treatments are conducted under supervision of experienced functional neurosurgeons and specialized neurologists. Furthermore, centers offering MRgHiFUS should have an adequate access to emergency neurosurgery and anesthesiology, and neurointensive or intermediate care. This means that centers offering MRgHiFUS treatment must be able to diagnose and treat emerging complications autonomously by providing permanent availability of experts from different disciplines and professions like neurology, neuroradiology, neuro-intensive care, and physiotherapy. Furthermore, an adequate patient selection requires a multidisciplinary team for the evaluation of the patient before the intervention, including assessment of motor and non-motor symptoms, e.g., cognitive impairment not allowing the adequate communication during MRgHiFUS.Hence, MRgHiFUS treatment should only be conducted in clinical settings where multiple treatment options can be offered and where therapy options can be evaluated by multidisciplinary movement disorder boards, which usually are located at specialized centers and university hospitals.Which patient groups could benefit most from MRgHiFUS?So far, all randomized controlled studies on MRgHiFUS selected patients who were in one way or another not suitable for DBS , 2.During its clinical introduction, MRgHiFUS has been considered an interventional treatment option mainly for patients who presented with contraindications for DBS. Such cases could include patients not tolerating electrode implants, patients not able to adequately handle the DBS devices, or patients with brain pathologies not eligible for insertion of electrodes and DBS. With clinical acceptance of the method, patient selection can start focusing on individual risk-benefit evaluations between MRgHiFUS, DBS and other modalities. Therapeutically, the main difference between MRgHiFUS and DBS-based modulation of neuronal circuits is the permanent nature of thermal lesions, which cannot be altered once they are set, respectively, can only be increased in size if not effective enough. Accordingly, patient selection should focus on patients where post-operative optimization of intervention outcome is not a priority or where the underlying pathology is not expected to require repeated adaptation of network modulation in the near future such as PD. In terms of indications, ET seems a good indication for MRgHiFUS because it mostly does not follow a course of multi-systemic progression such as PD and even improved albeit residual tremor can result in good quality of life (QoL). In terms of patient cohorts, MRgHiFUS could be an attractive option for elderly patients in general, where improving prominent symptoms might provide significantly increased QoL within the relevant time horizon. It should be noted that the lack of post-interventional effect tuning goes together with reduced clinical time and effort and accordingly reduced financial burden for the healthcare system.Which safety procedures and contraindications should be respected to ensure maximum patient safety during the procedures?The efficiency of focusing acoustic energy into a circumscriptive target inside the skull for thermal ablation depends strongly on the acoustic properties of the patient skull which have high inter-individual variability. A rough estimate of expected focusing quality is provided by the skull density ratio (SDR), which is calculated from preoperative computed tomography data of the patient's head. Typical SDR values for Caucasian patients are between 0.45 and 0.80, whereas SDR should not have values below 0.35 for thalamic targets and 0.4 for extra-thalamic targets such as the GPi. Low SDR values imply poor focusing quality, i.e., less precise lesion definition and compensating for associated low focusing gain will require higher acoustic energies, leading to more skull heating with the risk of unintended damage to skull bone-marrow. Ultimately, under-treated stereotactic targets because of insufficient total thermal dose are a risk . PatholoKnowing and respecting ultrasound physics and the limits of the technique is essential to prevent possibly harming the patients either by insufficient treatment, which then needs to be repeated with other techniques, or treatment side effects. Thus, MRgHiFUS should only be performed in a center providing the needed technical expertise and support.To the best of the authors' knowledge, there is no data available regarding the need for cooperation of the patients during MRgHiFUS treatment.MRgHiFUS interventions are conducted by repeating sonications with stepwise increased energy levels and resulting peak temperatures at target to allow constant evaluation of intended treatment effects and unintended side effects. Patient cooperation for neurological testing is therefore essential and reliable two-way communication between treatment team and patient is required.Treatments under general anesthesia would technically be possible, but the lack of neurological testing as a means of target verification might increase the risk for complications due to mistargeting or excessive lesion size. Therefore, such treatments are discouraged for the time being.The authors are not aware of any published experiences regarding MRgHiFUS under effective anticoagulation or antiaggregation. Microbleeds in the core of the lesion, on the other hand, are regularly observed by neuroimaging , 31.Although MRgHiFUS does not require a skin incision or skull trepanation, the energy applied to the skull and brain via ultrasound waves is invasive. Associated risks include cavitation effects to blood-vessels and to the margins of intracranial cysts like Virchow-Robin spaces. Consequently, though the risk of intracranial hemorrhage may be low, normal coagulation status and absence of any antiaggregatory or anticoagulative therapy must be assured preoperatively. The risk for convulsive epileptic seizures during the treatment should be low as such an event could endanger the patient when occurring with the head fixed in a stereotactic frame. Furthermore, the risk of severe nausea and vomiting due to sonic stimulation of somatosensory pathways in the thalamus should be considered and addressed properly. Administration of antiemetics prior to the treatment and ensuring a slow and careful passage of the patient into and out of the magnetic resonance imaging (MRI) scanner's static magnetic field are preventive measures. The presence of an anesthesiologist to immediately treat severe nausea by providing suction of the oral cavity in case of emesis is advisable as draining the water and taking the patient out of the treatment device may take too long. Lastly, lower-back pain, camptocormia, a short neck and obesity must be considered during evaluation of patients for MRgHiFUS therapy.Only few scientific evidences are available about MRgHiFUS contraindications due to local pathology. Yang et al. published a technical note and report of one patient who received MRgHiFUS thalamotomy for ET despite an implanted VP shunt. They found no adverse effects and only a small number of elements which had to be switched off to prevent damaging the shunt components .Obviously, all contraindications for high-field MRI apply, such as MRI-incompatible medical implants, claustrophobia, inability to lay prone for extended periods of time, etc. should be thoroughly considered. In addition, scars in the scalp may lead to trapped air bubbles on the skin surface hindering the treatment. Old trepanations, craniotomies or cranioplasties must be also considered during intervention planning and frame fixation to prevent accidental heating, skin burns, off-target tissue ablation and skull fractures or impressions. For cranial implants like CSF shunts, there is limited experience available so far, although such implants, if generally MR compatible, might not necessarily be a strict contraindication. The safety of the patients should be considered extremely carefully, cases be published and unusual conditions registered in a registry.Should MRgHiFUS lesioning apply the same stereotactic targets as currently established for DBS?There is a lack of comparative studies of DBS, RFA, MRgHiFUS, and radiosurgical ablations, but in a thorough meta-analysis of the published results of all these techniques, Dallapiazza et al. found comparable response rates and complication rates, indicating that indeed the same targets which apply for DBS may apply for MRgHiFUS .Functionally, MRgHiFUS lesions and DBS should elicit comparable neurological effects if placed identically in spite of the different mechanisms of action and even though further studies are needed to understand better specific mechanisms of action of DBS. However, the methods differ in their flexibility to access and cover functional targets and in their risk profiles for inducing side effects. If targets are within the accessible volume dictated by ultrasound physics, MRgHiFUS theoretically can create lesions of any size or form by combining successive point lesions and therefore could paint anatomical structures for optimal target coverage. However, doing so can be very time consuming and transient edema associated with thermally induced lesions might lead to intra- and postoperative adverse effects that resolve only after days or weeks. In addition, off-target tissues could accumulate enough thermal dose during the process to get permanent thermal damage. DBS on the other hand, can adapt the geometry of the stimulation field around the electrode to optimize treatment effect and over time can follow disease dynamics. Furthermore, new DBS technique using “closed loop” modulation of the stimulation (intensity and duration) could better adapt to symptoms variability , 34. StiThe efficacy of MRgHiFUS thalamotomy of the VIM in ET was shown in randomized controlled trials , 14, 35.Regarding essential tremor, in principle the same targets used for DBS could be also applied for MRgHiFUS. The VIM surely represents a suitable and established target with a favorable risk profile . AdditioBond et al. published the results of a randomized controlled trial on MRgHiFUS thalamotomy of the VIM for tremor dominant PD in 2017, showing good tremor response and low side effects .Aside from unilateral VIM lesioning for tremor-dominant PD, lesioning of the pallidothalamic tract (PTT), as an alternative to VIM, could lead to decreased responsiveness to L-Dopa medication. Therefore, even unilateral MRgHiFUS of that target may lead to freezing, dysarthria, and dysphagia in patients at risk.Lesioning the GPi is difficult as it is located more lateral and closer to the outer margin of the brain volume less accessible to the MRgHiFUS system. Patients with relatively low SDR (below 0.4) might not qualify for such a treatment.In summary, MRgHiFUS lesioning cannot be presently applied to all stereotactic targets as currently established for DBS. Only CCT and VIM can be considered as established targets and all others should be further investigated in prospective controlled clinical studies.Experiences with and risk evaluation for bilateral ablative treatment in terms of recommended indications and temporal staging?Technically, MRgHiFUS allows for bilateral treatments, both within the same treatment session, or in a staged manner during two consecutive sessions. Reasons for simultaneous bilateral treatments could be to increase therapeutic effects, f. i. for neuropathic pain, or to save patients from the burden of multiple hospitalizations. Staged bilateral treatments could be considered to extend therapeutic benefit to the contralateral side or in case of axial symptoms like head or voice tremor. In addition, multiple functional targets could be applied to try regulating pathologic brain function via multiple pathways.Current literature from RFA and DBS describes potential risks of bilateral treatments which are considered also relevant for MRgHiFUS . In partIf applied, treatments should be staged at least three, better even 6 months after initial MRgHiFUS and the second treatment only considered in absence of side effects from the initially treated side. Overall, bilateral treatments are still considered experimental. Risk-benefit evaluation will depend strongly on indications, targets to be assessed and symptomatic patient status.How should pre- and post-interventional examinations and assessments for MRgHiFUS patients be standardized?There is no relevant data and evidence available on this topic.For reasons of comparability and integration of registries pre- and post-interventional evaluations of MRgHiFUS patients should be carefully standardized using state-of-the art assessment tools and internationally accepted norms. Wherever applicable, MRgHiFUS and DBS interventions should be assessed using identical protocols except for intervention-specific data.What should we expect for long-term treatment outcomes after MRgHiFUS?MRgHiFUS lesioning is still a relatively new treatment and consequently, knowledge about long-term treatment outcome is limited. The longest follow-up data published so far demonstrated efficacy for up to 4 years , 35, 43.Continued evaluation of the patients who were enclosed in RCTs to learn about long-term efficacy of MRgHiFUS treatment is one important step but will not provide generalizable data easily. The method will also have to prove its worth in comparison with the treatment gold-standard. The best way to provide the required data for these observations is to include as many cases as possible in national or even international registries. One problem still unsolved is the heterogeneity of the used endpoints in studies and registries.The primary objective of the workshops and consensus meetings was to discuss in-depth the current status of MRgHiFUS and to provide guidance to those involved in MRgHiFUS therapy. Secondarily, it was aimed to evaluate the potential benefits, specific challenges, and distinctive risks of this novel technology, and to promote further clinical and scientific exchange of experience and outcome data. Finally, by establishing the Swiss MRgHiFUS Working Group, an interdisciplinary network for MRgHiFUS and future clinical and scientific collaboration was created.This article describes issues that are relevant and of potential interest not only for MRgHiFUS experts, but also for general practitioners, neurologists and neurosurgeons and provides consensus-based recommendations in key areas of uncertainty. A number of questions currently remain unaddressed or could not be completely answered and therefore corresponding studies are highly desired. A priority for this research regards experimental indications such as chronic neuropathic pain and OCD. Carefully designed clinical studies will be required to optimally integrate these interventions into currently available treatment strategies.It should be noted that the current recommendations were formulated by medical professionals practicing in the setting of the national Swiss healthcare system which might affect their generalizability to a broader patient population outside Switzerland. Still, Switzerland was among the first countries to introduce MRgHiFUS treatment and has already longstanding experience in that field. One more important limitation of the present consensus statement is that it does not provide a similarly high level of evidence such as a systematic review but relies on the experiences and opinions of a group of experts. The members of the Swiss MRgHiFUS workgroup think that at the given time the number of available RCTs is still too small to justify a systematic review. The consensus statement deemed to be the proper method as it added the experience of a group of experts, which is representative for Switzerland, to today's available evidence. As the number of publications on MRgHiFUS is growing exponentially, the consensus statements were outdated already when the manuscript was first drafted. To provide a timely statement, a third conference was held, which dealt with recent developments and publications.It is expected that the evidence included in the article and consensus-based recommendations will be helpful and provide valuable guidance for clinicians and healthcare professionals involved in the treatment of patients with MRgHiFUS.These recommendations  are baseIt also became apparent that DBS and MRgHiFUS should not be treated in separate communities and should not be played off against each other. Rather, they should be adopted as mutually complementing therapy options that should be appreciated for their distinct advantages and therapy potential to further extend the therapeutic spectrum and optimize as well as tailor patient treatment.The original contributions presented in the study are included in the article/LS and AK-L wrote, reviewed, and finalized the manuscript. All authors contributed to the workshop and to the manuscript in co-editing.The consensus meetings and workshops were organized and supported by the SMDS.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 RF declared a past co-authorship with several of the authors LS, CB, FB, PK, and SM 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.


Oropharyngeal squamous cell carcinoma is the most common human papillomavirus (HPV)-associated cancer in the UK, but little is known about the prevalence of oropharyngeal HPV in sexually active teenagers. We investigated reported HPV vaccination coverage  and prevalence of oropharyngeal HPV in sexually active students attending six technical colleges in London, UK.In 2017, we obtained mouthwash samples and questionnaires from male and female students taking part in the ‘Test n Treat’ chlamydia screening trial. Samples were subjected to HPV genotyping.Chlamydia trachomatis infection and 1.5% Neisseria gonorrhoeae and 5.0% were gay or bisexual. Only 47% (50/107) of females reported being vaccinated against HPV 16/18, of whom 74% had received ≥2 injections. HPV genotyping showed three mouthwash samples  were positive for possible high-risk human papillomavirus (HR-HPV), one  for low-risk HPV 6/11, but none  for HR-HPV. Four samples  were positive for HPV16 using a HPV16 type-specific quantitative PCR, but these were at a very low copy number and considered essentially negative.Of 232 participants approached, 202 (87%) provided a mouthwash sample and questionnaire. Participants’ median age was 17 years and 47% were male. Most (73%) were from black and minority ethnic groups, 64% gave a history of oral sex, 52% reported having a new sexual partner in the past 6 months, 33% smoked cigarettes, 5.9% had concurrent genitourinary Despite the high prevalence of oral sex and genitourinary chlamydia and low prevalence of HPV vaccination, the prevalence of oropharyngeal HR-HPV in these adolescents was negligible. In 2016, oropharyngeal squamous cell carcinoma in men overtook cervical cancer as the most common human papillomavirus (HPV)-associated cancer in the UK.In 2008, a school-based HPV16/18 vaccination programme to prevent cervical cancer was established in England for 12–13 year old girls, with ‘catch-up’ vaccination for those aged 14–18. HPV vaccination may also protect against oral HR-HPV.In 2017–2018, we explored the coverage of reported HPV vaccination , feasibility of obtaining mouth wash samples and prevalence of oropharyngeal HPV in ethnically diverse, sexually active male and female adolescents taking part in the ‘Test n Treat’ chlamydia screening study.The Test n Treat study assessed the feasibility of providing on-site same day testing and treatment for chlamydia to sexually active London further education/technical college students aged 16–24.Under the supervision of a research nurse, each participant gargled 10 mL of saline solution for 30 s and spat it back into the collection container. The container was then shaken to ensure an even distribution of cells and 2 mL was transferred to a Cobas PCR Media tube (for chlamydia/gonorrhoea testing using the urine protocol for the Roche 4800 machine). Samples were kept in a cool bag during the field visit and transferred to a −80 °C freezer the same day.Samples were centrifuged , washed with 5 mL phosphate-buffered saline  and resuspended in 1 mL PBS before nucleic acid extraction.Genotyping for HR-HPV (HPV16/18/31/33/35/39/45/51/52/56/58/59/68), possible HR-HPV (HPV26/53/66/70/73/82) and low risk (LR)-HPV (HPV 6/11) was conducted using a universal probe  and 21 type-specific probes against the indicated genotypes, as previously described,Of 232 Test n Treat participants attending follow-up, 202 (87%) provided a mouthwash sample. Participants’ median age was 17 years, 47% were male and 64% gave a history of oral sex. Six per cent had concurrent genitourinary chlamydia infection and 1.5% had gonorrhoea . They deLess than half  of the female participants reported being vaccinated against HPV (and 5% (5/107) did not know whether they had been vaccinated). All were in age groups eligible for the main vaccination rollout at age 12–13 (n=107) or the ‘catch-up’ vaccination (n=1). A higher proportion of white students reported receiving the vaccination than black and ethnic minority students, 62% (16/26) vs 41% (33/80). Half  of all vaccinated students received three injections, 24% (12/50) received two and 26% (13/50) were not sure how many injections they had received. No one reported only one injection. Of females who said they had not been vaccinated, 60% (31/52) reported attending a sexual health clinic in the previous 6 months.Of the 202 samples, three  were positive for possible HR-HPV, one of which  was also positive for LR-HPV6/11. No samples  were positive for HR-HPV. Four of the 202 samples  were positive for HPV16 using a HPV16 type-specific quantitative PCR, but this was at a very low level  and was considered essentially negative.Chlamydia trachomatis and Neisseria gonorrhoeae, one  was positive for chlamydia .Of 202 valid mouthwash samples tested for Despite high prevalences of oral sex and genitourinary chlamydia/gonorrhoea and low self-reported uptake of HPV vaccination , we found no HR-HPV.This is the first UK community-based study of oral HPV in ethnically diverse, sexually active adolescents. Many previous studies were of mainly white individuals or those attending healthcare facilities.The main limitation of this observational study is a lack of power due to the relatively small sample size which was determined to give adequate precision in the feasibility outcomes of the Test n Treat study.Our prevalences of HR-HPV and HPV16 were lower than the 2.3% and 0.8%, respectively, in under 25s in a systematic reviewLess than half the female participants reported HPV vaccination, which is lower than the average 70% coverage across London. However, half our sample were of black ethnicity, a group who are less likely to be vaccinated despite being at greater risk of both HR-HPV and cervical cancer.These findings could inform health policy. Although these adolescents had high prevalences of genitourinary STIs and engaged in oral sex, we found no cases of oral HPV16 infection. Despite the caveats of a small observational study, our findings are interesting and may partly reflect the impact of HPV vaccination on herd immunity.


Solid organ transplant (SOT) recipients are at increased risk of Human Papillomavirus (HPV) persistent infection and disease. This study aimed to evaluate HPV seroprevalence, cervical HPV prevalence, genotype distribution, and frequency of HPV-related cervical lesions in SOT recipients in comparison to immunocompetent women.®) and squamous intraepithelial lesions (SIL) by liquid-based cytology. A multiplexed pseudovirion-based serology assay (PsV-Luminex) was used to measure HPV serum antibodies.Cross-sectional study including SOT and immunocompetent women aged 18 to 45 years who denied previous HPV-related lesions. Cervical samples were screened for HPV-DNA by a polymerase chain reaction (PCR)-based DNA microarray system (PapilloCheckp = 0.112), but this difference was not statistically significant. High-risk (HR)-HPV was significantly more frequent in SOT than in immunocompetent women . Simultaneous infection with ≥2 HR-HPV types was found in 3.1% of SOT and 0.9% of immunocompetent women. HPV seropositivity for at least one HPV type was high in both groups: 63.8% of 105 SOT and 69.7% of 119 immunocompetent women (p = 0.524). Low-grade (LSIL) and high-grade SIL (HSIL) were significantly more frequent in SOT  than in immunocompetent women  (p = 0.001).125 SOT and 132 immunocompetent women were enrolled. Cervical samples were collected from 113 SOT and 127 immunocompetent women who had initiated sexual activity. HPV-DNA prevalence was higher in SOT than in immunocompetent women (29.6% vs. 20.2%, These results may reflect the increased risk of HPV persistent infection and disease progression in SOT women due to chronic immunosuppression. Cervical cancer (CC) is the fourth most frequent cancer in women in the world, with estimated 570,000 new cases each year and 311,000 deaths in 2018 [Virtually all CC and most high-grade cervical cancer precursor lesions are associated with persistent infection with high-risk (HR) oncogenic Human Papillomavirus (HPV). HPV types 16, 18, 33, 45, 52 and 58 are responsible for about 90% of CC [Solid organ transplant (SOT) recipients are at increased risk of persistent HPV infection and disease . SeveralSistema Único de Saúde, SUS) [Brazil ranks second among 35 countries in absolute numbers of kidney and liver transplantations, with about 95% of SOT financed by the public health system  . QuadrivWe conducted a prospective study to determine the safety and immunogenicity of HPV vaccination among adult female SOT recipients, compared to immunocompetent women of the same age in Sao Paulo, Brazil. In this paper, we present the cross-sectional analysis of HPV-related findings from study participants at enrolment in the cohort. This cross-sectional study aimed to assess the fraction of potentially vaccine-preventable disease by determining HPV seroprevalence, prevalence of cervical HPV-DNA, and HPV-related cytological lesions among SOT recipients compared to immunocompetent women of the same age.Centro de Referência para Imunobiológicos Especiais, CRIE) of the Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (HC-FMUSP), from July 12, 2017 to October 16, 2018.This cross-sectional study was conducted at the Reference Center for Special Immunobiologicals  vaccine in SOT women in comparison to immunocompetent women. Inclusion criteria for SOT women were: having had a transplantation at least six months earlier and being on immunosuppressive treatment. For immunocompetent women, the inclusion criteria were: absence of immunocompromise due to drugs or disease. Exclusion criteria for both groups were: pregnancy or breast-feeding; any other causes of immunocompromise, such as HIV/AIDS, cancer or primary immunodeficiency; biological therapy; heart, lung, liver, kidney or neurologic severe disease; history of genital warts or any HPV-related cervical, vaginal or vulvar lesions, as reported by the candidates; and previous HPV vaccination. Rapid HIV and pregnancy tests were performed for all candidates at enrollment.The sample size calculation was based on the comparison of HPV seroconversion rates for each 4vHPV vaccine type in SOT and immunocompetent women. Based on the design and results of a previous comparative HPV vaccination study conducted among SOT recipients and non-immunosuppressed women in Canada , a sampl™, TriPath, Burlington, NC, USA) and transported daily, at room temperature to the laboratory.The following data were collected: age, color, educational level, age at menarche, age at sexual debut, sexual orientation, number of sexual partners, number of pregnancies, history of contraception and previous sexually transmitted infections (STIs), known comorbidities and, for SOT women, immunosuppressive drugs and regimens used. Body mass index (BMI) was measured for all women. A 5mL venous blood sample was collected for HPV serology. For women reporting having initiated sex, a speculum examination was performed by a trained nurse or physician during which, a cytobrush was used to sample the ecto/endocervical junction and inserted into a liquid-based cytology (LBC) vial  laboratory, a public reference center for cytopathology. All cases were analyzed by cytotechnicians. In case of suspected abnormalities, they were reviewed by experienced cytologists and anatomopathologists. The results were reported according to the Bethesda System for reporting cervical cytology: negative for intraepithelial lesion or malignancy; atypical squamous cells of undetermined significance (ASC-US); atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (ASC-H); low-grade squamous intraepithelial lesion (LSIL) (encompassing: HPV/mild dysplasia/CIN 1) and high-grade squamous intraepithelial lesion (HSIL) ; atypical glandular cells favor neoplastic and endocervical adenocarcinoma in situ [LBC samples were vortexed for 20 seconds and processed by the fully automated BD Totalys in situ . All par® , a polymerase chain reaction (PCR)-based DNA microarray system that can individually identify 25 HPV types, of which 12 high-risk (HR) HPV genotypes  and 13 low-risk HPV types . The test cannot distinguish between HPV 55 and 44 due to cross-reactivity. The test was performed according to the manufacturer’s instructions at the Laboratory of Medical Investigation / Virology (LIM-52), FMUSP.Cervical samples were screened for HPV-DNA using PapilloCheckType-specific anti-HPV antibodies were detected using a multiplexed heparin-pseudovirion serology assay (PsV-Luminex). In this test, pseudovirions containing both L1 and L2 capsid proteins of eight HPV types  were used to detect type-specific anti-HPV antibodies, as previously described . The tesREDCap 9.8.5- © 2020  and the statistical analyzes was conducted using R for Windows, version 3.6.1. Chi-squared test or Fisher’s exact test was used, as appropriate, for comparisons of categorical variables  between the two groups. Logistic regression analysis was used to analyze risk factors for HPV in both groups. Associations are expressed as odds ratio (OR) and their 95%CI. Statistical significance was set at p<0.05.The data bank was built at The study was approved by the Ethics Committee for Analysis of Research Projects (CAPPesq) of the HC-FMUSP (CAAE 66795817.9.0000.0068). All participants provided written informed consent.We enrolled 125 SOT women  and 132 immunocompetent women aged 18 to 45 years. For SOT participants, the mean time since transplantation was 5.55 years. The conditions that led to organ failure and transplantation are described in the Cervical samples for HPV-PCR and liquid-based cytology were collected from 113 SOT and 127 immunocompetent women who had initiated sexual activity. Results of HPV PCR and HPV serology are shown in HPV serology was performed for 105 SOT and 119 immunocompetent women. Seropositivity for at least one HPV type was observed in most women: 63.8% of SOT and 69.7% of immunocompetent women . This dip = 0.013) and lower body mass index . SOT was not associated to a higher risk of HPV-DNA positivity in cervical samples in this study (p = 0.993).Multivariate logistic regression analysis of risk factors for cervical HPV with a variable identifying SOT and controls was performed. Only variables with p<0.2 in the bivariate analyses  were incIn SOT women, there were no statistically significant differences in cervical HPV detection according to time since transplantation , age at transplantation , type of organ transplanted  and immunosuppressive regimens  .This study compared SOT recipients and immunocompetent women aged 18 to 45 years, who denied previous HPV infection and related-lesions at the enrolment. HPV screening by PCR found a greater prevalence of HPV infection in SOT (29.6%) than in immunocompetent women (20.2%), but this difference was not statistically significant. But HR-HPV prevalence was significantly higher in SOT . In addition, HPV serology demonstrated that most women (63.8% SOT and 59.7% immunocompetent) were seropositive for at least one HPV type, suggesting previous HPV exposure, despite absence of previous HPV related infection/diseases as reported by the participants. We also found a 5-fold greater prevalence of LSIL and HSIL in SOT compared to immunocompetent women of the same age range (15% vs. 2.4%). These results suggest similar exposure / HPV infection in both group, with higher HPV persistent infection in SOT women, resulting in higher frequency of HPV-related precursor lesions.A systematic review (SR) with meta-analysis of the HPV prevalence in Brazil that excluded studies in people living with HIV/AIDS, found an overall cervical HPV prevalence of 25.4%  , comparaAnother Brazilian study evaluated HR-HPV and cervical cytological abnormalities in 32 kidney transplanted women aged from 31 to 70 years , ie. an Another study, in Denmark, evaluated cervical cytological abnormalities; HR-HPV prevalence, and HPV genotype distribution in kidney transplant recipients (n = 43), and found a lower prevalence of cytological changes  , compareAnother study, conducted in Poland  evaluateWe were not able to find any Brazilian study on HPV seroprevalence in SOT population. Three studies evaluated seroprevalence of HPV types included in 4vHPV vaccine in non-immunocompromised women in Brazil –21. Two The strengths of our study include enrolment of different types of solid organ transplant recipients ; one of the largest sample size than reported elsewhere in literature; the inclusion of an immunocompetent comparator group and HPV serology evaluation.A limitation of this study is also its sample size, which was calculated to inform the primary aim of the parent cohort study, ie. to determine the immunogenicity of 4vHPV vaccine in SOT recipients. However, considering the shortage of data on HPV prevalence in SOT recipients, this study may provide additional relevant information. Another limitation was the inclusion of women who verbally declared that they had not had a previous genital HPV-related lesion, without presenting previous tests. Women may have voluntarily omitted information on their prior disease so as to benefit from free HPV vaccination offered by enrolling in this study, since the vaccine were not available in the Brazilian public health system (SUS) for most of them, elevating HPV seroprevalence rates found in our study. It was not possible to analyze subgroups of SOT recipients due to the small number of liver, lung and heart transplant.Considering that immunosuppression increases the risk of persistent HPV infection and CC development, further studies on HPV prevalence in SOT recipients and other immunocompromised populations are needed. This study results demonstrate the need of public policies for screening HPV and HPV-related cervical lesions specific for this immunosuppressed population. In addition, HPV vaccination may be an essential preventive measure for immunosuppressed women.S1 Table*NSAID = non-steroidal anti-inflammatory drug.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table*Papillocheck test does not differentiate these HPV types.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


While many European Union/European Economic Area (EU/EEA) countries recently expanded human papillomavirus (HPV) vaccination to boys, HPV vaccine supply is currently limited for girls in low- and middle-income countries (LMIC) that are severely affected by HPV.Globally, about 50% of countries have introduced HPV vaccination. Some LMIC with high burden of cervical cancer have not yet introduced HPV vaccination, or are reaching suboptimal vaccination coverage. While WHO issued a call for cervical cancer elimination in 2018, a global shortage of HPV vaccines is currently predicted to last at least until 2024.We reviewed national policies of EU/EEA countries and recommendations of the World Health Organization (WHO) Strategic Advisory Group of Experts on immunisation to discuss current challenges and dose-sparing options. Several EU/EEA countries have extended HPV vaccination to boys and the European Cancer Organisation has issued a resolution for elimination of all HPV-associated cancers in both sexes. The European Centre for Disease Prevention and Control concluded in its 2020 guidance that cost-effectiveness of extending routine vaccination to boys depends on several context-specific factors. The extension of HPV vaccination to boys in EU/EEA countries may affect global availability of vaccines. Temporary dose-sparing options could be considered during the COVID-19 post-pandemic period. Human papillomavirus (HPV) infection is the most frequent sexually transmitted disease and the second most common cause of cancer attributable to an infectious agent globally . CervicaIn view of the large burden of cervical cancer attributable to HPV, immunisation strategies against HPV infection have initially focused exclusively on girls.From the growing body of evidence, including clinical trials, field trials, large cohort studies and ecological studies on the impact of HPV vaccination, HPV vaccines have proven to be generally safe and effective against persistent HPV infection, anogenital warts, and pre-cancerous lesions, in particular when administered to HPV naive subjects. There is also evidence of substantial indirect (herd) protection when vaccine coverage is above 50% ,4.We review and discuss the implications of the expansion of HPV vaccination to boys in the European Union/European Economic Area (EU/EEA) in light of the current cervical cancer elimination goals and of the global shortage of HPV vaccines, which is mainly affecting girls in low- and middle-income countries (LMIC).Globally, by 2020, about half of all countries have introduced HPV vaccination, covering about one third of the eligible population of girls. Low- and middle-income countries have the lowest rates of introduction of HPV vaccination and some are struggling with its introduction while experiencing a high burden of cervical cancer and HPV-associated illness . A recenA call for elimination of cervical cancer was issued by the World Health Organization (WHO) in May 2018, followed by the American Cancer Society in June 2018 ,8. In Seextend the benefits of vaccines to everyone, everywhere” and identifies vaccination ‘coverage and equity’ as one of its seven strategic priorities [The WHO considers vaccination fundamental for equity in healthcare and beyond. It is one of the key elements of primary healthcare and universal healthcare coverage. The proposed Immunisation Agenda 2030 aims to “iorities . All vacIn 2008, the European Centre for Disease Prevention and Control (ECDC) produced its first guidance on HPV vaccination in EU/EEA countries following the introduction of the first two vaccines against human papillomavirus . The guiSupplementary Table) and concluded that cost-effectiveness of extending HPV vaccination to boys depended on several factors, many of which are context-specific. The cost of the HPV vaccine was the main driver of the incremental cost-effectiveness ratio (ICER) when comparing a scenario of universal vaccination with a baseline scenario of vaccination of girls only. Notably, the extension of HPV vaccination to boys appeared more cost-effective compared with increasing vaccine uptake among girls in cases where vaccination coverage among girls is persistently lower than 75–80% [As part of the guidance, a systematic review of studies looking at cost-effectiveness of extending routine HPV vaccination to boys was performed  and the United Kingdom (UK) have extended, or have decided to extend in the coming years, HPV vaccination to boys, while other countries are discussing the opportunity to extend it soon , Table 2The WHO has highlighted that the current global HPV vaccination shortage, foreseen to last at least until 2024, might delay the introduction of HPV vaccination in the countries most in need . In partBetween 84% and 90% of all cervical cancers occur in LMIC and implementation of mass screening remains a major challenge in several of these countries. Recent projections showed that an estimated 11 million women from LMIC will be diagnosed with cervical cancer in the next 10–20 years. Many of these cervical cancers are diagnosed late and thus have poor prognosis . In thesAdditionally, it should be noted that the average individual health benefit obtained from a dose of vaccine administered to a boy in the EU/EEA is substantially lower than the average individual health benefit obtained from a dose of HPV vaccine given to a girl in LMIC where cervical cancer screening is not implemented or well-established. The WHO’s Strategic Advisory Group of Experts (SAGE) on immunisation recently recommended a temporary pause in the implementation of HPV vaccination of boys in those countries that are considering doing so . It was In light of the limited evidence available, the use of a single dose of an HPV vaccine is currently not recommended. However, a substantial amount of research is ongoing. Non-randomised data from post-hoc analyses of two randomised controlled trials (RCTs) from Costa Rica and India (the latter now converted into a cohort study), using bivalent and 4-valent recombinant HPV vaccines respectively, showed that immunogenicity was still observed years after a single dose of HPV vaccination, although inferior to that observed after two or three doses ,26. ReceAlternative dose-sparing options are also being considered. Vaccination schedules could be modified in order to spare doses and gain time during the current vaccine shortage. Different options were proposed at the SAGE meeting in October 2019, however, it was concluded that the feasibility of the suggested alternative schedules would need to be carefully assessed in each different context . One cosOther gradual approaches may also be contemplated. The Immunisation Committee from the Province of Quebec in Canada recently recommended an off-label mixed schedule using the 9-valent HPV vaccine as a first dose, and the bivalent HPV vaccine as a second dose, until evidence of effectiveness of a single dose of HPV vaccine becomes available ,32. The The COVID-19 pandemic has affected national routine immunisations and may influence the future priorities of vaccination programmes in many countries. A modelling study estimated that, because of the impact of the pandemic on HPV vaccine uptake, over 130,000 cases of genital warts and between 2,882 and 6,487 cases of cervical cancer will not be avoided in the US over the next 100 years, with over 60% of them occurring in the next 50 years .Too many girls living in LMIC with a high burden of cervical cancer are not vaccinated against HPV, hence the global call for action for cervical cancer elimination. In high-income countries, including several EU/EEA countries, the priority has shifted towards the prevention of all HPV-associated illness and the equity of access to HPV vaccination for all genders thanks to successful vaccination and screening strategies against cervical cancer. In view of the current global HPV vaccines shortage which could be affected by the current COVID-19 pandemic in different ways, and given the growing global demand for HPV vaccines, there are different aspects to consider including equity of benefit, equity of access globally, current and future vaccine supply, national and international health needs and priorities, costs and political imperatives. The SAGE issued clear and sensible temporary recommendations, however, countries that have already extended HPV vaccination to boys, or have already decided to extend it, may face important challenges to provisionally reverting or modifying their decision after long debates on the opportunity to do so.The dose-sparing options suggested by SAGE represent possible temporary solutions, in particular the extension of the time interval between the first and the second dose of the HPV vaccine. However, the communication and implementation of such temporary decisions may be difficult and might affect HPV vaccination confidence and uptake of the full two-dose schedule. The potential impact of the COVID-19 pandemic on supply and uptake of other vaccines suggests postponing any further change of the current vaccination schedules to the post-pandemic phase.


Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen. Babesia bovis is a highly pathogenic tick-transmitted apicomplexan protozoon causing bovine babesiosis, which affects the welfare and productivity of cattle in vast tropical and subtropical regions of the world [he world ,2,3.Babesia belongs to the order Piroplasmida, which is characterized by a dixenous life cycle, and alternating asexual reproduction in a vertebrate and sexual and asexual reproduction in an ixodid tick host. Similarly to other related apicomplexans, B. bovis is an obligate intracellular parasite, and thus the processes of invasion and egress from host cells are essential for its survival [B. bovis, Rhipicephalus microplus is the most common vector worldwide [B. bovis, pertaining to Babesia sensu stricto (Clade VI) [The genus survival . In the orldwide . In the lade VI) ,3, thrivlade VI) ,6.B. bovis infection is the accumulation of infected erythrocytes within capillaries, provoking ischemia, which leads to nervous signs and respiratory shock [In naive adult cattle, the ensuing parasitemia is accompanied by fever and anemia. A particular feature of ry shock . Infectiry shock ,7.B. bovis with its bovine and tick hosts remain unknown, yet elucidation may potentially lead to the development of novel control strategies [Many of the details of the mechanisms of pathogenicity and interactions of rategies ,9.Plasmodium falciparum and Toxoplasma gondii, nine ROM subfamilies altogether, designated ROM1, 2, 3, 4, 6, 7, 8, 9 and 10, have been described, although not all are present in both microorganisms. Of these, ROM4, a subfamily confined to Apicomplexan parasites, has attracted particular interest, since it has been shown to be involved in adhesin cleavage—that is, dismantling adhesive junctions formed between parasite and host cell membranes, allowing invasion of the host cell [Proteases of several parasitic protozoa have been described as key players in host–pathogen interactions . Among tost cell ,13,14,15ost cell .B. bovis contains five ROM4-coding paralog genes [P. falciparum counterpart. Recent transcriptomic studies showed that two of the five B. bovis rom4 genes display increased expression (highly significant) in the tick hemolymph compared to bovine blood stages. This finding suggests that ROM4′s functions in B. bovis extend beyond erythrocyte invasion, and may be associated with processes that occur in the tick milieu [We have previously shown that og genes . Functiok milieu . rom4 gene paralogs of B. bovis, including their genomic positions, topologies and polymorphisms among geographic isolates. In addition, we analyzed whether a differential expression pattern, similar to the one found in bovine blood and tick stages, could also be observed in in vitro cultured and in in vitro induced sexual stages of B. bovis. The present study was designed to characterize B. bovis strain T2Bo contains five paralogous genes within a 36 kb-segment of chromosome (chr) 2, encoding four rhomboid serine proteases (ROMs), which we have designated ROM4.1, ROM4.2, ROM4.3, ROM4.4 and ROM4.5 (B. bovis rom4 genes have 3–7 introns (data not shown). Rom4.3, 4.4 and 4.5 genes are tandemly arranged head to tail, separated by 678 and 748 bp intergenic regions, respectively. A 6 kb segment, which contains two orfs, one of which corresponds to a subtilisin-like protease (XP_001610126), separates the oppositely-oriented rom4.1 and rom4.2 genes. On the other hand, rom4.2 and rom4.3 genes are separated by a 19 kb segment, where 11 orfs are encoded, only one of which corresponds to a 20S proteasome-related threonine protease (XP_001610115). In addition, immediately upstream of rom4.5 lies a gene encoding ubiquitin fusion degradation protein (XP_001610110), also associated with proteasome function. Rom4.2 is encoded in the opposite strand to the remaining rom4 gene paralogs  than the other two (747 bp each), 467 bp fragments conserved in the 5′ regions of rom4.4 and 4.5 IGs were compared to rom4.3 IG. Levels of identity were 83% (rom4.4 vs. rom4.5 IGs), 75% (rom4.3 vs. rom4.5 IGs) and 80% (rom4.4 vs. rom4.5 IGs). A highly polymorphic, roughly 120 bp-long segment starts 514 bp upstream of the start codon of rom4.3 and 231 bp upstream of the start codons of rom4.4 and 4.5  upstream of the three most closely similar and tandemly arranged genes,  and 4.5 .B. bovis T2Bo ROM4 paralogs showed that ROM4.3 and 4.4 encode almost identical protein sequences, which display higher levels of identity with ROM4.5 than with the remaining two ROMs (rom4 genes and the other protease-related genes that lie in close proximity to them in the genome. Comparison of the predicted amino acid (aa) sequences of the five two ROMs . The higtwo ROMs . No sequB. bovis ROM4 protein contains 5–7 predicted TMDs. In addition, these proteins present hydrophilic regions in the N and C termini. In all cases, the active site lies in the region of TMDs, within the rhomboid family domain (cl21536) (Each cl21536) . ROMs doB. bovis ROM4 paralogs are predicted to be active and bear the conserved functional aa signatures corresponding to these families of proteins: (Y/F)R in Loop 1, HxxxN in TMD2, GxSG in TMD4 and (A/C/I/S)H and GxxxG in TMD6 , Babesia sensu lato (s.l.), Theileria s.s., T. equi and Cytauxzoon felis. Syntenic paralogs to rom4.2 could only be found in B. divergens and Babesia sp. Xinjiang, both of which represent s.s. Babesia. Interestingly, rom4.4 and 4.5 are jointly syntenic to a single gene in other Babesia s.s. species, a situation referred to as co-orthology. With the exception of rom4.1, no synteny was found for the remaining B. bovis rom4 genes for Babesia s.l., Theileria s.s., T. equi or C. felis. We analyzed the syntenic relationships of lineages . The romIn vitro cultured B. bovis T2Bo merozoites were exposed to xanthurenic acid at 26 °C for 24 h (a) or incubated for the same time under normal culture conditions in the absence of this reagent (b). At the end of the incubation period, parasites were observed outside or inside of erythrocytes in (a) and (b), consistent with the presence of SS and AS, respectively. topoisomerase (BBOV_III004820) and one pair of primers each for actin (BBOV_IV009790) and gadph (BBOV_II002540) genes were selected. Plasmid DNA or SS and AS cDNA samples were used as templates  targeting emplates . All priemplates . Actin pemplates . Howeversmid DNA . rom4.1, rom4.2, rom4.3/4.4 and rom4.5 were evaluated by qPCR using cDNA obtained from SS and AS parasite suspensions. Transcription of all genes was verified in both types of preparations. Data were normalized with respect to the transcription level of topoisomerase and compared. The transcript levels of rom4.1 and rom4.3/4.4 were increased 18 and 218-fold in SS compared to AS (p < 0.01), respectively (relative SS/AS log2 ratio of 4.17 and 7.77). In addition, a non-significantly increased transcription level (p > 0.05) in SS compared to AS was observed for rom4.2 (1.9-fold). Finally, rom4.5 showed non-significant differences (p > 0.05) in AS as compared to SS  sequence of each ROM4 paralog of the reference strain B. bovis T2Bo available in Genbank was compared with the experimentally analyzed corresponding ROM4 sequences of the B. bovis R1A vaccine strain from Argentina. For both ROM4.1 and ROM4.2, peptide sequences showed 100% identity between these two strains. The rom4.3 and 4.4 gene paralogs were amplified and analyzed together, since they showed identical aa sequences . An average dN/dS value of 0.24 was obtained, strongly indicative of negative selection (The average ratio of non-synonymous over synonymous substitutions (dN/dS) was calculated for the repeat region, using the nucleotide sequences encoding the different repeat variants found among election . B. bovis Mo7 from Mexico and A15 from Australia, two geographic isolates of very distant origins.Apart from the diversity in the number of repetitive sequences and the slight differences found in the repeat sequence of Israel and Australia, the remainder of the N-terminal regions had little or no polymorphisms. As an example, Drosophila melanogaster, in which they were shown to be involved in the epidermal growth receptor signaling pathway. Soon after, ROM representatives were found in all kingdoms of life, and recognized as part of an ancient, large family of intramembrane proteases containing members with and without protease catalytic activity [T. gondii, where five different members that received the denomination ROM1 to ROM6 were described. ROM4 and 5 are paralogs and both belong to the ROM4 family (sometimes referred to as ROM4/5), whereas ROM2 is confined to T. gondii. It was later observed that the ROM repertoire varies considerably among members of the Apicomplexa phylum [P. falciparum has not only orthologs of T. gondii ROM1, 3, 4/5 and 6, but contains four additional members of this family, which received the denomination ROM7 to 10 [P. falciparum the presence of ROM4, 6, 7 and 8, but ROM1, 3, 9 or 10 are absent in the first. Notably, while P. falciparum has only one ROM4 member, piroplasmids have an expanded number of two to five ROM4 paralogs, with B. bovis showing the highest number of five paralogs among the studied organisms [ROMs were first discovered in activity ,15,18. Tactivity ,19. In Aa phylum . Indeed,M7 to 10 . Moreoverganisms . P. falciparum and T. gondii have been demonstrated to cleave adhesins on the parasite surface, a prerequisite to allowing parasite internalization into the host cell [B. bovis in vitro cultures, but the protease involved in this cleavage has not yet been identified [B. bovis ROM4 paralogs, ROM4.1 exerts this role, since it is the ortholog of ROM4 of P. falciparum and T. gondii [As mentioned before, the ROM4 of ost cell ,14. The entified ,22. We h. gondii .rom4 locus to a gene family of five paralogs in B. bovis is intriguing. The fact that all are transcriptionally active in AS and SS and in bovine and tick stages suggests their functional relevance. It remains to be explored if these five paralogs exert different roles associated with the interaction of the tick and mammalian host cell. The tandemly arranged rom4.3, 4.4 and 4.5 have a single syntenic counterpart in other Babesia s.s. parasites which, together with their high sequence similarity, suggests they have expanded recently into this rom4 subgroup in B. bovis, representing true in-paralogs. Importantly, the presented synteny data confirm the orthology results obtained by bidirectional best hit (BBH) using BLAST and phylogenetic analysis in a previous study [rom4 paralog with syntenic counterparts in all piroplasmids is rom4.1, we can speculate that this corresponds to the ancestral rhomboid 4 gene of B. bovis, which, through duplications and subsequent sequence divergence, gave rise to the currently existing paralogs. Likely, the tandemly arranged rom4.3, 4.4 and 4.5 originated from the most recent of these duplication events. The particular expansion of the us study . Given trom4.1 and rom4.3/4.4 transcription levels are significantly increased in xanthurenic acid-induced parasite suspensions as compared to non-induced ones. Xanthurenic acid is a metabolite present in the gut of mosquitoes that induces gametogenesis in P. falciparum [B. bigemina, B. bovis or B. ovata with this compound has been shown to yield extracellular suspensions of parasites, which are morphologically different from merozoites and express sexual stage-specific markers. These parasite stages are considered to represent haploid gametocytes and diploid zygotes. Accordingly, xanthurenic acid-induction protocols have been used to study the life cycle of Babesia spp. and antigen expression patterns in SS [In the present study, we show that lciparum ,24. Corrns in SS ,26,27,28rom4 expression in xanthurenic induced SS and non-induced AS cultures observed by RT-qPCR showed some agreement with those in tick and merozoite stages observed by RNAseq in the study of Ueti et al. [B. bovis tick stages show a dramatic increase in transcription of rom4.1 of 1.3 × 104-fold as compared to bovine stages, and in the present study, we observed the same tendency but a more modest increase in 18-fold in the transcription of this gene, analyzed by RT-qPCR in xanthurenic acid-induced SS as compared to non-induced AS. It is possible to hypothesize that certain processes in which ROM4.1 is involved and that are critical for the parasitic stages that take place in the tick milieu, such as gamete formation and fusion, are elicited in vitro by exposure to xanthurenic acid. Additionally, in the case of rom4.5, expression was increased 14-fold in asexual bovine stages as compared to sexual tick stages [p > 0.05). In vivo and in vitro results of the expression of the other rom4 paralogs did not correlate. Our study showed a significant increase in rom4.3/4.4 transcription levels in SS as compared to AS. However, rom4.4 was found to be upregulated in bovine stages compared to tick stages by RNAseq. No significant differences were found for rom4.3. Finally, tick stages showed upregulation of rom4.2, though no significant differences in transcription of this gene were found in our study between AS and SS [Babesia sp. s.s. parasite stages present in tick hemolymph are likely dominated by kinetes, with activated mechanisms for tick cell invasion and asexual replication [B. bovis parasite suspensions, on the other hand, are characterized by the presence of gamete-like forms with projections and surface ruffles, which can fuse, forming multinucleated syncytia. Completion of kinete formation with nuclear fusion, which is assumed to be eventually followed by meiosis, has not been observed in this type of suspensions so far [B. bovis will help to elucidate the functional relevance of ROM4 paralogs in the parasite life cycle [The patterns of i et al. . B. bovik stages . In our S and SS . Our stus so far . These dfe cycle . rom4.3, rom4.4 and rom4.5 genes in spite of overall high sequence identity among their upstream intergenic regions. However, the presence of a distinctive, ~120 bp polymorphic region could be connected to gene-specific regulatory protein binding sites. Other mechanisms that guarantee the specific expression control of each of these genes, such as enhancers, cannot be ruled out. It is interesting to note that a differential regulation was observed for in vivo and in vitro AS and SS for the tandemly arranged Notably, ROM4.5 contains a highly conserved 29 aa sequence bearing a predicted B-cell epitope expanded to up to eight repeats in the analyzed strains, in a hydrophilic, surface-exposed and accessible region of the protein. We thus hypothesized that this region is subject to positive selection pressure of the host immune system. To test this hypothesis, we analyzed the average ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site, as an indication of selective pressure exerted on the coding sequence of the repetitive region. An average dN/dS value of 0.24 was estimated, which does not support the hypothesis of the existence of positive selection in this region. However, analysis of a larger number of isolates would be important to strengthen this conclusion. B. bovis and other piroplasmid species [rom4.5 alleles might provide a tool for strain genotypification. It can be speculated that the repeats region acts as a decoy for the host immune defense system, diverting the responses against other functionally relevant molecules required for invasion of the host cell by the parasite, as has been suggested for surface antigens reported in  species ,30. Howerom4 paralogs in the bovine asexual stage of B. bovis did not differ significantly between the virulent T2Bo strain and an attenuated strain derived from it by serial blood passages in splenectomized cattle. This indicates that the pathogenic/attenuated phenotype does not depend on a differential transcription of these genes [As for other protease-coding genes, transcription of se genes . B. bovis and in all other piroplasmids analyzed [B. bovis ROM6 (XP_001609020) is the only piroplasmid rhomboid not exclusive to apicomplexans. It is encoded in chromosome 1 (chr1); has mitochondrial localization; and participates in various physiological processes within this organelle, including apoptosis, electron transport chain and homeostasis [B. bovis; or the inactive ROM8 (XP_001610508), encoded in chr4 of B. bovis. Since their presence is shared by Plasmodium spp. and piroplasmids, their function may be related to intraerythrocytic processes taking place in the Aconoidasida, to which both groups of protozoa belong [B. bovis possesses two members of the “derlin” subfamily, XP_001612151 and XP_001611988, encoded in chr4 . This type of catalytically inactive rhomboid was discovered in yeasts, and later found to be represented in many different lower and higher eukaryotes. Their function appears to be related to retrotranslocation of misfolded proteins from the endoplasmic reticulum to the cytoplasm, where they are ubiquitinated and degraded in the proteasome [In addition to ROM4, ROM6, 7 and 8 have been described in analyzed . B. bovieostasis . No funcOM6 XP_0009020 is oteasome .B. bovis are based on suspensions of erythrocytes infected with live attenuated parasites. Although generally effective and safe in younger animals, these vaccines can cause disease in adult cattle, can co-transmit other pathogens and have a cumbersome production process. Thus, the search for alternative vaccination strategies and formulations is an active field of research. A substantial amount of information about B. bovis antigens expressed in the vertebrate stage that could serve in vaccine formulations against sporozoites and merozoites has accumulated during the last two or three decades. The search for possible antigens has mostly focused on proteins that fulfill relevant roles in host–pathogen interactions of the merozoite stage, which are able to elicit an adequate humoral and cellular immune response in the bovine host [Currently, the only available vaccines against ine host ,34. On tine host . Howeverine host ,36,37,38Eimeria tenella ROM4 elicited a cellular and humoral response and high protection levels against challenge with the homologous E. tenella strain [T. gondii ROM4, or with a combined vaccination scheme of T. gondii rom4-DNA plasmid followed by a ROM4 peptide, showed humoral and cellular responses, increased survival rates and fewer brain cysts upon challenge with a virulent T. gondii strain [Importantly, ROM4s of other Apicomplexan parasites have shown potential as vaccine candidates. Indeed, vaccination of chicken with a recombinant form of a strain . Similari strain ,41. SincP. falciparum invasion of erythrocytes [ROMs are also considered attractive targets for chemotherapeutic measures against different pathogens, and several ROM-specific inhibitors have been developed ,43,44 Twhrocytes . B. bovis, suggesting several possible roles in host–pathogen interactions. This knowledge may facilitate the development of novel control tools against bovine babesiosis and other piroplasmid-caused diseases of domestic animals and humans.In summary, this study presents new information regarding the structures, transcription levels and sequence polymorphisms of ROM4 paralogs and their products in rom4 paralogs, and the presence of other protease-coding genes in the proximity of rom4 genes, were analyzed in the NCBI website  for the sequenced B. bovis T2Bo strain [services.healthtech.dtu.dk/service.php?TMHMM-2.0), and the presence of a signal peptide was investigated with SignalP 5.0 . Discovering the location of the active site and prediction of catalytic activity were carried out in the MEROPS server (www.ebi.ac.uk/merops/). Multiple sequence alignments were performed by Clustal omega . Percentages of identity and similarity were calculated at the SIAS website (imed.med.ucm.es/Tools/sias.html). Synteny analysis was carried out at Piroplasma DB (piroplasmadb.org/piro/app). Conservation of amino acids in the repeat sequences was visualized using Weblogo (weblogo.berkeley.edu/logo.cgi). B-cell epitopes were predicted in the following web servers: ABCpred (crdd.osdd.net/raghava//abcpred/); Emboss Antigenic server (www.bioinformatics.nl/cgi-bin/emboss/antigenic); Bepipred Linear Epitope prediction 2.0 (tools.immuneepitope.org/bcell/). Accessibility was evaluated with the Emini Surface Accessibility Prediction (tools.immuneepitope.org/bcell/). The average ratio of non-synonymous vs. synonymous substitutions (dN/dS) in the repeat region variants was calculated with SNAP v2.1.1. (hcv.lanl.gov/content/sequence/SNAP/SNAP.html).Locations, lengths and introns of o strain . TransmeB. bovis T2Bo strain merozoites were in vitro propagated in microaerophilic stationary phase (MASP) culture following established procedures [2 incubator at 37 °C, to obtain SS or AS, respectively, as described in [® Kit (Ambion), following the manufacturer’s recommendations and quantified in a NanoDrop  equipment. The removal of genomic DNA was confirmed by RT-PCR targeting a 259 bp fragment of rom4.3/4.4 using primers F: TCAGCCAGAGCAACCCGA and R: GCCTATGAATGAAAGTGTC, with and without addition of reverse transcriptase, and with AS and SS RNA as template, following standard conditions  following the manufacturer’s recommendations. ocedures . Infecteribed in . Suspensnditions . cDNA waB. bovis T2Bo rom4 paralog, and for the housekeeping genes coding for topoisomerase II (BBOV_III004820), actin (BBOV_IV009790) and gadph (BBOV_II002540), were designed using the PrimerExpress 2.0 software (Applied Biosystems), considering the following parameters: a length of 20–22 nt, an amplicon size of ~60–240 bp, a hybridization temperature between 50 and 60 °C, ~50% GC content and a hybridization target within gene exons  and quantified spectrophotometrically. Recombinant plasmids containing each target sequence were prepared to be used in the qPCR set-up. To this end, the corresponding DNA fragments were amplified by direct PCR using DNA from the T2Bo as template in a final volume of 25 μL containing 2.5 mM of each dNTP, 20 μM of each primer, 2.5 U PFU polymerase  with its corresponding buffer and 6.6 ng DNA as template or water as negative control. The cycling conditions consisted in a denaturing period at 95 °C for 3 min; 35 cycles of denaturing at 95 °C for 30 s, hybridization at 58 °C for 30 s and extension at 72 °C for 1 min; and a final extension period at 72 °C for 10 min. Amplicons were visualized in ethidium bromide-containing 1% agarose gels and band sizes were assessed by comparison with a 1 kb Plus DNA marker (Invitrogen). Amplicons were cloned in CloneJet (Thermo Fisher Scientific), and recombinant plasmids were used to transform competent ROXdye (Thermo Scientific) as a passive reference control to normalize PCR-unrelated fluorescence variations. Each reaction was run in parallel with a negative control containing 1 μL ultrapure water instead of DNA. Amplification was carried out in a StepOne Plus thermocycler (Applied Biosystems) with a cycling protocol of 5 min at 95 °C, followed by 40 cycles of 95 °C for 1 min, 55 °C for 30 s and 72 °C for 40 s. The qPCR protocol was designed following MIQE international rules [qPCR reactions were carried out using the SYBR Green qPCR Master Mix (Thermo Scientific) in a final volume of 10 μL, containing 0.2 μM of each primer, 1 μL of cDNA or plasmid template and 0.2 μL al rules . Primer al rules . The spep < 0.05) [Once the primer efficiency and reaction specificity had been verified and a housekeeping gene target had been chosen see , transcr < 0.05) ,50. B. bovis geographical isolates from Argentina , Brazil (B4), Mexico (Mo7), Australia (A1 and A15), Israel (I3) and South Africa (SA2) were used as templates for PCR amplification of rom4 alleles. Data on these strains are included in DNA samples of rom4 paralogs from different B. bovis geographical isolates were amplified by PCR, cloned and sequenced for polymorphism analysis. The whole sequences of rom4.1, rom4.2, rom4.3/4.4 and rom4.5 of the R1A Argentine vaccine strain; and the N-terminal hydrophilic-coding sequences of rom4.5 from different geographical isolates were analyzed. Primers were designed to amplify overlapping segments of ~500–1500 bp for each gene (Alleles of ach gene . PCR amp