Tuberculous meningitis (TBM), the most severe manifestation of tuberculosis, has poorly understood immunopathology and high mortality and morbidity despite antituberculous therapy. This calls for accelerated clinical and basic science research in this field. As TBM disproportionally affects poorer communities, studies are often performed in resource-limited environments, creating challenges for data collection and harmonisation. Comparison of TBM studies has been hampered by variation in sampling strategies, study design and choice of study endpoints.  Based on literature review and expert consensus, this paper provides firstly, practical recommendations to enable thorough diagnostic, pathophysiological and pharmacokinetic studies using clinical samples, and facilitates better data aggregation and comparisons across populations and settings. Secondly, we discuss clinically relevant study endpoints, including neuroimaging, functional outcome, and cause of death, with suggestions of how these could be applied in different designs for future TBM studies. However, there is a clear need to further TBM research by standardising and potentially aggregating clinical and laboratory data across multiple study sites. The International TBM Research Consortium previously published recommendations for standardised clinical data collection2. However, standardised methods for sample collection and processing have not been established. Moreover, the approach to TBM outcome assessment is critical for the interpretation of clinical trials. In this paper we discuss the timing and handling of clinical samples, clinically relevant study endpoints and study design, with the aim of facilitating improved future TBM studies and data sharing.Tuberculous meningitis (TBM) is the most severe form of tuberculosisBaseline sampling. Baseline research samples are ideally obtained at the time of initial routine diagnostic sampling. This can either be residual material from the diagnostic work-up, or extra sample volume, depending on the patient consent and clinical circumstances (refer to ‘Ethics’ section). Corticosteroids, routinely administered to all TBM patients3, exert genomic effects within hours of administration, and non-genomic effects even faster4. This is relevant to immunological studies because of changes induced in gene transcription, which can influence cytokine production and cell counts. It is, therefore, important to know the time frame between corticosteroid administration and sampling. Samples should preferably be taken before or within the first hours after administration of the first corticosteroid dose. Similarly, exposure to antituberculous drugs reduces diagnostic accuracy5, and the timing of pre-study treatment relative to sampling needs to be noted. Given the potential risk of delayed treatment, patients may be started on treatment before reaching the study site. There are no strong data supporting a particular cut-off for a maximum number of days of antituberculous pre-treatment for study inclusion: a shorter duration will decrease patient heterogeneity at the cost of fewer inclusions. Studies have used a maximum of two6, five (ISRCTN 15668391), six7 or seven8 days of pre-treatment prior to sampling as part of their eligibility criteria.Follow up sampling. The frequency and timing of follow up samples will be influenced by sample type, e.g. blood versus cerebrospinal fluid (CSF), sampling method, e.g. lumbar puncture versus ventricular drain, and whether sampling is exclusively for research or combined with clinical activities. In centres using indwelling ventricular catheters, more frequent sampling can take place, with care to maintain sterility of the catheter valves9. In other centres, given the invasive nature of CSF sampling and potential associated risks, follow up CSF samples will commonly be collected during clinically indicated procedures9, or at a timepoint for pharmacokinetic or drug-adverse event monitoring6. In some settings, follow up CSF sampling to reassess diagnosis is routinely performed.10 of its maximum, which is reached at approximately day 2411.Pharmacokinetic sampling from blood and/or CSF is typically performed early after treatment initiation (within 2–5 days) to analyse drug exposures in the critical initial period of illness, and to maximize sample size because of high early mortality. Subsequent sampling adds information on exposure and can be performed around day 10–14 of treatment. At that point, hepatic auto-induction by rifampicin is estimated to be at 70–80%12, CSF cytokines before day seven9 and CSF chemistry before day 7–1013. Due to high early mortality. data from later time points may be missing. Therefore, where possible and safe, we recommend an early (day 2–3) and later (day 7–14) CSF sample for both immunological and pharmacokinetic studies. In case of clinical worsening during treatment, classified as a paradoxical reaction14, repeated CSF and blood sampling could provide valuable information. For immunological studies, it may also be worthwhile repeating blood sampling after corticosteroids have been tapered3 or after antituberculous treatment has been completed.Immunological studies found that CSF cells start to normalise already before day twoConsent for sampling, storage and future use. Ethical considerations depend on whether samples are taken specifically for research or during clinical procedures. CSF samples are most commonly collected during diagnostic and therapeutic lumbar punctures or neurosurgery. CSF collection has the rare but serious potential of bleeding, nerve damage or infectious complications. This risk, although extremely small15, together with the additional discomfort, warrants careful consideration of the balance of risk and benefit when sampling is for research only, and this requires specific consent6. Discomfort from research-related procedures should be minimised; for example, heparinised extension sets, which can remainin situ for a full day, can be used to avoid repeated phlebotomy. Collaborative studies involving the transport of patient samples may require material transfer agreements to ensure the ethical storage and processing of samples and sharing of associated outputs. SeeBox 1. GeneralConsent should cover the purpose of the research, risks and benefits, details of samples being collected, information about genetic testing, patient confidentiality, and voluntary participation. Of note, specific consent for biobanking for future research and genetic testing must be obtained. If applicable, patients must be informed that genetic data will not be shared with them, and that intellectual property generated will not accrue to them.In some countries it may be required to inform patients about possible shipment of samples overseas for collaborative research and appropriate material transfer agreements may be required.For patients with an altered level of consciousness and compromised decision-making capacity, consent can be requested from family members. Patients should be re-consented when they regain mental capacity. Consider seeking approval from local ethics committees to waive consent for participants who do not regain the ability to consent, who demise, or whose families are not contactable, as this will enhance inclusion of more severely ill patients.Paediatric studies16.Age of consent and assent may differ based on local regulationsConsent is required from the legal guardian of minors, to ensure children are protected from potential risks.17.Assent is required from minors as developmentally appropriate, to demonstrate respect for their participation in researchRegardless of the analyte being investigated, general principles for sample handling apply . RecommeBox 2. TimingBaseline samples: preferably before treatment initiation; record timing relative to starting corticosteroids and antitubercular treatment.Follow up samples: aim to collect during routine procedures.QualityProcess swiftly after collection.Consider sample volume relative to collection tube. Avoid over- or under-filling tube.For CSF, avoid using the initial pass of the sample, and document the appearance of the sample before and after centrifugation.Avoid freeze-thaw cycles. Work over dry ice if necessary.CompartmentNote if CSF is from lumbar or ventricular source.in situ.Ventricular CSF may be obtained from TBM patients with external ventricular drainsLumbar CSF volumes may be limited in patients with spinal arachnoiditis due to the presence of exudate in the spinal subarachnoid space.BiobankingBiobank small aliquot volumes at -80°C. If an ultralow freezer is not available at the site of collection, consider using a -20°C freezer for a short interim.Label with study number, date, sample type, project information.Proteins. Different methods are available for proteomic analysis of blood and CSF, including tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS)15, antibody-based multiplex (Luminex)18 or antibody-based methods using a PCR-based multiplex proximity extension assay19. The concentration of CSF proteins can differ by orders of magnitude in the diseased state, which will favour choosing an analytical method with a wide dynamic range15. Of note, it is necessary to validate the methods for the use of CSF, which may use a different background matrix to plasma or serum. Polypropylene rather than polyethylene tubes are preferable for storage due to their low protein binding nature. Centrifugation is important for sample purity and quality control. Samples that appear haemolysed after spinning should preferably be disregarded to avoid measuring proteins of erythrocyte origin, see15. When samples are transported on dry ice, CO2 may enter the container’s headspace and cause acidification of the samples. After transport, it is recommended to leave the samples in a -70 or -80°C freezer to allow the CO2 to dissipate before samples are analysed20. Freeze-thaw cycles can cause conformational changes in proteins, including cytokines, which can affect measurement by antibody-based techniques. These effects have mostly been studied in plasma and serum, and protein concentrations can both increase or decrease21 because of evaporation or protein denaturation, respectively. In some instances one freeze-thaw cycle is enough to influence measured concentrations in blood22, but this first cycle is generally unavoidable because most advanced measurements cannot be performed at the bedside. Data is lacking for many CSF proteins15, but it is advisable to reduce the number of freeze-thaw cycles by biobanking samples in small aliquots (e.g. 100–200 μl).Metabolites. Metabolomics is a powerful technique that can be applied batchwise in small volumes of bodily fluids, which makes it suitable for studying CSF. Nuclear magnetic resonance (NMR)23, gas chromatography-mass-spectrometry24 and liquid chromatography-mass spectrometry25 have been applied successfully on CSF of TBM patients.26. Metabolomics identified isopropanol and propylene glycol in CSF, possibly originating from disinfection prior to lumbar puncture and the CSF collection tubes, respectively. However, both are also produced in the human body and, therefore, they cannot be definitively considered artefacts27. Ongoingex vivo, in-tube cell metabolism affects metabolite concentrations. Blood will be especially vulnerable given the relatively high cell counts28, but also some CSF metabolites can already be affected when the sample is left at room temperature for 30 minutes, and this impact increases in the next 90 minutes29. We therefore recommend not delaying centrifugation by more than one hour after sampling and ideally, samples should be kept at 4°C in the meantime. Centrifugation speed can influence metabolite levels and should be consistent across all samples28. Of note, concentrations of many metabolites are different (higher) intracellularly compared to plasma or CSF and haemolysis in blood samples has been shown to influence measurements and variability of blood metabolites26. It is therefore preferable to obtain cell-free plasma and CSF supernatant and discard haemolytic blood and CSF samples. When CSF is sampled, preferably do not use the first tube for metabolomics as metabolite levels may be influenced by blood contamination.Metabolomic studies gain quality with careful sampling strategies. For blood, ethylenediaminetetraacetic acid (EDTA)-plasma is preferred over serum, as EDTA inhibits enzymes and clotting is not activated28. In blood, the influence of a few freeze-thaw cycles seems to be relatively small26. It is unclear whether sample storage at -80°C for more than 2.5 years influences metabolite concentrations28.When a -80°C freezer is not available in the processing lab, an acceptable strategy is to freeze samples at -20°C and transfer them to -80°C within a week to a monthPharmacokinetics. Pharmacokinetic sampling can follow different approaches. Intensive pharmacokinetic sampling involves collecting a large number of samples in a relatively limited number of individuals, yielding detailed information for each participant. Alternatively, sparse pharmacokinetic sampling involves a limited number of samples, which makes it feasible for use in a larger group of patients, enabling so-called population pharmacokinetic modelling. Pharmacokinetic time points depend on the approach used and the pharmacokinetic characteristics of the drugs involved. For example, catching the peak concentration with intensive pharmacokinetic sampling requires multiple samples around the time to that peak concentration; for drugs with a long elimination half-life, sampling can sometimes be limited to trough levels. It is important to note the exact timing of preceding drug doses and food intake to improve pharmacokinetic modelling, if possible including the treatment patients received before study recruitment. Where possible, take CSF and blood samples simultaneously to enable calculation of drug penetration ratios.30. Isoniazid is especially unstable with a decline in concentration seen after one hour at room temperature31. Blood requires centrifugation, while CSF can be collected directly from the lumbar needle or drain into cryovials at the bedside, or aliquoted in the laboratory after collection without centrifugation. Samples can be stored at -80°C, unless stability has been validated at higher temperatures for a specific drug.Stability studies specific to the research setting need to be part of the bioanalytical method validation. These studies define the sample type (EDTA or lithium-heparin plasma or serum) and the processing conditions. Samples should be processed and frozen as soon as possible after collection32.Of course, intralaboratory bioanalytical method validation needs to be performed, providing information on measures such as accuracy, precision, selectivity and limits of quantitation. Participation in an interlaboratory proficiency testing program is recommended2. Assays for simultaneous measurement of multiple drugs make efficient use of sample volume and may need as little as 100 µL of plasma or CSF to measure concentrations of antituberculous drugs. If CSF to plasma concentration ratios are assessed based on the measured concentrations, they should be based upon drug plasma concentrations that are corrected for protein binding, as only the protein-unbound fraction of plasma is able to penetrate into the CSF2.Plasma assays need careful validation before they can be used to measure CSF drug concentrationsFlow cytometry. Ideally, flow cytometry is performed within hours of sample collection. However, in most field sites this is not achievable, especially not outside working hours. An alternative strategy is storing samples until flow cytometry can take place within 24 hours after sampling. Blood leukocytes thrive best when stored as whole blood in heparin or EDTA-tubes at room temperature. CSF is toxic to leukocytes, especially neutrophils and monocytes33, and a delay in processing samples can cause underestimated cell counts. This can be reduced by early centrifugation, preferably within 30 minutes, and resuspension in cell-culture medium. Centrifugation must be gentle in order not to activate cells, for example at 300 × g. When the volume of CSF is known and microparticles are added to the solute, cell counts can be calculated. This strategy has been applied in TBM, storing CSF cells at 4°C until flow cytometric measurement the next day12. As an alternative strategy, cells can be fixed and cryopreserved34; this has been successfully piloted in CSF by one of the authors but it remains to be established whether all leukocyte populations can be successfully quantified. It is important to consider that cell fixation methods can influence later antibody-fluorochrome binding, and should be validated beforehand.Transcriptomics. The purpose of RNA-sequencing (RNA-Seq) is to elucidate differential gene expression between at least two groups, i.e. different phenotypes, or longitudinal changes following disease onset or treatment. In TBM, transcriptomics has been applied on both blood35 and CSF36. RNA should be stabilised immediately after collection in order to preserve RNA integrity, prevent degradation and minimise non-specific gene induction for downstream applications. For RNA stabilisation of blood, PAXgene RNA tubes (Qiagen) have been developed and used successfully35. Vigorously shaking the PAXgene tube and leaving it at room temperature for at least two hours can increase RNA yield. The tube may even be left at room temperature overnight. The PAXgene tube is under vacuum for 2.5ml of blood and adding a minimum of 1ml is advised.15. RNA has been extracted successfully from CSF collected in PAXgene tubes, which contain RNA-stabilising additive, using standard extraction methods. The high protein content of lumbar CSF renders reagents containing guanidinium thiocyanate such as Tri-Reagent or TRIzol less effective in dissociating nucleic acids from their associated proteins. These reagents, however, have been applied successfully in stabilising the ventricular CSF, which has a lower protein content. The CSF needs to be spun down immediately after collection (approximately 800 × g) and TRIzol added to the pellet before cryopreservation (0.5ml). The TRIzol must be well mixed after it is added and left at room temperature for at least 10 minutes before freezing. If there is still some fluid above the pellet, TRIzol LS, which is designed for liquids, can be used. Tempus RNA tubes also contain RNA-stabilising additive and may potentially be used for lumbar CSF, but further testing of efficacy is required. Regardless of collection method, RNA-stabilised CSF may be stored in -20°C and preferably -80°C, and extraction is best performed within six months of collection for optimal RNA yield, but PAXgene tubes can be stored for up to two years. Due to the overall low RNA yield from CSF, DNase treatment during RNA extraction is highly recommended as DNA contamination will significantly impact RNA-Seq quality and specificity.RNA extraction from CSF is more challenging. Leukocyte counts are approximately 20–1000 times lower than in blood and, therefore, cell-associated RNA concentrations are low. RNA released from damaged brain cells in extracellular membrane vesicles may also be measurableEx vivo induced cytokine response. Blood leukocyte cytokine responses are commonly studied in two different models. When peripheral blood mononuclear cells (PBMC) are isolated, calcium-binding EDTA-tubes can be used. The EDTA is washed away during the isolation of PBMC, which are subsequently resuspended in medium, resupplying the calcium ions that are necessary for the function of many immune receptors. In contrast, in a whole blood stimulation model, blood is used without centrifugation. EDTA should then be avoided and heparinised blood tubes can be used instead. Commercial heparin tubes can be contaminated with endotoxin, so it is recommended to run a nil control and regularly verify that unstimulated samples are free of cytokine production. Additionally, stimulated cells can be fixed for flow cytometry. SeeMicrobiological studies. The threshold of detection is a key principle for microbiological tests. As TBM is a paucibacillary condition, the diagnostic process needs to be optimised. The volume of CSF tested is independently associated with microbiological confirmation where a volume of >6ml increases the likelihood of a positive culture and microscopy result37. To improve sensitivity, centrifugation of a large volume CSF is recommended38. Most laboratories concentrate CSF by centrifugation at 3000 × g for 10–15 minutes38. All but approximately 2ml of supernatant should be drawn off and the pellet is resuspended by vortexing for 15–20 seconds. The resuspended cell pellet can then be divided acrossM. tuberculosis culture, acid-fast bacillus microscopy and nucleic acid amplification tests39. If there is insufficient CSF available for testing, use of unprocessed CSF for Xpert MTB/RIF or repeated lumbar puncture in 48 hours is likely a better strategy. For batchwise proteomic or metabolomic studies aimed at detecting mycobacterial compounds for diagnostic purposes, both the sample pellet (cell fraction including proteins and lipids) and supernatant (metabolites) can be used.M. tuberculosis load in the CSF may be valuable in the interpretation of immunological results, and could possibly be used as a pharmacodynamic endpoint as Xpert MTB threshold cycle predicted new neurological events (but not death) after starting treatment in one study40. It should be noted that this strategy detects both live and dead bacilli. Alternatively, time-to-culture-positivity can be used.An estimation of theOur current knowledge of TBM pathophysiology stems largely from observational studies. To evaluate new therapeutic and diagnostic interventions for TBM however, clinical trials are required. Definitions of relevant endpoints for smaller, exploratory trials are needed to allow progression to larger phase 3 trials, and comparison across studies. Selection of trial endpoints is influenced by study aim, expected effect size of the intervention, feasibility, budget and ability of surrogate markers to predict clinical outcomes. Here we review various clinical endpoints that have been explored in TBM studies and suggest endpoints, tools and timepoints that may improve standardisation across future trials.42 and predicts poor functional outcome and mortality46. The association between hydrocephalus and outcome is mixed48, probably because this is also a function of the treatment of raised intracranial pressure. The value of meningeal enhancement as a predictor of clinical stage or outcome is unclear50, and enhancement may not fully resolve despite effective treatment53.The classic neuroimaging triad described in TBM is 1) basal meningeal enhancement, 2) hydrocephalus, and 3) cerebral infarction. Cerebral infarction, see54; however, the supporting data is limited, and T1-weighted imaging remains the preferred sequence55. Three-dimensional MRI sequence with 1mm or thinner slices has superior yield compared to conventional two-dimensional spin echo for detecting miliary tuberculomas, including both primary lesions noted before treatment and those associated with paradoxical worsening after treatment.Pre- and post-contrast magnetic resonance imaging (MRI) is the imaging modality of choice for TBM – seeBox 3. Pre-contrast T1 weighted imaging for normal anatomy, hydrocephalus, and for comparison with post-contrast imaging.T2-weighted and fluid-attenuated inversion recovery sequence (FLAIR) to detect established infarcts, hydrocephalus, and inflammation.Diffusion-weighted imaging (DWI) to detect acute or evolving infarcts and abscesses.Post-contrast T1-weighted imaging to detect breakdown of the blood brain barrier due to tuberculomas, abscesses, inflammation, meningeal and other enhancement.56, especially those that are acute (evolving), small (e.g. lacunar infarcts), or located in the posterior fossa. If MRI has been obtained previously, subsequent imaging should also be with MRI, when feasible, as comparisons between CT and MRI are suboptimal.If MRI is not accessible, CT with contrast can be obtained, if available. Consideration should always be given to the risk of radiation exposure with multiple CT scans, especially in paediatric patients. Additionally, CT is generally less sensitive to detect infarcts than MRICerebral infarcts. Cerebral infarcts in TBM may be subclinical and discovered only on imaging or at autopsy57. The onset of neurological deficits may be gradual45; this, combined with the altered sensorium common in TBM, can render it challenging to detect subtle neurological deficits. Thus, for research purposes, imaging for infarcts needs to be obtained at prespecified timepoints, even in the absence of new neurological signs or symptoms. The timing depends on the clinical question. Obtaining an MRI at baseline and again at two to three months after initiation of treatment is essential, as new infarcts are frequently detected within this critical time period56. For infarct-related questions, obtaining a third MRI toward the end of the treatment period has low yield53, although late imaging may be appropriate for other questions, such as the treatment response of tuberculomas or relationship between structural abnormalities and neurocognitive impairment. Structured data extraction from neuroimaging for infarcts is important, seeMeningeal enhancement. A formal grading system for meningeal enhancement has not been developed or validated in TBM, although scoring systems have been devised for use in research studies58. Poor interrater reliability of previously proposed criteria highlights the need for standardization and validation59. A simple rating system that includes the presence60, location 53 and severity of meningeal enhancement61 would allow for better standardization of radiological outcomes across studies.Hydrocephalus. Hydrocephalus is rarely seen in isolation in TBM and is typically accompanied by meningeal enhancement. Evan’s ratio—the quotient of the transverse diameter of the anterior horns of the lateral ventricles and the greatest internal diameter of the skull— can be used to standardise hydrocephalus evaluation. An Evan’s ratio of 0.3 or greater is considered abnormal62, but no specific reference values exist for TBM. Similarly, a grading system for periventricular lucency  has been used in TBM61 but not validated.Historically, the primary endpoint in most TBM trials has been six or nine-month mortality. This hard endpoint does not capture the full extent of TBM’s disabling neurological sequelae and more nuanced assessment of functional ability is needed.63. There are a variety of assessment tools for each of these domains. Properties that differentiate useful tools are validity (correlation with other tools and future outcomes), reliability (consistency of scoring between and within assessors), responsiveness to change (ability to detect meaningful change over time), and feasibility or acceptability64. Context and study population must also be considered when selecting a tool.The World Health Organization International Classification of Function (WHO-ICF) describes function in terms of impairment, activity (formerly disability) and participation (formerly handicap)65. To our knowledge it has not been used in a TBM trial and may not offer much in addition to the mRS. The Barthel Index is intended to measure performance in activities of daily living and is used to monitor progress during rehabilitation. It has been used in stroke clinical trials79 and reported in a number of TBM observational studies. The only scale specifically designed for use in brain infections is the 15-item questionnaire known as the Liverpool Outcome Scale80. It was designed to describe outcomes amongst children at the time of hospital discharge following encephalitis, and has predicted which children are likely to be dependent65. It asks specific questions about seizures, bladder and bowel control and behaviour, which may be missed by a global scale like mRS. This has not been studied in TBM but may be a promising tool, though more time-consuming to complete. Greater detail on assessment of functional outcome and on neurocognitive impairment in adult and paediatric TBM is the topic of another article in this Tuberculous Meningitis Consortium collection78.The modified Rankin Scale (mRS) is a commonly used tool for measuring neurological disability and dependence in people having suffered a stroke or other neurological pathology. The mRS is vulnerable to interobserver variability, though this can be reduced by using a structured short questionnaire . Althoug81. Other markers that associated with outcome, which need validation, include normalisation of CSF leukocyte count, glucose and lactate82, CSF brain injury markers83 or CSF metabolism25. Of note, the association of baseline CSF cytokine levels with outcome show inconsistent results, as reviewed in reference84.Baseline risk factors with established prognostic value for mortality include higher disease severity as indicated by British Medical Research Council (MRC) grade or low admission Glasgow Coma Scale (GCS), positive HIV-status, CSF culture positivity, and low CSF to blood glucose ratio85.Very limited data is available on the prognostic value of the above parameters when re-measured during the course of the disease. In the Vietnamese patient population, however, higher GCS during the course of treatment was associated with better prognosis, with good internal validity in a time-updated modelKnowledge of the cause of death can improve the interpretation of pathophysiological studies. Mortality that can be directly attributed to TBM, such as brain herniation or ischemia, suggests different mechanisms compared to mortality as a consequence of neurological  or immunological  sequelae. Post mortem studies provide the most accurate estimates of cause of death. However, feasibility can be hampered for religious, logistic or financial reasons.86. The quality of verbal autopsy information varies depending on the skills of the interviewer and memory of the respondents87. Therefore, it is necessary to keep in contact with the patients or their close family members after the patient is discharged from the hospital and to obtain the information close after the patient’s death. For the purpose of establishing cause of death in patients, the WHO has released standard questionnaires. A focussed version has been used to assess the cause of death in an immunological TBM study12 and is presented inVerbal autopsies, based on interviews with caregivers, can be used to estimate cause of death outside the hospital setting. The underlying assumption of verbal autopsies is that each cause of death has a set of observable features that can be accurately recognized, recalled, and reported by lay respondents1.           Name of verbal autopsy interviewer:2.           Name of verbal autopsy respondent:3.           What is your relationship to the deceased?             □ father □ mother □ spouse □ sibling □ other relative (specify) □ no relation4.           What was the name of the deceased?5.           Is the date of death known?6.           When did the deceased die? 7.           Where did death occur?             □ at home □ in hospital □ at work place □ other (specify) □ don’t know8.           Could you tell me about the illness/events that led to the death of the deceased? Did an injury or (road) accident occur?9.           For how long was the deceased ill before passing away?10–18.   Did the deceased have fever? Night sweats? A cough? Any breathing problems? Severe headache? A stiff, painful neck? Mental confusion or decreased level of consciousness? Convulsions? Motor impairment?19–20.   Primary (direct) and secondary (indirect) cause of death 1 and 2 according to respondent21.        Did the deceased finish the full TB treatment regimen?22.        If the TB treatment was ongoing, at what stage was it? (specify no. months and days)23.        Where did the deceased get the anti-TB drugs from?            □ referral (study) hospital site □ private practice □ community clinic24.        If the treatment was stopped, what was the cause?            □   drug adverse event            □   did not know it had to be minimum six months of treatment without interruption            □   did not want to continue the treatment            □   other (specify)25.        In the last month before death, how was the daily activity of the deceased?            □   normal daily activity, no symptoms at all            □   able to carry out all usual duties and activities            □   requiring some help but able to look after own affairs without assistance            □   requiring some help but able to walk without assistance            □   unable to walk without assistance and unable to attend to own bodily needs without assistance            □   bedridden, incontinent and requiring constant nursing care and attention26.        In the final days before death, did the deceased travel to  a hospital or health facility?27.        If yes, for what reason? When? How long?28.        What did the doctor/health provider do for the deceased?88 Verbal Autopsy Sample Questionnaire 3 “death of a person aged 15 years of above”, used in adapted form in a study that included cause of death in adult TBM patients in Indonesia12. For patients below 15 years of age, an adoption could be made from the respective WHO template.* Adapted from the WHO** Adaptation from the modified Rankin Scale.*** Answer options: □ Yes (if applicable specify intensity and duration) □ No □ Don’t know.Phase II trials and pharmacokinetic studies. Smaller early phase interventional trials are used to select optimal treatment regimens. These studies typically use surrogate markers for major clinical events as endpoints, usually measured within the first six months of study entry  using two-month mortality as an end-point, particularly with interventions that are expected to have a large effect size (> 30%). Two large phase III TBM trials set the primary mortality endpoint at nine months, aligning with standard of care treatment duration67, which increases chances of reliable follow up and will allow for better monitoring of disability in survivors. As the trajectory of neurocognitive disability in TBM is not well defined, and reducing disability is a key objective of TBM management, there is, however, also a need for longer term (1–2 years) assessments of neurocognitive function to be included in TBM trials to assess excess mortality that could still be the consequence of loss of functionality in TBM survivors.TBM causes a considerable burden of disease, necessitating a better understanding of pathophysiology and improved treatment regimens. This requires rigorous study, ideally combining comparable data from multiple centres. Based on published data and expert consensus, this paper offers practical recommendations to standardise clinical sample collection and analysis across multiple platforms. A similar evidence base for study endpoints is lacking and needs to be established, but considerations are discussed and suggestions offered.Radiology images inNo data are associated with this article. Rohlwink and colleagues present a comprehensive summary of a proposed standardised approach to the investigation of TB meningitis which can be applied in future studies. They highlight the importance of testing of the cerebrospinal fluid and particularly the importance of this being obtained rapidly before corticosteroids impact critical parameters and also the utility of sampling across time-points. In the ethics section it may be worth including a specific mention of Material Transfer Agreements. Very clear guidance on the practical considerations to sampling are provided in Table 1. Can the authors expand on the sensitivity/specificity of the fix/cryopreserve approach relative to early traditional flow cytometry? This has many practical advantages if this were to be established. Can the authors please advise as to the utility of Diffusion Tensor Imaging in TBM to distinguish cytopathic from vasogenic oedema in terms of prognosticating and directing corticosteroid treatment? Also, is there a role for early angiography (e.g. CTA/MRA) in the prediction of those at risk of subsequent cerebral infarcts, in whom prophylactic antiplatelet might be considered? Especially given the consequences of cerebral infarction on outcome. Is there any role for patient-reported outcome measures in comparison to the routine ones described? As this may vary across the study settings. Otherwise, a very helpful manuscript.Does the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:Neurological Infectious DiseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you - Dr William P. Howlett and Dr Benedict D. Michael - for your critical appraisal and for raising many interesting points. Some of the more specific issues we choose to answer below. We appreciate the reviewer’s insightful questions on the imaging section of the paper.  Data are extremely limited regarding the utility of advanced imaging  in guiding the management of TBM. A few studies have examined the association of microstructural white matter injury on DTI with CSF inflammatory markers, expression of matrix metalloproteinase-9, and cognitive function . Changes in DTI in response to anti-TB therapy have also been explored. However, to our knowledge, no studies have been published investigating the utility of DTI to distinguish cytopathic from vasogenic edema and to direct glucocorticoid therapy.  The handful of studies that have performed CTA or MRA in patients with TBM have observed a high prevalence of abnormal intracranial vessels. One study (n=67) found that a higher proportion of patients with abnormal MRA compared with normal MRA had evidence of a cerebral infarct on an MRI from the same timepoint . Among 18 patients in this study, 5 of 12 patients (42%) with abnormal MRA at baseline developed new infarcts on an MRI obtained at 3 months compared with 0 of 6 patients with a normal MRA. These results, albeit from a small sample, suggest that patients with an abnormal baseline MRA may benefit most from prophylactic antiplatelet therapy. However, in this same study, a large number of patients had an infarct without corresponding MRA abnormalities, which may speak to the limitations of angiography in identifying small vessel disease that may also be treated with antiplatelet therapy. Small trials examining the benefit of aspirin in TBM have found a trend toward a reduction in brain infarcts in patients who have received aspirin . Whether a baseline CTA or MRA can be used to risk stratify patients and identify those who might benefit the most from antiplatelet therapy is an open question that merits further investigation. clinically relevant study endpoints” starting onPage 7 at “Clinical Endpoints” and ending onPage 13 at the end of “Conclusion”.  My comments are under the following headings General CommentsSpecific comments/suggestionsDetailed review/comments/suggestions highlighted   General CommentsWhile I have read the paper as a whole, I will confine my comments to the second objective of the review “ This “editorial” Open Letter is extremely well written comprehensive and timely addressing the need for a standardised approach for methodologies for clinical sampling and clinical endpoint ascertainment’s for studies in tuberculous meningitis. This informed standardised approach is particularly important for TBM because of its late clinical presentation, difficulties in clinical laboratory and radiological diagnostic certainty coupled with its poor clinical outcome and prognosis. TBM because of its chronic and prolonged clinical course more than any other form of meningitis illustrates a need for a standardized methodological approach in future studies in order to better understand and optimise its treatment and management.  The authors emphasise the considerable burden of TBM and the need for a better understanding of its pathophysiology and improved treatment regimes. This requires rigorous study ideally combining data from multiple centres.  The present paper offers practical recommendations to standardise clinical sample collection and analysis across multiple platforms. I agree that the study endpoints for clinical, neuroradiological and functional outcomes are clearly presented and well discussed in the paper with preferences suggested and recommend that the paper should be published with minor changes listed below.Specific Comments 9, Paragraph headingHydrocephalus. Line 3.  Evan’s ratio.  A description ofEvan’s ratio and its grading into mild ,moderate and severe would be useful to the reader.Page11 Paragraph heading:Possible early surrogate markers for long term outcomes. Line 3. The term High Medical Research Council (MRC) grade is used here incorrectly as it measures muscle power on a scale of 0-5 with 5 being normal. The correct term is British Medical Research Council stage (MRC 1-3).* with 3 being the most severe.PageAscertainment of death and most likely cause of death.  As patients < 15 yrs. are the main age group affected with TBM perhaps a short comment here on whether the very detailed verbal autopsy form/questionnaire available for the those TBM patients aged 15 yrs. and above could possibly be adapted/used for those <15 yrs.Page 11, Paragraph heading Detailed Review/Comments Minor suggestions are as followsClinical endpoints. page 7, Neuroimaging pages. 7, 8 and 9. The need for clear clinical end points for the standardization of intervention study outcomes is well justified. Neuroimaging is particularly well covered in this paper addressing as it does the classic triad in TBM of basal meningeal enhancement, hydrocephalus and cerebral infarction all of which are illustrated clearly in Fig 2. While the use of computerised tomography (CT) with contrast is discussed including its limitations a clear preference for magnetic resonance imaging (MRI) is indicated not least because of increased sensitivity to detect infarcts. Proposed MRI sequences are presented in Box 3. The indication for neuroimaging at baseline and repeated again 2-3 months after the initiation of treatment is emphasised the latter in order to detect subclinical neurological sequalae e.g. infarcts or hydrocephalus. The possibility of a third MRI at the end of treatment is mentioned to answer specific questions related to structural CNS damage and cognitive function and the outcome of tuberculomas Cerebral infarction is known to be associated with a poor prognosis and a very detailed list of end point data are presented in Table 2.Evan’s ratio: maximal width of frontal horns/maximal width of inner skull >0.30.  A potential grading system for periventricular lucency is also mentioned but not described as it is considered to less useful prognostically. Meningeal enhancement is mentioned and the need for a standardized rating system which is currently unavailable with the authors proposing a simple system based on presence, location and severity. Hydrocephalus is one of most frequent major neurological complications in TBM  and has a poor prognosis. While the authors suggest the Evan’s ratio for the standard evaluation of hydrocephalus the method of measurement is not described. A description and its grading into mild ,moderate and severe would be useful to the reader.Evan’s ratio. The indications for neuroimaging as endpoints in TBM studies are very clearly addressed and stated with a preference for MRI over CT and including preferred MRI sequences and the optimum timing of scanning. The authors include a Table with a detailed list of itemized end points for infarction. They also propose a simple scale for meningeal enhancement based on presence, location and severity. Mention is made of the measurement of the degree of hydrocephalus by a recognised scaleSuggestion  Page 9, Paragraph headingHydrocephalus. Line 3  Evan’s ratio. A description ofEvan’s ratio and its grading into mild ,moderate and severe would be useful to the reader.Functional outcomes. pages 10, 11 The authors make a very valid point that the standard primary end points for most TBM trials to date have been 6 or 9 month mortality. They emphasise the need for the inclusion of a method for assessment of TBM morbidity, in particular functional ability as measured in terms of disability and handicap. In order to achieve this they have listed, described and evaluated the main available functional outcome scales in Table 3. These scales are evaluated under a number of properties including validity, reliability, responsiveness to change and feasibility or acceptability, terms which  are defined in the subtext in Table 3. In their evaluation of the different scales a first mention is made of the most commonly used scale i.e. the modified Rankin Scale (mRS). Its use in measuring disability in stroke and other neurological disorders is described in particular its high validity, feasibility and reference to its previous use in TBM while also mentioning a vulnerability to interobserver variability some which is modifiable. While the extended Glasgow outcome scale has been used more in brain injuries it is considered unlikely to offer more than (mRS) and has not been used in TBM. The Barthel Index is designed to measure performance in activities of daily living and its progress over time particularly during rehabilitation but has not been used to date in TBM studies. The WHO disability assessment schedule is designed for adults and has not been used in TBM. The Liverpool Outcome Scale (LOS) is discussed in more detail as it was designed to describe outcomes amongst children at discharge from hospital following encephalitis and specifically answers questions concerning seizures, continence and behaviour, complications that occur post TBM and not addressed by the more commonly used (mRS). The LOS is described as valid, reliable, simple and designed to identify neurological disorders missed by (mRS). Limitations include that it not yet assessed In TBM and is time consuming to apply. For greater detail on assessment of functional outcome and neurocognitive impairment the reader is referred at the end of the discussion to a specific article written by the same TBM consortium which is currently under review and titled “neurocognitive and functional impairment in adult and paediatric TBM” . The paper addresses the importance of measurement of functional outcomes after TBM and briefly discusses and assess the main scales available to document and measure those outcomes. While it identifies the modified Rankin Scale (mRS), the most commonly used scale as valid, moderately reliable and brief to apply, it favours the Liverpool Outcome Scale as being equally valid, reliable and simple but as being more comprehensive in ascertainment of neurological sequalae of TBM whilst aware of its limitations that it not yet assed In TBM and is time consuming to apply. For a more detailed discussion the reader is referred for to a referenced article currently under review.No SuggestionsPossible Early Surrogate clinical markers for longer term outcomes. Page 11.High Medical Research Council (MRC) grade*Low admission Glasgow Coma ScaleHIV SeropositivityCSF TB culture positiveLow CSF/Blood glucose ratio Other markers are mentioned with very limited data on their prognostic value in TBM in particular when remeasured during the course of the disease but would need validation. These includeNormalization of CSF: leukocyte count, glucose, lactateCSF Biomarkers of brain injury and metabolism including cytokine levels. The authors have correctly listed some of the main early surrogate clinical markers for long term outcomes in TBM. These include level of consciousness as measured by Glasgow Coma Scale (GCS 3-15) and the presence of neurological disorder and alteration in GCS as measured by the Medical Research Council scale (MRC 1-3).* The role of HIV in TBM and its deleterious effect on prognosis is listed and  the poor prognostic burden of high mycobacterial load in CSF as manifested by CSF positive culture and low CSF glucose levels. Other CSF biomarkers assessing the course of the infection are mentioned but need validation. The authors list possible early surrogate clinical markers for longer term outcomes. They itemise the base line risk factors with known or proven prognostic value for increased mortality which include higher disease severity as follows:Suggestion11 Paragraph heading:Possible early surrogate markers for long term outcomes. Line 3 The term High Medical Research Council (MRC) grade is used here incorrectly as it measures muscle power on a scale of 0-5 with 5 being normal. The correct term is British Medical Research Council stage (MRC 1-3).* with 3 being the most severe. See below Page*British Medical Research Council (BMRC) staging* system. Stage I:  Glasgow Coma Scale  of 15 with no focal neurologic signs. Stage II:  Glasgow coma scale 11-14. signs of meningeal irritation, slight or no alteration of sensorium , minor neurological deﬁcit  or no deﬁcit Stage III: Glasgow coma scale = or <10. severe alteration of sensorium, convulsions, focal neurological deﬁcit, involuntary movementsAscertainment of death and most likely cause of death.  Page 11. The authors discuss the importance of knowing the cause of death and its role in improving the interpretation of pathophysiological studies. They mention clinical examples of different causes of  mortality in TBM e.g. 1) herniation or ischaemia  2) pressure sores  3) concurrent immunosuppression. While it is evident that post-mortem autopsy studies are the gold standard,  they acknowledge that for religious, logistical or financial reasons they may not occur. Verbal autopsies based on interviews with care givers can be used as an alternative to estimate the cause of death outside the hospital. This assumes that each cause of death has a set of observable features that can be accurately recognised, recalled and reported by respondents, The quality of the information depends on the skills of the interviewer and the memory of the respondent. After discharge close follow up with family members and patient carers is therefore recommended. For the purpose of establishing the cause of death in patients above the age of 15 yrs.  the WHO verbal autopsy questionnaire form is recommended and is presented in detail in Box 4. The authors adequately address the question of trying to ascertain the cause of death and outline the reasons why autopsies may not take place. The need for autopsies is discussed and an emphasis is placed on the value of a verbal autopsy. Qualifications and limitations of its use are well discussed and the detailed WHO verbal autopsy form for 15 yrs. and above is presented. However no mention is made of the use of a verbal autopsy form/questionnaire for the those TBM patients aged <15 yrs., the major group with TBM.Suggestion. A short comment on whether the very detailed verbal autopsy form/questionnaire for the those TBM patients aged 15 yrs. and above could be adapted for those <15 yrs.Endpoints for Intervention trials. Page 12. These are presented and discussed in detail in two paragraphs and study design considerations for different study types are presented clearly in Table 4. In phase II trials and pharmacokinetic studies potential endpoints, sample size and endpoint timing in days are listed under the different study types. In pharmacokinetic trials endpoints such as drug exposure and probability of pharmacokinetic /pharmacodynamic target attainment coupled with exposure-response relationship for efficacy and/or safety, drug dose response and optimal route of administration for bioavailability can measured. In Phase 11a safety trial the occurrence of adverse events is related to the intervention including serious clinical events and immune reconstitution inflammatory syndrome (IRIS) and paradoxical reactions. In phase IIb efficacy trials involving 100-150 patients for 14-180 day clinical markers of treatment response are used including early mortality or functional status, composite of early mortality and disability, improvement of GCS, Neuroimaging results and disease relapse. Phase III trials typically involve a large number of patients 250-400 and last from 180-365 days and beyond and are very costly. The need for these trials is emphasized as being critical to inform guidelines and change practice in TBM. Their importance is in underlined by their ability to determine overall mortality and resulting neurocognitive function and functional status.   The section is well written, clearly presented and summarized in Table 4.No suggestionsDoes the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:I am a general physician and neurologist who has worked in Africa for over 25 years. My area of expertise in clinicalI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


A theoretical model that does not evolve with new information deriving from scientific research, by changing the assumptions from which it was born, becomes a philosophy; the scientist becomes a scholarch. Cranial manual osteopathic medicine is very controversial, although it is commonly practiced, from the clinician to the nonmedical health worker. The article, divided into two parts, reviews the assumptions with which the cranial model was created, highlighting the scientific innovations and new anatomical-physiological reflections. In the first part we will review the synthesis and movement of cerebrospinal fluid (CSF), the movement of the central and peripheral nervous system; we will highlight the mechanical characteristics of the meninges. The aim of the article is to highlight the need to renew the existing cranial model. The osteopathic cranial manipulative medicine (OCMM) was born around 1898, when a student of the American School of Osteopathy observed a disjointed skull in the museum of the same institute and noticed that the temporal bones resembled the gills of fish . This thProduction of CSFAccording to OCMM, cranial movement begins with the synthesis and distribution of CSF -8. The pMovement of the CSFAnother concept that creates a dichotomy with the osteopathic model of PRM concerns cerebral and spinal fluid motion. The circulation of the CSF is not unidirectional and its flow  is not uniform. The flow does not correspond to the amount of CSF produced, and can be bi-directional and oscillatory . We coul Furthermore, the CSF can penetrate the perivascular space thanks to the pores of the vessels (in particular at the level of the subarachnoid space), called stomata Figure  21]..21].The CSF, having penetrated into the arterial perivascular space, enters the cerebral interstitium, thanks to the regulation of aquaporin proteins (aquaporin 4 - AQP4) of the water channels (CSF is 99% water); in these channels are found the terminal portions of the astrocytes or glial cells, which constitute the outer wall of the perivascular space . In thisPulsation of the CSFThe major forces that are documented to determine the movement of the CSF are the heartbeat and diaphragmatic breathing; the heart and the diaphragm muscle create the pulsations of the CSF. During an inhalation, the CSF is pushed cranially , while with an exhalation, the movement is pushed caudally . The intThe inherent motility of the central nervous system and spinal cordAccording to the concept of OCMM, the central and peripheral nervous system would have an intrinsic or inherent movement, capable of moving the CSF and the cranial meninges -3. TheseThe mobility of the cranial and spinal meningeal membranesThe cranial meninges are the falx of the brain, the tentorium of the cerebellum, the falx of the cerebellum and the pituitary tent -8. The dThe meningeal system is rich in lymphatic vessels, which, from a developmental point of view, is formed only after birth; they follow the course of the venous sinuses and the course of some arteries, such as the middle meningeal artery . The menThe OCMM relies on palpation of the skull to assess and possibly resolve changes in movement, shape, and orientation of the cranial bones. The review of the article, divided into two parts, discusses recent data on the synthesis and movement of CSF, highlights the movement of the central nervous system and the mechanical characteristics of the cranial and spinal meninges. From the first part of the article it is possible to deduce new reflections, such as that of naming the PRM with the name of SRM, and the possibility that the movement perceived by the osteopath's hands placed on the skull, derives from the movement of the brain (thanks to the heart and respiratory diaphragm), filtered by the damping of the cranial meninges.


Cerebrospinal fluid (CSF) rhinorrhea refers to the loss of CSF through the nasal cavity. Its causes can be classified as either spontaneous or non-spontaneous. Spontaneous causes of CSF rhinorrhea include congenital anatomical defects and are extremely rare, accounting for less than 4% of reported cases. Following failure of conservative management, definitive treatment most commonly involves an endoscopic transsphenoidal repair of the defect. We present a case of spontaneous CSF rhinorrhea in a previously well 52-year-old female, which required surgical intervention due to failure of conservative management. The causes of cerebrospinal fluid (CSF) rhinorrhea can be classified as either spontaneous or non-spontaneous. Spontaneous or non-traumatic causes include congenital anatomical defects related to the temporal bone, skull base, or dura mater . Non-spoA 52-year-old female presented with a six-month history of progressive rhinorrhea from the left nostril. There was a six-month history of headaches, which aggravated by bending over, but never with any associated fever, neck stiffness, photophobia, or trauma. The remainder of the patient’s history was insignificant for any known medical co-morbidities, except an elevated body mass index (BMI) of 34.5. On examination, clear fluid poured from the left nostril when the patient moved from the supine to prone position. On fundoscopy, we noted a normal cup-to-disc ratio with the absence of papilledema. We were unable to access a beta-2 transferrin assay study to confirm the identity of the fluid, but biochemical analysis of the draining fluid showed that the glucose concentration of the fluid compared with that of blood glucose favored CSF.Computed tomography (CT) scan of the head at this time showed the presence of an empty sella with no evidence of skull fractures or obvious bone defects, but magnetic resonance imaging (MRI) with gadolinium enhancement revealed a CSF leak through the sphenoid bone between the left greater wing and the body Figure , with noThe patient returned one week later with bilateral CSF rhinorrhea accompanied by bouts of coughing. Open surgery with a left coronal incision was performed under general anesthesia, and protrusion of the medial temporal lobe was noted in keeping with the temporal lobe encephalocele depicted on imaging scans, and the harvested fascia lata graft was employed to repair the defect in the sphenoid bone. The temporal lobe was then allowed to fall over the fascia lata to ensure that its position was retained. Before securing the bone flap and closure of skin, duraplasty was employed to create a watertight closure and the lumbar drain was discontinued. Post-operation regime included the use of broad-spectrum antibiotics, acetazolamide, and strict bedrest with head elevation. The post-operative period was uneventful, and the patient displayed no evidence of recurrent CSF leak on further follow-up.CSF rhinorrhea occurs due to an abnormal communication between the subarachnoid space and a defect in the skull base, leading to the loss of CSF through the nasal cavity . SpontanThe gold standard for detecting the presence of CSF is by testing for beta-2 transferrin or beta-2 trace protein ,8. Beta-Radiological diagnostic techniques include the use of high-resolution CT and MRI scans. High-resolution CT and MRI scans are the most reliable means of differentiation between spontaneous and nonspontaneous CSF rhinorrhea. CT and MRI can assist in localization of leaks, especially when associated with fractures of surrounding bone or tumors, but do not demonstrate the leakage itself ,7. CT/MRTreatment of CSF rhinorrhea usually involves an initial conservative approach, which, if failed, is followed by a surgical approach. Conservative treatment of CSF leaks consists of the use of acetazolamide along with prolonged bed rest with elevation of the head, which can decrease intracranial pressure . SurgicaEarly diagnosis and prompt treatment of CSF rhinorrhea are important to prevent complications such as meningitis, intracranial sepsis, and abscesses, which are associated with high mortality rates. The overall rate of ascending meningitis associated with CSF leaks is 19% . CurrentSpontaneous CSF rhinorrhea is a rare condition usually associated with an elevated BMI and ICH. The gold standard for the detection of CSF in secretions is beta-2 transferrin testing; however, CT/MR cisternography is the imaging test of choice as it can accurately identify the CSF leak. Prompt diagnosis and treatment of CSF leaks are necessary to prevent complications including ascending meningitis, which is associated with high mortality rates.


Idiopathic nephrotic syndrome has a relapsing-remitting course in the majority of pediatric patients, demanding vigilant monitoring and self-management. A novel, expert-designed, user-informed mobile application (app), UrApp©, was created to support management tasks, including home urine protein monitoring.ClinicalTrials.gov, NCT04075656) is a randomized trial comparing UrApp-supported nephrotic syndrome management with standard-of-care with parallel process evaluation of the intervention delivery. Sixty caregivers of children with newly diagnosed, steroid-sensitive nephrotic syndrome will be randomized 1:1 to UrApp-supported care or standard-of-care. Follow-up will be 1 year, with primary outcomes of adherence to urine monitoring and medications assessed at 6 and 12 months. Secondary outcomes at 6 and 12 months include self-efficacy, quality-of-life, hospitalizations and delayed relapse diagnoses. A mixed-methods approach will evaluate UrApp engagement, use retention, features used, user perceptions, and contextual barriers and facilitators of UrApp use. User behavior will be assessed for relationships to the primary and secondary outcomes. A Stakeholder Committee of volunteer trial participants, clinicians, and engineers will examine the trial results and design a pragmatic UrApp-enhanced nephrotic syndrome intervention with potential for wide implementation. The final UrApp intervention will be tested in a user-centered hybrid effectiveness-implementation trial designed with stakeholder input.The UrApp Pilot Study (The UrApp Pilot Study examines the efficacy of a novel app designed specifically for nephrotic syndrome. The protocol involves dual efficacy and process evaluation aims to increase efficiency and incorporates the stakeholders’ perspective in formative assessment to inform intervention redesign and the design of a future user-centered trial.ClinicalTrials.gov, NCT04075656. Registered on September 2, 2019, https://clinicaltrials.gov/ct2/show/NCT04075656 Idiopathic nephrotic syndrome (NS) is one of the most common chronic kidney diseases in children, with a prevalence of approximately 16 cases per 100,000 children –5. Durinbefore the development of overt symptoms such as edema. When alerted of a new relapse, providers can prescribe corticosteroids or other medications to treat the relapse and prevent acute disease complications and hospitalizations. Patients and their family members can also track urine protein for resolution so that treatments can be stopped or reduced to minimize toxicity ), retention (use over time [dose]), and app functions used and frequency of use (depth of use [fidelity and dose]). A second main purpose of process evaluation is to understand the contextual influences (i.e. barriers and facilitators) and user perceptions on an intervention’s future implementation and success.Process evaluation examines the extent to which an intervention is implemented as intended and the influences on its effectiveness, and is increasingly recognized as integral to both the development and assessment of health promotional programs . Our UrAued use) , 17. We frequency of use, frequency of use over time, and specific app functions used and frequency. These measures will be downloaded at 6 and 12 months among participants in the UrApp arm (n = 30).User behavior data will be captured by software analytics and include information on: User perceptions of UrApp features and contextual barriers and facilitators of UrApp use will be collected via surveys and interviews of the 30 participants randomized to the UrApp arm at 6 and 12 months of trial participation. Interviews will be conducted either in person or over the telephone with audio recording. Interviews will follow a semi-structured guide and will last approximately 30 min. Questions will target perceptions of the usefulness, satisfaction, and ease of use of UrApp and its specific features. Barriers and facilitators of UrApp use will be queried, as well as recommendations for changes to UrApp. The interviewers will lead with open-ended questions, with follow-up questions probing for understanding. Participants will receive $50 for completed interviews.general perceptions of mobile health apps will be obtained at baseline. A survey on the perceived usefulness, satisfaction, and ease of use of UrApp will be administered to caregivers at 6 and 12 months in the UrApp arm. Surveys items will be adapted from the USE Questionnaire, a survey designed specifically to measure user perceptions of mobile apps and other user support products [Participant surveys on the products .A priori, we define adequate engagement as accessing UrApp at least two times per week during the first 3 months, poor retention as a 50% decrease in weekly use over time, and adequate depth of use as transmission of data to providers for all episodes of disease relapse/remission when prompted. The relationship between user behavior and primary and secondary outcomes (detailed under Aim 1) will be assessed via Chi-square test/Fisher’s exact test or t-tests or Wilcoxon rank-sum tests, as appropriate.Survey results will be tabulated and described for each study time-point. Baseline perceptions of mHealth apps and caregiver demographic characteristics will be examined for associations with UrApp perceptions at 6 and 12 months using Chi-square tests and two-sample t-tests or Wilcoxon rank-sum tests, as appropriate.Analysis of interviews will involve multiple steps to ensure data validity and reliability . The intreciprocal relationships (including partners as key personnel), partnership , co-learning , transparency-honesty-trust  [Stakeholder engagement is an effective framework for ensuring that research is patient-centered, with results that have greater relevance and impact for the intended end users . In thisication) . This wiStakeholder members will include four pediatric nephrologists, three research engineers with human-computer interaction expertise who developed UrApp and are intimately aware of technical possibilities/restrictions, and five patient caregivers in the UrApp arm who have completed 12 months of follow-up. Participants in the UrApp arm at the lead site (CHOA) will be approached for interest in joining the stakeholder committee during the pilot trial. Four face-to-face stakeholder meetings with a telephone conference option for members who cannot be present will be conducted. Meetings will be held every 4–6 months, with the first meeting starting at the end of pilot trial recruitment, when we anticipate having 6-months follow-up data on at least half of the trial participants.partnership). Meeting 1 will focus on introducing members to each other and building relationships . We will outline the goals of the stakeholder committee to refine UrApp and plan a future trial (co-learning). A summary of the trial results to date on UrApp efficacy and process evaluation will be shared , and members will be invited to share their perspectives and interpretation of the trial results (co-learning). Meetings 2–4 will include a review of updated trial results. Informed by the trial results, the committee will iteratively plan UrApp refinement . Caregivers will provide input on outcomes most important to users. The investigators will use the feedback to iteratively develop measures/procedures to be reviewed at subsequent meetings . Caregivers will provide feedback on the protocols to improve recruitment and retention.Caregiver members will be compensated $75 for each meeting, and food/drinks will be provided .An external Data Monitoring Committee (DMC) has been created prior to the randomization of the first patient. The DMC will perform safety reviews every 6 months unless otherwise requested by the Chair of the DMC. The DMC will also receive reports on a regular basis on all serious adverse events (SAEs) reported for this trial. One interim analysis and one final analysis are planned. The study would be stopped if the DMC and the lead PI believe that there is a significant safety issue or that the interim analysis indicates that completing the study is not appropriate.A SAE is defined as: death; life-threatening event; requires or prolongs hospitalization; results in disability significant, persistent, or permanent; pregnancy with a resultant birth defect; causes cancer; or overdoses of a study medication. In addition to the completion of the appropriate forms, any serious adverse event occurring during the study will be reported to the primary investigator , the DMC, the local IRB, and the participating sites. The PI will make an assessment of whether the event constitutes an unanticipated problem posing risks to subjects or others. This assessment will be provided to the lead site IRB, which, in turn will make a final determination and notify the appropriate regulatory agencies and institutional officials.Adverse events that do not meet criteria for SAE will be reported using the Adverse Event Form during the follow-up visit. Information regarding the diagnosis, date of occurrence, severity of the event, and therapy will be recorded. A determination of the relationship of the adverse event to study participation will be made by the site PI. Should a relationship be found, results will be reported to the lead PI DMC within 1 business week.The present protocol represents one of the first formative efforts to rigorously test and refine a technically innovative mobile health app designed specifically for nephrotic syndrome management. UrApp® was designed by an expert panel with input from patients and caregivers and demonstrates excellent analytic validity and usability . Our goa


In conclusion, we highlight that CSF biomarkers, which are easy to collect, can provide rapid insights as diagnostic tools. Early alterations in endogenous NE machinery in atypical Parkinsonism may represent a specific risk trait in forms characterized by a worse prognosis.Academic centers utilize sequential clinical and neuroimaging assessments, including morphometric ratios, to obtain an unequivocal diagnosis of the non-synucleinopathic forms of Parkinsonism, such as progressive supranuclear palsy (PSP), however, a 1–2 year follow-up is required. The on-going long-lasting trials using anti-tau antibodies for PSP patients might therefore be biased by the incorrect enrollment of Parkinson’s disease (PD) patients manifesting early axial signs. This perspective study aimed at achieving two major goals: first, to summarize the established biomarker candidates found in cerebrospinal fluid (CSF) in probable PSP patients, including low p-tau and altered neurofilaments. Second, we share our recent data, from CSF samples of well-selected PSP subjects, attributable to both main variants (and revisited in light of MDS criteria), who were followed for 1 year before and 2 years after lumbar puncture. We found a significantly high level of noradrenaline (NE) in these patients, similar to controls, when compared to PD patients. In contrast, CSF samples, in PD, showed a significant reduction in CSF NE and its major metabolite, which confirmed that PD is a multi-system disease involving several endogenous pathways. The NE axis impairments were prominent in PSP featuring worse NPI. It might represent a counterpart to the early and peculiar psycho-pathological profiles that are observed in  Despite the fact that pathological mechanisms, in experimental models of PSP, appears well delineated  biochemical markers may be crucial to achieving a more accurate diagnosis of PSP.For decades, the scientific community has recognized total tau (t-tau), phosphorylated tau (p-tau), and Aβ42 (Aβ42) as biomarkers for Alzheimer’s disease (AD) diagnosis , primarily caused by neuronal injury driven by different isoforms of tau proteins due to abnormal phosphorylation at different amino acid groups, the cascade of mechanisms that induce neuronal loss remains unclear and the search for consistent CSF biomarkers continues.In this article, we discuss recent findings that have shed new light on diagnostic markers for tauopathies.In PSP, neurodegeneration affects various brain regions, including the cortex and subcortical structures, such as the basal ganglia, cerebellum, and brainstem. Pathological changes are the result of extensive synaptic loss and dysregulation in several neurotransmitter systems, principally the dopaminergic and cholinergic networks  than in controls; further, that p-tau did not differ between iNPH and PSP  to provide specific and prognostic insights in different diseases [consider Lu et al.  for sporIt is worth considering that CSF Nfl has been associated with increased mortality in both brain neurodegenerative disorders that feature alpha-synuclein dysregulation [PD and multiple system atrophy (MSA)], and syndromes characterized by abundant tau protein deposition /dopamine ratio was higher in advanced PD patients manifesting dyskinesia than in Goldstein et al.  have focHere, we report the preliminary data from our center. We recruited 15 patients with PSP, who were followed-up with, at our site, for at least 3 years (including 2 years after CSF sample collection). The Ethical Committee of the University of Tor Vergata approved this study, including formal consent released by each patient , including all procedures (imaging and CSF sampling).The diagnosis of PSP was made by clinical findings and corroborated by imaging in most cases see . SpecifiWe applied the new diagnostic criteria released by the MDS-study group ; two are deceased, to date.-five patients were classified as PSP-Parkinson (PSP-P) with a dominant parkinsonian syndrome showing some LD response  or a negligible response at 6 months (A2).-two patients manifested with a mixed clinical syndrome with features reminiscent of CBD (early levitation and asymmetric apraxia); however, it was associated with repeated falls in the last 18 months (criteria P1).In addition, the majority of patients with PSP showed midbrain atrophy . In previous articles, we have documented that patients afflicted by peripheral neuropathies  in fact represent a reliable control group, given their CSF catecholamine content  were discontinued at least 2 days before; levodopa (LD)/carbidopa or LD/benserazide were held overnight.The first 4 ml of CSF were used for routine laboratory analyses. The next 2 ml was obtained for research. The CSF was aliquoted into 2 × 1,000 μl aliquots in plastic sample tubes. We added 10 μl of 0.01 M perchloric acid to each 1,000 μl aliquot. The samples were frozen at −80°C until further analyses were performed within 2 weeks of collection.We determined the concentration of catecholamines and derivatives in CSF using HPLC with electrochemical detection, as described previously , norepinephrine (NE), dopamine, and 3,4-DHPG CSF levels in the three groups, PD, PSP, and control . In addition, CSF NE levels were significantly higher in CON when compared with PD  scores to measure an ample panel of behavioral symptoms in PSP patients , and PSP with pure akinesia, whose definition requires a multi-disciplinary approach. Several PSP patients manifested signs reminiscent of concomitant synucleinopathy. Similar to idiopathic PD, for instance, PSP patients may have an altered cortical plasticity, as shown by Conte et al.  utilizinIn 2015, Magdalinou and co-authors proposed a panel of nine biomarkers to discriminate atypical PD from PD with dementia. In recent years, different reports have suggested more specific biomarkers to improve PSP diagnosis. The significance of lower CSF p-tau and higher CSF and plasma Nfl levels in patients with PSP, when compared with controls was documented. Aside from p-tau and Nfl concentrations, other studies are investigating to what extent biomarkers implied in different cascades involved in neuroinflammation and synaptic efficiency may also play relevant roles in PSP pathogenesis.Furthermore, as we have presented in this study, patients with PSP exhibit higher concentrations of CSF NE when compared with patients with PD.Hence, a limited, but already enriched panel of biomarkers is currently available, at relatively low cost, to improve the accuracy of early differential diagnosis between PD and PSP, when clinical presentation is not unequivocally distinguishable.With regard to PD, we have previously shown the stage-dependent increase of CSF t-tau levels  rater. MP and SB: writing and editing. GF: competence in CSF data analysis (HPLS) and CSF sample collection. RM: responsible for biochemistry lab and data analysis. NM: Provided strong support in the development of the study and in the revision, facilitating the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.