Background: Tuberculosis kills more people than any other bacterial infection worldwide. In tuberculous meningitis (TBM), a common functional promoter variant (C/T transition) in the gene encoding leukotriene A4 hydrolase (LTA4H), predicts pre-treatment inflammatory phenotype and response to dexamethasone in HIV-uninfected individuals. The primary aim of this study is to determine whether LTA4H genotype determines benefit or harm from adjunctive dexamethasone in HIV-uninfected Vietnamese adults with TBM. The secondary aim is to investigate alternative management strategies in individuals who develop drug induced liver injury (DILI) that will enable the safe continuation of rifampicin and isoniazid therapy. Methods: We will perform a parallel group, randomised (1:1), double blind, placebo-controlled,  multi-centre Phase III non-inferiority trial, comparing dexamethasone versus placebo for 6-8 weeks in addition to standard anti-tuberculosis treatment in HIV-uninfected patients with TBM stratified by LTA4H genotype. The primary endpoint will be death or new neurological event. The trial will enrol approximately 720 HIV-uninfected adults with a clinical diagnosis of TBM, from two hospitals in Ho Chi Minh City, Vietnam. 640 participants with CC or CT- LTA4H genotype will be randomised to either dexamethasone or placebo, and the remaining TT- genotype participants will be treated with standard-of-care dexamethasone. We will also perform a randomised comparison of three management strategies for anti-tuberculosis DILI. An identical ancillary study will also be perfomed in the linked randomised controlled trial of dexamethasone in HIV-infected adults with TBM (ACT HIV). Discussion: Previous data have shown that LTA4H genotype may be a critical determinant of inflammation and consequently of adjunctive anti-inflammatory treatment response in TBM. We will stratify dexamethasone therapy according to LTA4H genotype in HIV-uninfected adults, which may indicate a role for targeted anti-inflammatory therapy according to variation in LTA4H C/T transition. A comparison of DILI management strategies may allow the safe continuation of rifampicin and isoniazid. M. tuberculosis bacteria in cerebrospinal fluid (CSF). Rifampicin (R), isoniazid (H), pyrazinamide (Z) and ethambutol (E) are the first-line antibiotics for all forms of drug-susceptible tuberculosis. These agents, used daily and in combination, kill the bacteria and prevent the emergence of resistance. There is concern that the blood-brain barrier reduces the concentrations of anti-tuberculosis drugs in the brain which may attenuate both bacterial killing and clinical response1. The most recent Cochrane meta-analysis and systematic review of adjunctive corticosteroids for TBM2 concluded that corticosteroids likely reduced death from TBM in HIV-uninfected individuals in the short term (up to 18 months of follow-up), but had no clear impact on longer term neurological disability or survival.Tuberculous meningitis (TBM) is the most severe form of tuberculosis, resulting in death or neurological disability in approximately 40% of HIV-uninfected sufferers, and 70% of those co-infected with HIV. Effective anti-tuberculosis chemotherapy has been available for more than 50 years, yet tuberculosis kills more people than any other single bacterial infection worldwide. Diagnosing TBM is difficult and depends upon detecting low numbers of3, however little evidence exists to guide their optimal management. Drug induced hepatitis associated with anti-tuberculosis treatment occurs in around 10% of HIV-uninfected patients4, and can be attributed to rifampicin, isoniazid or pyrazinamide. Determining which drug is responsible is difficult. Corticosteroid therapy leads to hyperglycaemia, and during treatment of TBM dexamethasone may worsen, or reveal, diabetes. Severe hyponatraemia (low plasma sodium), high temperature and CO2 retention all exacerbate raised intracranial pressure and require active management. The role of corticosteroids in adrenal suppression in TBM requires further investigation.Hydrocephalus, stroke, and tuberculoma formation are important and common complications of TBM6. In humans, LTA4H rs17525495 allele homozygosity (TT and CC respectively) was associated with susceptibility to mycobacterial infection, albeit with opposite inflammatory states - high for the TT-genotype and low for the CC-genotype5. Heterozygotes (CT) had an intermediate inflammatory response, and were more likely to survive TBM5. A retrospective study, analysing HIV-uninfected Vietnamese adults with TBM enrolled into an earlier randomised controlled trial of adjunctive dexamethasone7 and prospective TBM observational studies, found that the survival benefit of dexamethasone was restricted to the hyper-inflammatory LTA4H TT-genotype patients, with possible harm suggested in hypo-inflammatory CC-genotype patients5. This suggests LTA4H genotype may be a critical determinant of inflammation and consequently of adjunctive anti-inflammatory treatment response.How corticosteroids improve survival in HIV-uninfected patients with TBM, and whether they do so in all HIV-uninfected patients, remains uncertain and is the focus of the LAST ACT trial. A common functional promoter variant (C/T transition) in the gene encoding LTA4H, which determines the balance of pro- and anti-inflammatory eicosanoids, appears to predict pre-treatment inflammatory phenotype and response to dexamethasone in HIV-uninfected participants2. Previous data strongly suggests hyperinflammatory LTA4H TT-genotype patients with TBM benefit from dexamethasone, and that adjunctive dexamethasone may not benefit, and may cause harm, when given to patients with LTA4H CT or CC-genotype.Our primary hypothesis is that dexamethasone has no benefit in patients with CC or CT genotype for adults with TBM. We regard the benefit of dexamethasone in patients with LTA4H TT-genotype as established and will hence not randomise them. In contrast, patients with CT or CC-genotypes will be randomised in a practice-defining randomised controlled trial (RCT) to assess whether placebo is non-inferior or even superior to dexamethasone in these patients. Our secondary hypothesis is that current guidelines for the management of anti-tuberculosis DILI in those with TBM result in the premature cessation of rifampicin and isoniazid  and place patients at unnecessary risk of TBM-related death and disability.Dexamethasone has been shown to improve survival in HIV-uninfected individuals with TBMOur primary aim is to determine whether LTA4H genotype, defined at randomisation, determines dexamethasone’s clinical effectiveness when added to the first 6–8 weeks of anti-tuberculosis treatment of TBM with dexamethasone duration depending on Medical Research Council (MRC) grade  at the sOur secondary aim is to investigate alternative management strategies in a subset of patients who develop DILI that will enable the safe continuation of rifampicin and isoniazid therapy whenever possible. The trial will be set in two hospitals in Ho Chi Minh City, Vietnam. The trial will enrol approximately 720 HIV-uninfected adults (≥18 years) with a clinical diagnosis of TBM, as judged by the attending physician, requiring immediate anti-tuberculosis treatment. Amongst these, 640 participants with CT or CC- LTA4H genotype will be randomised to either dexamethasone or placebo. The remaining participants (approximately 80) with TT-genotype will be treated with standard-of-care dexamethasone. The trial schema is shown inLAST ACT is a parallel group, randomised (1:1), double blind, placebo-controlled, multi-centre Phase III non-inferiority trial, comparing dexamethasone versus placebo for 6-8 weeks in addition to standard anti-tuberculosis treatment in HIV-uninfected patients with TBM stratified by LTA4H genotype.M. tuberculosis. An identical ancillary study will also be performed in the linked RCT of dexamethasone in HIV-infected adults with TBM (ACT HIV)8. The results from both trials will be reported together, after the primary results from both trials have been published. This trial schema is shown inAn ancillary DILI strategy study will perform a randomised comparison of management strategies in DILI early in anti-tuberculosis treatment (the intensive phase). We will perform an open, randomised comparison of three management strategies with the aim of demonstrating which strategy results in the least interruption in rifampicin and isoniazid treatment. All patients enrolled in the main trial will be eligible to take part in this study, with the exception of those known to have TBM caused by isoniazid resistant or MDRSeven ancillary studies will be conducted within the trial. The studies are as follows:Ancillary study 1: A randomised comparison of management strategies in response to DILI (as above). (All patients.)Ancillary study 2: Host and bacterial genetic determinants of treatment response. We hypothesise that the LTA4H gene, and genes involved in related inflammatory pathways, may influence participant inflammatory state, TBM severity, and treatment response. We will also examine genetic variants associated with the development of DILI. (All patients.)Ancillary study 3: Impact of dexamethasone on CSF inflammation and gross cerebral pathology . We will investigate how dexamethasone influences the resolution of inflammatory markers in the CSF and the gross pathological consequences of TBM on the brain .  patients only.)Ancillary study 4: Influence of diabetes mellitus on presentation and response to treatment. We will investigate whether diabetes mellitus influences clinical presentation and CSF inflammatory phenotype (linking with ancillary study 2), and how it impacts upon treatment outcomes. (All patients)Ancillary study 5: Influence of Strongyloides infection on presentation and response to treatment. We will determine whether Strongyloides co-infection alters clinical and/or CSF inflammatory phenotype at TBM presentation (linking with ancillary study 2) and treatment response. (All patients.)Ancillary study 6: Pathophysiology and treatment of hyponatraemia and raised intracranial pressure. We will investigate the pathophysiology of TBM-associated hyponatraemia, enabling a better understanding of the causes of hyponatraemia, the relationship between plasma sodium and elevated intracranial pressure, and the best management of severe hyponatraemia. (HTD patients only.)Ancillary study 7: Dexamethasone induced adrenal suppression. We will investigate whether the use of corticosteroids in the doses prescribed in infectious diseases is associated with significant adrenal suppression. (HTD patients only.)The primary endpoint is death or new neurological event  by ≥2 points for ≥2 days from the highest previously recorded GCS (including baseline) or the onset of any of the following clinical adverse events: cerebellar symptoms, focal neurological signs, or new seizures) during 12 months from randomisation. Survivors without a new neurological event known to be alive at 12 months will be censored at that time-point and subjects who withdrew or were lost to follow-up before 12 months will be censored at the date they were last known to be alive.In the DILI strategy study the primary endpoint is the proportion of time in the 60 days following randomisation during which neither rifampicin nor isoniazid are given (or the subject is dead).The secondary endpoints of the main trial are as follows:a)Overall survival during a follow-up period of 12 months. Overall survival is defined as the time from randomisation to death, during a follow-up period of 12 months. Survivors known to be alive at 12 months will be censored at that time-point and subjects who withdrew or were lost to follow-up before 12 months will be censored at the date they were last known to be alive.b)Neurological disability at 12 months. Disability will be assessed by the modified Rankin scale  on days c)Severe (grade 3&4) and serious adverse events. Comparison of the frequency of severe and serious adverse events between treatment groups will form an important part of the study analysis.d)Requirement for ‘rescue’ corticosteroids. Neurological complications occurring after the start of anti-tuberculosis chemotherapy for TBM are common. The cause varies, but includes hydrocephalus, infarcts, tuberculoma formation and hyponatraemia. If the symptoms are thought to be caused by tuberculomas, doctors may re-start or increase the dose of corticosteroids. In this trial, any re-start or dose increase of corticosteroids during the 12-month follow-up will be defined as ’rescue’ corticosteroids.The secondary endpoints of the DILI strategy study are as follows:a)Development of acute liver failure >1.5 and hepatic encephalopathy) after randomisation.b)ART interrupted due to drug-related injury .c)Time to new neurological event  or death from randomisation until the 12 month follow-up of the main trial.d)Overall survival, i.e. time to death from any cause, until the 12 month follow-up of the main trial.e)Neurological disability at the 12 month follow-up of the main trial.Study participants for LAST ACT must be adults (aged 18 years or older), HIV-uninfected, with a clinical diagnosis of TBM  and anti-tuberculosis chemotherapy either planned or started by the attending physician. Participants will be considered eligible for enrolment in this trial if they fulfil all the inclusion criteria and none of the exclusion criteria. The inclusion and exclusion criteria apply at the point of consent, just before the patient’s blood is sent for LTA4H genotyping. Therefore, it is possible that when the LTA4H genotype result returns and the patient is randomised, the patient will have had >3 days of corticosteroids and/or >6 days of anti-tuberculosis treatment. This is acceptable, although the total duration of corticosteroid and anti-tuberculosis treatment before randomisation must be recorded. The time for an LTA4H result to return is not anticipated to be >3 days (e.g. taken on a Friday afternoon and processed on a Monday morning). As TBM is a serious infection requiring hospitalisation, all eligible participants will be treated in hospital, at least for the initial 3 weeks of their illness.9, and less than 90 days of anti-tuberculosis drugs given.Study participants for the DILI strategy study must be receiving first-line anti-tuberculosis drugs and fulfil the definition of drug-related liver injury: elevation of blood transaminase concentrations ≥3 times the ULN with symptoms and signs of hepatitis , or ≥5 times the ULN or a rise in serum bilirubin >2.0mg/dL (>34 µmol/L) without symptomsExclusion criteria for the main trial are:a)An additional brain infection (other than TBM) confirmed or suspected: positive CSF Gram or India Ink stain; positive blood or CSF Cryptococcal antigen test; cerebral toxoplasmosis suspected and attending physician wants to give anti-toxoplasmosis treatment with anti-tuberculosis treatment.b)M. tuberculosis immediately before screening.More than 6 consecutive days of two or more drugs active againstc)More than 3 consecutive days of any type of orally or intravenously administered corticosteroid immediately before randomisation.d)Dexamethasone considered mandatory for any reason by the attending physician.e)Dexamethasone considered to be contraindicated for any reason by the attending physician.f)Patient has previously been randomised into the trial for a prior episode of TBM.g)Lack of consent from the participant or family member (if the participant is incapacitated by the disease).Exclusion criteria for the DILI strategy study are:a)M. tuberculosis or standard first-line anti-tuberculosis drugs unable to be given for any reason other than DILI.TBM known to be caused by isoniazid resistant or MDRb)Signs of chronic liver disease of any cause .c)Lack of consent from the participant or family member (if the participant is incapacitated by the disease).d)Elevation of blood transaminase concentrations ≥3 times the ULN with symptoms and signs of hepatitis , or ≥5 times the ULN or a rise in serum bilirubin >2.0mg/dL (>34 µmol/L) without symptoms, at baseline (day 0).8.).Recruitment activities will only occur in an inpatient hospital setting . The target sample size of around 720 participants will be enrolled within an anticipated accrual rate of 4 years. Once discharged from hospital the participants will be contacted by phone to remind them of their next visit. Patients who miss a visit will be contacted by phone for a maximum of three times after which a maximum of three home visits will be conducted. As part of routine clinical care participants with suspected TBM will have an HIV test, a lumbar puncture, and a GeneXpert MTB/RIF test on CSF. When possible, participants will be screened for eligibility on the day their CSF results return and at the time the decision is made to start anti-tuberculosis chemotherapy for suspected/confirmed TBM. For the DILI strategy study, based on enrolment of all consenting and eligible patients, we anticipate a total sample size of 170 participants  will be performed. Randomisation will occur once the result of the LTA4H genotyping is available. LTA4H CC- and CT-genotype participants will be randomised to two parallel groups in a 1:1 ratio, receiving either dexamethasone or placebo for 6–8 weeks (according to disease severity). TT-genotype participants will be treated with open-label dexamethasone for 6-8 weeks (according to disease severity). All patients will receive standard anti-tuberculosis chemotherapy. Randomisation will be stratified by participating hospital/site, LTA4H genotype (CC or CT), and modified MRC disease severity grade  assessedThe randomisation list will be computer-generated based on random permuted blocks with variable block size following Oxford University Clinical Research Unit (OUCRU) standard operating procedures. The OUCRU biostatistician in charge of randomisation list preparation will set up statistical code to generate the randomisation list and transfer it to the Study Pharmacist. The Study Pharmacist will then change the random seed, i.e. the initialisation of the random numbers generator, in the statistical code in order to blind the biostatistician and then run the code to prepare the final randomisation list. The generated randomisation lists will be securely incorporated within the trial database. A reliable manual back-up system will also be available. Randomisation to the three strategies for DILI will be 1:1:1 with stratification by initial randomisation (dexamethasone or placebo).All participants and investigators will be blinded to the treatment allocation. OUCRU clinical trials unit (CTU) pharmacists will create blinded drug packages (in fully made-up and labelled treatment packs) containing either active drugs or identical placebo sufficient for 6–8 weeks of treatment (dependent on the MRC grade of the participant) according to the prespecified randomisation list, and pre-ship them to the sites. After randomisation, the ward or trial research nurse will take the completed prescription form to the site pharmacy; they will dispense the trial-number specific pack containing the study drug. Unused drug will be returned to the site pharmacy if a participant withdraws from treatment. In ancillary study 3 MRI brain images will be read by an independent neuroradiologist, blinded to the treatment allocation and outcomes of the participant.If, in the opinion of the local clinician, it is important for good clinical care to unblind treatment the documented request will be discussed with the site Principal Investigator (PI) and Chief Investigator (CI). If it is agreed that knowledge of treatment allocation is essential for the best management of the patient, the unblinding code will be provided by the study pharmacist holding the randomisation list at OUCRU CTU upon documented request from the CI. Generalised clinical deterioration is not sufficient for unblinding, given equipoise about the evidence base supporting the use of dexamethasone regardless of clinical severity. All instances of unblinding will be recorded and reported to the Data Monitoring Committee (DMC) and Trial Steering Committee (TSC).An individual participant may stop study drug early for any of the following reasons: Participant no longer believed to have TBM and all anti-tuberculosis treatment stopped; Unacceptable toxicity or adverse event; Intercurrent illness that prevents further treatment; Any change in the participant’s condition that justifies the discontinuation of treatment in the treating physicians opinion and after discussion with the site PI; Inadequate compliance with the protocol treatment in the judgement of the treating physician; Withdrawal of consent for treatment by the participant.All participants will receive the standard of care anti-tuberculosis drugs according to national guidelines. Each participant will be LTA4H genotyped. The enrolment and randomisation of the participant into the trial will be finalised according to genotype. TT-genotype participants will be treated with anti-tuberculosis drugs and open label dexamethasone . LTA4H CParticipants for the DILI strategy study will be randomised to one of three strategies : Strateg1)Observe: measure transaminases, bilirubin, and INR every 3 days; do not change/stop anti-tuberculosis drugs unless transaminases rise to ≥10x normal, or total bilirubin rises >2.5mg/dl (>43 µmol/L), or INR >1.5 or symptoms of hepatitis worsen , in which case go to Strategy 3.2)Stop pyrazinamide alone. Observe, measuring transaminases, bilirubin, and INR every 3 days. If transaminases do not fall to <5x ULN by day 5, or total bilirubin rises >2.5mg/dl (>43 µmol/L), or INR >1.5 or symptoms of hepatitis worsen at any time , go to Strategy 3.3)9,Current standard of care , isoniazid , pyrazinamide  and ethambutol  will be given for at least the first 2 months of treatment, provided drug resistance is not suspected or proven. Pyrazinamide will then be stopped and rifampicin, isoniazid and ethambutol (at the same doses) will then be given until at least 12 months anti-tuberculosis treatment in total has been given. The best treatment of TBM caused by isoniazid-resistant tuberculosis is uncertain, and the attending physician should decide which option to take. For participants with MDR tuberculosis, second-line treatment should be given as soon as possible, following national guidelines and local policies.Participants with abnormal liver function tests (LFTs) at screening are eligible to enter the trial. These patients should be given standard first-line anti-tuberculosis treatment unless blood transaminase concentrations are ≥3 times the ULN with symptoms and signs of hepatitis , or ≥5 times the ULN or a rise in serum bilirubin >2.0mg/dL (>34 µmol/L) without symptoms. In these participants, initial anti-tuberculosis treatment should consist of levofloxacin, ethambutol, and an aminoglycoside . LFTs should be monitored every 3 days and rifampicin started as soon as blood transaminases are <5x the ULN and/or the symptoms and signs of hepatitis resolve. Once the participant is tolerating a rifampicin-containing regimen isoniazid can be introduced and, if isoniazid is tolerated, the aminoglycoside can be stopped. If pyrazinamide is not used in treatment, at least 12 months of anti-tuberculosis treatment must be given. Participants with liver dysfunction at the start of treatment that require modified initial anti-tuberculosis treatment regimens are ineligible for the DILI strategy study.11. In order to ensure the safety of patients we will:Treating participants infected with hepatitis B (HBsAg positive) with corticosteroids carries a very small risk of reactivation and flare of hepatitis B infection1)Test all enrolled participants for HBsAg at baseline (day 0).2)After randomisation (of CT or CC- genotypes) to dexamethasone or placebo, all HBsAg positive participants will be unblinded with respect to the study drug.3)HBsAg positive participants randomised to dexamethasone (CT- or CC- genotypes) will receive tenofovir therapy at 300mg once daily. Tenofovir therapy will be for the duration of dexamethasone therapy, and then for a further 12 months after completion of dexamethasone therapy.4)HBsAg positive participants (of any LTA4H genotype) randomised to placebo will NOT receive tenofovir therapy. Unblinding of HBsAg positive participants is necessary to identify those requiring tenofovir.5)HbSAg positive TT- genotype participants, all of whom will receive open label dexamethasone, will also receive tenofovir therapy at 300mg once daily, according to the current standard of care for HBsAg positive patients as issued by the Vietnam Ministry of Health. Tenofovir therapy will be for the duration of dexamethasone therapy, and then for a further 12 months after completion of dexamethasone therapy.6)LFTs and hepatitis B viral load testing will be performed in those participants with hepatitis B who are receiving dexamethasone. LFTs will be performed at least weekly until discharge, and then every 3 months for the full duration of the study. Liver function tests will be performed more frequently if there is a clinical need. Hepatitis B viral load testing will be performed every 3–6 months  for the full duration of the study. Participants who are HBsAg negative, but HBcAb positive (i.e. evidence of past hepatitis B infection) will not receive tenofovir and will therefore not be unblinded, but they will be followed carefully for evidence of episodes of hepatitis. The monitoring of HBcAb positive participants  will be as for HBsAg patients.Participants with hepatitis C infection (positive hepatitis C antibodies) will not be unblinded. However, they will be carefully monitored with liver function tests performed at least weekly until discharge, and then every 3–6 months for the full duration of the study. Liver function tests will be performed more frequently if there is a clinical need.All other concomitant medications essential for participant management are permitted at enrolment, subject to the exclusion criteria of no contraindications to the use of dexamethasone in the judgement of the attending clinician. If use of a concomitant medication that cannot safely be used with dexamethasone becomes essential after randomisation, then the study drug should be stopped and the concomitant medication used without unblinding. Drugs which increase the risk of gastrointestinal bleeding, such as non-steroidal anti-inflammatory drugs (NSAIDS), should be used with caution. Any other oral or intravenously administered corticosteroids are not permitted, unless deemed essential, in which case the study drug should be stopped and replaced by the chosen corticosteroid. Normal standards of clinical care should be followed.Corticosteroid use is not routine for the treatment of hydrocephalus or stroke, however for neurological deterioration secondary to tuberculomas most physicians recommend using corticosteroids. In this situation open-label dexamethasone is recommended. High-dose intravenous dexamethasone (0.4mg/kg/24hrs) should be prescribed, and the study drug stopped (if the study drug course is still ongoing).The trial assessment schedule for the main trial is outlined inClinical assessment will include conscious level by GCS, new or ongoing focal neurological deficit, clinical treatment response, all serious adverse events, all adverse events of any grade leading to modification of anti-tuberculosis treatment or its interruption/early discontinuation (and clinician-assessed likelihood of relationship of adverse event to dexamethasone), and adherence to drugs (study drug and anti-tuberculosis drugs). Assessment of disability by the modified Rankin scale will be performed at day 30 from randomisation, monthly until completion of anti-TB drugs, and at month 12.GCS and new focal neurology will be recorded daily during the participant’s hospital admission. Participants will be visited by one of the research team at screening, baseline, randomisation (when LTA4H genotype returns), and at least every 3 days for the first 4 weeks of treatment (unless they are discharged or die before 4 weeks) and then at least every 7 days whilst they remain in hospital. Formal trial clinical assessments will occur on day 0, 3, 7, 10, 14, and weekly thereafter until discharge (+/- 1 day).After discharge clinical assessments will occur monthly until 12 months. Some of these assessments can be made by phone. Formal outpatient review will occur monthly (+/- 7 days) for at least the first 2 months following hospital discharge. The patient should have formal outpatient review at least every 2 months until month 12 after randomisation. The endpoints of survival and neurological disability by 12 months (-0/+1 month) should be assessed by formal outpatient review whenever possible.Alanine transaminase (ALT) and bilirubin will be measured to evaluate liver toxicity every 7 days until discharge and at each subsequent follow-up visit until anti-tuberculosis treatment stops.EDTA blood will be taken for HBsAg, HBcAb, hepatitis B viral load, and hepatitis C antibodies. Serum will be stored for later DNA extraction when consent has been given.HbA1c and fasting blood sugar will be measured at baseline and at 60 days from randomisation. To enable more detailed phenotyping of diabetes we will also measure C-peptide and blood lipids at baseline and store serum for future diabetes related auto-antibody testing.All enrolled participants will be tested for serological evidence of Strongyloides infection at baseline (past or latent infection), and with stool examination for evidence of active infection at baseline and at 21–30 days from randomisation, depending on discharge date. When infection is detected, treatment with ivermectin will be provided. Stool will be examined for Strongyloides larvae at the end of study drug to determine whether reactivation alters TBM treatment responses.We will compare adrenal responsiveness at 3 weeks after randomisation and at the end of study drug treatment (6 or 8 weeks depending upon disease severity) in 100 consecutive patients using the short Synacthen test. The patient’s background cortisol level is measured by drawing 2mls of blood at 0900hrs. 250mcg of Synacthen is then administered intravenously; 3ml samples of blood are taken at thirty minutes and sixty minutes to measure the cortisol level after this adrenal stimulation. The Synacthen test will be repeated at day 60 after randomisation.M. tuberculosis culture will be performed on all CSF taken. As part of an ancillary study of the impact of dexamethasone on CSF inflammation and gross cerebral pathology we will measure concentrations of a variety of inflammatory mediators in the CSF  at baseline and on days 30 and 60 after randomisation to determine how dexamethasone influences their expression.Lumbar puncture should be performed, unless clinically contraindicated, as part of routine clinical care for the baseline assessment, and on days 30 and 60 after randomisation to assess treatment response. Opening pressure should be measured, at least 5mls of CSF should be taken for mycobacterial investigations alone, and assessments of cell count and differential, protein, glucose, and lactate should also be performed on 1–2 mls of additional CSF. Ziehl Neelsen stain, GeneXpert, and13. We will also use portable ultrasound to measure the optic nerve sheath diameter, which has been shown to be a reliable and non-invasive measure of raised intracranial pressure15. These additional measurements will be assessed at days 0, 3, and 7, and weekly until discharge. Plasma cortisol will be measured at day 0.The rapid diagnosis of intracranial hypertension is challenging and urgently required in TBM. In the subset of participants enrolled to HTD, Ho Chi Minh City, Vietnam, we will investigate the pathophysiology of TBM-associated hyponatraemia by serial assessments of fluid balance, paired plasma and urinary sodium and osmolality, and intravascular volume by Doppler ultrasound assessment of inferior vena cava collapsibility indexChest Xray will be performed at screening and on day 60 after randomisation. Brain imaging by MRI (or CT if the participant cannot tolerate an MRI) will be performed at baseline (+/- 7 days), 60 days (+/- 7 days), and at 12 months (-0/+1 month). We will investigate whether dexamethasone influences the incidence and outcome of hydrocephalus, infarcts and tuberculoma formation in these participants.Specific procedures will be followed when notifying and reporting adverse events (AEs) or adverse reactions (ARs). The definitions of the EU Directive 2001/20/EC Article 2 based on the principles of ICH good clinical practice (GCP) apply to this trial protocol. All AEs and ARs will be assessed as to whether they are serious or not. If the event is serious and not only related to TBM, or is fatal, then a serious adverse event (SAE) form must be completed and the OUCRU CTU notified within 24 hours. All AEs and ARs (serious and non-serious) should be graded using toxicity gradings.Causality of all SAEs or serious adverse reactions (SARs) in relation to the trial therapy (dexamethasone) will be assessed. There are five categories: unrelated, unlikely, possible, probable, and definitely, related. If the causality assessment is unrelated or unlikely to be related, the event is classified as an SAE. If the causality is assessed as possible, probable or definitely related, then the event is classified as an SAR. If there is at least a possible involvement of the trial treatment (or comparator), the investigator must assess the expectedness of the event. An unexpected adverse reaction is one not previously reported in the current Summary of Product Characteristics (SPC) at the time the event occurred, or one that is more frequent or more severe than previously reported. If a SAR is assessed as being unexpected, it becomes a suspected unexpected serious adverse reaction (SUSAR). Investigators should always check the current version of the SPC.The OUCRU CTU is responsible for the reporting of SUSARs and other SARs to the regulatory authorities and the research ethics committees. The following events will be reported to the relevant authorities in Vietnam: All unexpected SAEs, all SAEs judged to be related or possibly related to the trial intervention, and all deaths. All SAEs will be reported to OxTREC  in the annual review form and to the DMC in accordance to the DMC charter. An independent DMC will oversee the safety of the trial.Interim analyses are planned 6-monthly during the first two years of recruitment and yearly thereafter until the completion of the trial but the DMC has the authority to modify the frequency of interim analyses. At these interim analyses, the DMC will receive a report including unblinded summaries of baseline characteristics, the primary endpoint, overall survival, and adverse events by treatment arm as well as conditional power curves for the primary endpoint. Statistical summaries will be provided both for all randomised subjects and for the subgroup of participants with CC and CT-genotype. The DMC may recommend termination or modification of the study in the circumstances that dexamethasone is harmful (placebo superior), or that dexamethasone is beneficial. The Haybittle-Peto boundary, requiring p < 0.001 at interim analysis to consider stopping for efficacy, should be used as guidance in both directions. This boundary may apply to the overall population with CC or CT genotype combined or either genotype alone. Importantly, a DMC recommendation will not be based purely on statistical tables, but will also require clinical judgment. The DMC will also review data from those enrolled into the drug-induced liver injury sub-study, in particular the incidence of acute hepatic failure in each of the management strategy arms.4, 73/189 (38.6%) of HIV-negative subjects with CC-genotype and 69/214 (32.2%) with CT-genotype experienced a neurological event or died during the 9 month follow-up period. Only few additional events are expected between months 9 and 12 of follow-up. All subjects in this trial received dexamethasone. In principle, administration of dexamethasone in CC and CT- genotypes would be discouraged if placebo could be shown to be non-inferior to dexamethasone. However, as the benefit of dexamethasone in the TT-genotype is undisputed and personalised administration of dexamethasone in HIV-uninfected subjects would necessitate rapid genotype testing, some evidence of harm of dexamethasone in the CC/CT population  is required. Therefore we have opted for a hybrid trial-design approach which assumes a modest harm of dexamethasone and aims to prove non-inferiority of placebo first but also allows claiming superiority of placebo in case dexamethasone proves to induce substantial harm16. Moreover, as it is possible that harm of dexamethasone only applies to the CC-genotype, the trial should allow dropping the CT group at an interim analysis but continue randomisation of the CC group. To protect the one-sided overall familywise error rate of 2.5% for the analysis of the primary endpoint across the two co-primary populations (the full CC/CT population and the CC population), we will assign a multiplicity-corrected one-sided significance level of 2% to the full population and 0.87% to the CC population exploiting the correlation between test statistics on the two populations using the Spiessens and Debois method17.In a previous TBM trial19, only a very low number of subjects will be excluded from the per-protocol population, hence only minimally reducing power in this population. Of note, with 184 neurological events or deaths , the trial also has 80% power to prove harm of dexamethasone in the CC/CT group or the CC group in case the true HR is at least 1.54 in all subjects or 1.96 in CC patients, respectively. Our recent trial enrolled 468 HIV-uninfected adults with TBM in 30 months and 90% of them had CC or CT-genotype. Assuming similar recruitment rates, enrolment will be complete within 4 years.We set the non-inferiority margin in favour of dexamethasone at a hazard ratio (HR) of 0.75 and assume a true HR of 1.15 in the CC/CT population. Assuming an absolute risk of a neurological event or death in the dexamethasone group by 12 months of 35%, a HR=1.15 corresponds to risk of 31.2% on placebo, and the non-inferiority margin implies that we can exclude an absolute risk increase of placebo of (at worst) +8.7%. Under these assumptions a total of 184 neurological events or deaths would be required to obtain 80% power at the one-sided 2% significance level. Assuming an overall event risk of ≥32%, and an 11% sample size increase to compensate for loss-to-follow-up and reductions in power due to the allowance for stopping due to futility, a total of 640 HIV-negative subjects with CC or CT-genotype will be randomised into the trial. Based on experience from our earlier TBM trials4 gave the following data: the median (interquartile range (IQR)) onset date of the drug-related liver injury was 50 (15–84) days from initiation of anti-tuberculosis treatment, the median (IQR) duration of the rifampicin and isoniazid interruption was 16 (12–24) days, and 12 subjects subsequently died (8 of them within 60 days). Of note, the duration of the treatment interruption was <t;30 days for 32 (89%) of the 36 subjects and the remaining 4 subjects never re-started rifampicin or isoniazid but continued to receive alternative anti-tuberculosis treatment for >100 days.A review of 36 subjects who interrupted rifampicin and isoniazid because of clinical hepatitis or jaundice events from our previous trialWe hypothesise that strategies 1 and 2, respectively, will result in a relative reduction in the duration of the treatment interruption of 50% for subjects with interruptions <30 days, but that they do not affect longer interruptions (as the corresponding subject might have permanent intolerance to rifampicin and isoniazid) or mortality. Based on simulations of hypothetical trials using re-sampling from the data described above, the hypothesised treatment effect, and the Wilcoxon rank sum test for analysis, we determined that the power to detect such an effect size with a sample size of at least 50 subjects per arm is >85%. Of note, given this is an ancillary and essentially ‘opportunistic’ study, we have chosen a liberal (i.e. not multiplicity corrected) two-sided significance level of 5% for each of the two primary comparisons of strategies 1 and 2, respectively, versus strategy 3.20 will be applied to all enrolled participants at the end of the study when all mycobacterial culture results are available  subjects are expected to be enrolled in the nested trial with roughly similar numbers from both arms, because the efficacy of the different management strategies is unlikely to depend on whether the patient received dexamethasone or not as it tests a very different intervention, and because the anticipated effect of the management strategy on neurological events and deaths is relatively small. However, in a supplementary analysis, we will also compare the primary endpoint between the treatment policies “dexamethasone treatment plus standard of care management of drug-related liver injury” vs. “placebo treatment plus standard of care management of drug-related liver injury” using an inverse probability weighting based analytical frameworkAll patients in the DILI strategy study will be analysed according to their randomised arm as an intention-to-treat (ITT) analysis. The two primary comparisons are the comparisons of strategies 1 and 2, respectively, versus strategy 3 (with tests conducted at the unadjusted two-sided 5% significance level) and comparisons between strategies 1 and 2 will be exploratory only.The primary endpoint of this trial is the time to death or a new neurological event during 12 months of follow-up. The primary endpoint will be analysed using a Cox proportional hazards regression model with treatment as the only covariate and stratification by TBM MRC severity grade at enrolment  and LTA4H genotype (CC or CT). The primary effect measure is the resulting hazard ratio comparing dexamethasone vs. placebo with a corresponding confidence interval and p-value. Non-inferiority of placebo in the entire population or the CC-genotype subgroup will be established if the corresponding test  rejects the null hypothesis that dexamethasone decreases the hazard of the primary endpoint by 25% or more. Superiority of placebo will additionally be established, if the null hypothesis that dexamethasone does not affect the hazard of the primary endpoint can be rejected against the one-sided alternative that dexamethasone causes harm. The distribution of the primary endpoint will also be visualized using Kaplan-Meier plots and explicit survival estimates at 3, 6, 9, and 12 months of follow-up will also be calculated.The proportional hazards assumption will be formally tested based on scaled Schoenfeld residuals and visually assessed by a plot of the scaled Schoenfeld residuals versus transformed time. In case of a significant test, a formal comparison of the absolute risk of an event by 12 months between the two groups will also be performed . The homogeneity of the treatment effect on the primary endpoint across subgroups will be assessed by subgroup analyses and formal tests of interactions between treatment and the following grouping variables: genotype (CC or CT), TBM MRC severity grade at enrolment , and drug resistance pattern . To obtain an adjusted treatment effect estimate and to assess the effect of other covariates, the primary endpoint will also be modelled using a multivariable Cox proportional hazards regression model including the following covariates (in addition to the treatment group): genotype, TBM MRC severity grade at enrolment, and drug resistance pattern.For the analysis of the primary endpoint of the DILI strategy study, the non-parametric Wilcoxon rank sum test will be used for pairwise comparisons. An additional adjusted analysis  will be also be performed treating the outcome as an ordinal outcome and using a proportional odds logistic regression model (which can be interpreted as an extension of the Wilcoxon rank sum test).Overall survival will be analysed in the same way as the primary endpoint . Neurological disability  at 12 months will be compared between the two arms with a proportional odds logistic regression model with the treatment assignment as the main covariate and adjustment for genotype and TBM MRC severity grade. The result will be summarised as a cumulative odds ratio with a corresponding 95% confidence interval and p‐value. Patients with a missing 12 month disability assessment will be excluded from the main analysis but an alternative analysis based on multiple imputation (including disability assessments at earlier time points in the imputation model) will also be performed. The number and proportion of subjects requiring ‘rescue’ corticosteroids will be summarised by treatment arm. Comparisons will be based on logistic regression with the treatment assignment as the main covariate and adjustment for genotype and TBM MRC severity grade.The number of patients with any adverse events and specific events, respectively, will be summarised and informally compared between the two treatment arms based on Fisher’s exact test. The total number of adverse event episodes per patient will also be summarised and informally compared based on a quasi-Poisson regression model with treatment as the only covariate. The following subgroups of adverse events will also be separately summarised: grade 3&4 adverse events; serious adverse events; serious adverse events possibly, probably, or definitely related to the study drug; adverse events leading to TB treatment interruptions. Grade 3&4 laboratory abnormalities will be summarised in the same way as clinical adverse events.Baseline characteristics will be summarised as median (lower and upper quartiles) for continuous data and frequency (percentage) for categorical data. The amount of missing data for each baseline characteristic will also be displayed.Participants’ confidentiality will be maintained throughout the trial. Participants will be assigned a trial identification number and this will be used on case report forms (CRFs); participants will not be identified by their name. The investigator will securely keep a participant trial register showing identification numbers, surnames and date of birth. The unique trial number will identify all laboratory specimens, case record forms, and other records and no names will be used, in order to maintain confidentiality. Data submitted to OUCRU CTU and samples sent to central testing facilities will be identified only by the trial number and participant initials.Written informed consent must be obtained in order to enter into the trial and be randomised. If a participant lacks capacity, written consent must be obtained from a person with responsibility (e.g. family member/relative), in their own language before enrolment by the site PI or an appropriately trained doctor. All potential participants (or their families) will be given a participant information sheet clearly listing the risks and benefits of the trial. All potential participants (or their families) will be able to discuss participation with their consulting doctor who will be able to address questions not covered or arising from the participant information sheet. Incapacitated adults with TBM will be eligible to enter the trial. These adults have more severe disease and therefore may benefit most from adjunctive dexamethasone. We anticipate around 70% of participants with TBM will lack capacity at the start of treatment. An option will be given to patients to enrol in the main study, but not the ancillary studies.If consent is provided by a relative, the participant should be consulted and consent recorded if and when they have the capacity to do so. If they are happy to remain in the trial, the participant should complete a participant consent form at this time. If they wish to withdraw from the trial, no further trial-related procedures will be performed, but data to this point would be used in analysis. Data from any participant who dies before regaining capacity (but whose family member has provided consent) will be included in analysis.The trial protocol has been approved by the Oxford Tropical Research Ethics Committee, the Ethics Committees of the Hospital for Tropical Diseases Pham Ngoc Thach Hospital and the Vietnam Ministry of Health.All deviations from protocol will be addressed in source documents and reported to the OUCRU CTU.A participant (or their relative) is free to refuse to participate in or withdraw from all or any aspect of the trial, at any time and for any reason. If a participant chooses to discontinue their trial treatment they should always be followed up (providing they are willing) and they should be encouraged not to leave the whole trial. If they do not wish to remain on trial follow-up however, their decision must be respected and the participant will be withdrawn from the trial. Participants may change their minds about stopping trial follow-up at any time and re-consent to participation in the trial.Clinical data and clinical laboratory data will be entered into CliRes, a 21 CFR Part 11-compliant data capture system provided by the OUCRU information technology department. The data system includes password protection and internal quality checks, such as automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate. Trial data will be recorded onto paper CRFs and entered into CliRes. The participants will be identified by a unique trial specific number and/or code in any database. The name and any other identifying detail will not be included in any trial data electronic file. CRFs, clinical notes and administrative documentation will be kept in a secure location and held for 15 years after the end of the trial. Clinical information will not be released without written permission, except as necessary for monitoring, auditing and inspection purposes. Electronic data will be kept for at least 20 years at the OUCRU CTU.A SPIRIT checklist for this trial protocol is attached .A Trial Management Group (TMG) will be formed to conduct the day-to-day management of the trial at the OUCRU CTU. This will include the CI, Head of OUCRU CTU, Trial Statistician, Clinical Project Manager, Trial Manager and Data Manager. The group will meet at least once per month, although may meet more or less often as required. The TSC has membership from the TMG plus independent members , Professor Ben Marais , DrTruong Huu Khanh ), including the Chair ).The role of the TSC is to provide overall supervision for the trial and provide advice through its independent chair. The ultimate decision for the continuation of the trial lies with the TSC. The DMC , Professor Graeme Meintjes  and Professor Nina Ruslami ), will advise the TSC and can recommend premature closure or reporting of the trial, or that recruitment be discontinued or modified. The DMC will advise the TSC and can recommend premature closure or reporting of the trial, or that recruitment be discontinued or modified. The DMC is independent from the sponsor. Access to interim data and results will be confidential and strictly limited to the DMC and results (except for the recommendation) will not be communicated to the outside and/or clinical investigators involved in the trial. This trial is sponsored by The University of Oxford  1865 282585).Manuscripts arising from the trial will, wherever possible, be submitted to peer-reviewed journals which enable Open Access via UK PubMed Central within six months of the official date of final publication. In line with research transparency and greater access to data from trials OUCRU’s clinical trials are registered at ClinicalTrials.gov and a data sharing policy is in place. Data exchange complies with Information Governance and Data Security Policies in all of the relevant countries.12. Previous data strongly suggests hyperinflammatory LTA4H TT-genotype patients with TBM benefit from dexamethasone, and that adjunctive dexamethasone does not benefit, and may cause harm, when given to patients with LTA4H CT or CC-genotype5. How corticosteroids improve survival in TBM in HIV-uninfected patients, and whether they do so in all HIV-uninfected patients, remains uncertain and is the focus of the LAST ACT trial. Adjunctive dexamethasone is currently standard of care for HIV-unaffected patients with TBM. This study may identify a role for using LTA4H genotype to guide adjunctive anti-inflammatory therapy.Dexamethasone has been shown to improve survival in HIV-uninfected individuals with TBMst February 2018. Estimated time for recruitment is 4 years.Trial protocol version 1.6. Estimated recruitment start date 1The trial has ethics approval from the Oxford Tropical Research Ethics Committee , the Ethics Committees of the Hospital for Tropical Diseases  and Pham Ngoc Thach Hospital , and the Vietnam Ministry of Health.No data are associated with this article.NCT03092817), which has also been submitted toWellcome Open Research8. ACT HIV is a parallel group, randomised (1:1), double blind, placebo-controlled multi-centre Phase III trial, comparing the effect of dexamethasone versus placebo on overall survival in HIV-infected patients with TBM. The 7 ancillary studies in LAST ACT are also recruited to through the ACT HIV trial. As such the hypotheses, design, methods, sample size justification; analysis plans and endpoints of these ancillary studies will also be described in the ACT HIV trial, and will appear identically here. LAST ACT follows the same OUCRU protocols and local / national guidelines as ACT HIV, therefore randomisation, blinding and unblinding procedures, adverse event and safety reporting, ethics and confidentiality sections also appear identically here for LAST ACT as for the ACT HIV trial.Due to the linked nature of this trial, some sections of this protocol also form part of the linked RCT ACT HIV : the numbers anticipated in the HIV-uninfected patients is n~70, to be analysed according to the 3 strategies. This may mean that the numbers in these analyses might be relatively low. Since the DILI strategy study will also be run in the ACT-HIV study, where anticipated numbers are n~100, will the two groups be potentially combined for analysis? It is acknowledged that this will present a number of limitations with respect to drug interactions when considering ART in the HIV-infected patients.Related to ancillary study 2: will genetic variants associated with development of DILI be examined related to the host or bacteria, or both?Related to ancillary study 3: the influence of dexamethasone on the resolution of inflammatory markers in the CSF will be examined. This should be done in parallel with blood, although I see no blood collection for such studies in Table 3.Considering that there is no LTAH4 genotyping at weekends, and a patient recruited on a Friday may have been already on anti-TB treatment for 5 days with adjunctive dexamethasone. Therefore, by the time the genotyping result is returned on Monday with a CC genotype, the patient already received 8 days of potentially harmful dexamethasone. Assuming that this patient is randomised to placebo, the harm is potentially already done, therefore having a negative effect on the placebo arm. Is there a way to control for this, by potentially not recruiting on a Friday or introducing weekend genotyping for the duration of the trial.HIV testing is done at baseline: would there be benefit in further HIV testing at 3 and/or 6 months? Questions:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Denying corticosteroids to a subgroup of patient, will it be ethically appropriate? Among   CC or CT- LTA4H genotype or TT- genotypes which one have inherently poor prognosis and need corticosteroids?  “LTA4H genotype may be a critical determinant of inflammation and consequently of adjunctive anti-inflammatory treatment response” --- outcome among varied genotypes should have been additional factor determining suitability for corticosteroids.This is wonderfully planned almost flawless study. I have two questionsI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Incorporating selenium or tellurium into photoactive species imparts new photophysical properties that may be exploited in materials applications. Sulfur-containing compounds, particularly derivatives of thiophene, are well studied for organic optoelectronic applications. Incorporating selenium or tellurium in place of sulfur imparts different physical properties due to the fundamental differences of these atoms relative to their lighter analogues. This has a profound influence on the properties of molecules and materials that incorporate chalcogens that may ultimately lead to new opportunities and applications. This mini-review will focus on the quantitative and qualitative photophysical characteristics of organic materials containing selenium and tellurium as well as their emerging applications as molecular photoactive species, including light-emitting sensors, triplet sensitizers, and beyond. In the polymer electronics communities substantial attention has focused on the synthesis and properties of polythiophenes and their derivatives.221–3 (S1–T1) transition.The photophysical properties and optical applications heavier organochalcogens is also an emerging field, yet it has received somewhat less attention. This is in part because heavier organochalcogens are not traditional luminescent materials: the fluorescence that occurs in lighter chalcogens is often quenched upon substituting heavier elements. Improved spin orbit coupling resulting from the heavy atom effect promotes intersystem crossing (ISC) to rapidly populate the triplet state.2.While the number of studies utilizing heavy chalcogen-containing organic materials for specific applications is growing ,25,26 onet al. directly compared the photophysics of chalcogen-containing polymers with substitutions from sulfur to selenium to tellurium.1) with identical solubilizing side chains: poly(3-hexylthiophene) (P3HT), poly(3-hexylselenophene) (P3HS), and poly(3-hexyltellurophene) (P3HTe). The PLQY of P3HT is 0.30, while those of P3HS and P3HTe are drastically reduced to 4.2 × 10–3 and 1.4 × 10–4, respectively. Furthermore, using transient absorption spectroscopy, this study reported simulated emission decay time constants of 600 ps for P3HT, 26 ps for P3HS, and 1.8 ps for P3HTe, suggesting rapid population of the triplet state. This study demonstrates that incorporating a heavier chalcogen results in faster ISC in organochalcogen polymers.One study from Pensack et al. directly compared the rate of ISC in oxonine, thionine, and selenine small molecule dyes (2) using both experimental observations and quantum mechanics.5 s–1.8 s–1,9 s–1 and 1.66 × 108 s–1, indicating that triplet state population was an order of magnitude faster than fluorescence. The population of triplet states is also supported by a singlet oxygen quantum yield of 0.53 in water. Using density functional theory, the rate of ISC in selenine was estimated to be around 1 × 1010 s–1 while the rate of fluorescence was estimated to be 1.6 × 108 s–1. This report demonstrates that in changing from sulfur to selenium, the rate of triplet formation may improve on the scale of an order of magnitude.28Another study by Rodriguez-Serrano et al. demonstrated that the effect of changing from sulfur to selenium can be considerably more extreme.3) underwent rapid ISC on sub-picosecond time scales, with selenoguanine triplet population being about three times faster than that of thioguanine. Due to the heavy atom effect of selenium, however, selenoguanine relaxed from the triplet state with a time constant of 1.7 ns, more than 830 times faster than thioguanine with a time constant of 1420 ns. In this case, it was hypothesized that the yield of singlet oxygen generation would decrease due to triplet decay outcompeting the diffusion of oxygen.31More recently, Farrell These studies demonstrate that it is important to increase the rate of triplet population when considering incorporation of selenium and tellurium for various applications, as faster triplet population is advantageous for materials that harvest triplet excitons. In addition, it has been highlighted that the rate of triplet decay must also be considered, as it is not desirable for non-radiative emission to outcompete other photophysical processes.3.While many organochalcogens undergo population of the triplet state, others have been reported to display suppressed intersystem crossing and instead emit from the singlet state. This emission may be tailored to turn on or off upon proximity to another analyte which can quench fluorescence. This is often controlled through a photoinduced electron transfer (PET) rather than through conversion to a triplet state. In PET, upon excitation an electron is transferred from the donor to the acceptor, quenching fluorescence .32 Throu4) quenches fluorescence due to photoinduced electron transfer, which was detected using spectrofluorimetry. Additional experiments showed that when the selenium is oxidized by –OCl, PET is inhibited, allowing the dye to fluoresce again. However due to the insolubility of the selenide-modified BODIPY in water, it also displays aggregation induced emission without having been chemically oxidized.5) by X-ray diffraction, which greatly enhances fluorescence.6) which selectively demonstrates turn-on fluorescence in the presence of –OCl.7), resulting in a probe with eleven times the brightness upon oxidation of selenium.40The presence of ROS in cells is undesirable and indicates stress,8) to monitor the release of an anti-tumor agent. ROS levels are elevated in cancer cells. The oxidation of the selenide was used to dissociate drug-carrying capsules in triple-negative breast cancer cells, with ROS-induced fluorescence serving as an indicator that the drug had been released.et al. showed that a diketopyrrolopyrrole-substituted tellurophene (9) undergoes PET in the unoxidized state, then upon oxidation of the tellurium, the dye fluoresces selectively in the presence of H2O2 due to inhibition of PET. This study also reported that the opposite process may be used to indicate the presence of glutathione as the fluorescence is quenched upon its reduction of tellurium.42The photoluminescence change of certain organochalcogens upon exposure to ROS may be used as a turn-on sensor to indicate the release of chemicals. The Li group synthesized an organoselenide coupled to a polycarbonate  produces singlet oxygen in 21% yield, which is useful in photodynamic therapy.versus 1420 ns), resulting in an expected decrease in singlet oxygen yield due to triplet decay being much faster than oxygen diffusion. However, the triplet state may react with a nearby nucleobase more easily through π–π stacking, which would improve photosensitized damage to DNA. DFT has been used to understand that the photophysical mechanism resulting in this pronounced increase in excited state decay involves transitions between five electronic states.48Once organochalcogens undergo ISC into the triplet state, the triplets may be harvested rather than allowed to phosphoresce or decay non-radiatively. The triplet can be transferred to another molecule either to begin a reaction cascade (often to create singlet oxygen for photodynamic therapy)10) (11) and found that FRET was more efficient over a shorter distance, with the covalent linkage resulting in a smaller transfer distance.et al. confirmed that while the emission spectrum of the complex is dominated by the emission of the dye, the scale of its time-gated fluorescence is determined by the persistence of the triplet state of the sensitizer.Triplet sensitizers can additionally help to address signal convolution, which is a large issue in fluorescent sensors for biological applications. Many fluorescent tags used to indicate the presence of biological analytes are short-lived and their emission overlaps with those of fluorescent biomolecules. Longer-lived phosphorescent tags would be advantageous because their emission would outlast that of biological autofluorescence, reducing signal convolution. However, it is challenging to design phosphorescent tags that are organic, water soluble, and luminescent at room temperature. Instead, the Uri and Enkvist groups have synthesized improved fluorescent tags by covalently linking them to triplet sensitizers. Energy from the triplet sensitizer upon excitation can be transferred to the fluorescent dyes through Förster Resonance Energy Transfer (FRET), increasing the fluorescence decay time and allowing it to persist at time scales beyond the nanosecond fluorescence of biomolecules.10) .50 The s5.i) TADF complex (12) resulted in dual emission from both TADF and phosphorescence.13), also resulting in dual emission.Much of the work taking advantage of the photophysical properties of heavy organochalcogens has been directed toward biological applications. The improved spin–orbit coupling resulting from selenium or tellurium substitution may also improve the performance of lighting elements or displays such as organic light emitting diodes (OLEDs) when organochalcogens are used in the emitting layer. The population of triplet states and their subsequent photoluminescence could contribute to higher efficiency phosphorescent OLEDs because fewer electrons are lost to fluorescence or non-radiative decay.Tellurium substitution is expected to affect the photophysics of organochalcogens to a greater extent than selenium substitution. Due to an increase in the heavy atom effect, tellurium incorporation leads to more efficient and faster intersystem crossing to the triplet state compared to lighter organochalcogens.14).Aromatic tellurium heterocycles – tellurophenes – are more synthetically accessible, and their photophysical properties have been studied by the Rivard group.15). When the compound is photoexcited, the halogens are photoreductively eliminated.Just as organotellurium compounds may sensitize other compounds, they may themselves be useful photoactive reagents,6.While the optical applications of sulfur-containing organic compounds are reasonably well-understood, selenium- and tellurium-containing compounds have emerged as photoactive species, yet the photophysical mechanisms are more complex. The rapid population of triplet states and possible subsequent transfer to another molecule may amplify a signal from a fluorescent probe, transduce a signal, induce a reaction, or generate a reactive new species. The continued development of the synthesis of both selenium- and tellurium-containing materials is sure to enable even more applications beyond the examples pointed out above such as sensors and OLEDs. Future studies on the photophysics of heavy organochalcogens, including experimental determination and quantum mechanical prediction of photophysical time constants of singlet and triplet population and decay, will assist in reliably predicting their properties and will contribute to the rational design of new photophysical materials. Broadening synthetic methodologies to incorporate heavier chalcogens and gaining insight on how to manipulate photophysical time constants are both essential to unlocking the potential of selenium and tellurium in photoactive applications.There are no conflicts of interest to declare.


The colour and appearance of liquid laundry capsules have been implicated in the risk of attracting the attention of toddlers, and therefore contributing to poisoning incidents in the home by encouraging interaction. This research set out to explore if differences in colours and contrasting colour designs used in mono and multi-coloured capsules result in different levels of attractiveness. This was performed using two study settings: a laboratory setting (out of context), and by comparison, a more ecologically valid setting, mimicking the real-world. Capsule attractiveness to toddlers was measured by visual attention (measured through eye tracking) and grasping choice . Results from the research in the out of context setting showed statistically significant differences in visual attention and grasping choice between colours and contrasting designs. In the visual attention study a preference for multi-coloured capsules was shown. In the grasping choice study, in addition to multi-coloured, mono-coloured white or purple capsules were also preferred. In the more ecologically valid setting, there were no statistically significant differences in the visual attention or grasping choice between any of the capsules. These results were consistent with each other and in line with market data reflecting poisoning incident rates, which show no change with colour or contrasting colour design. We suggest that the results from out of context studies might not be a reliable indicator of real world behaviour. Given the importance of toddler home safety, using a methodology that is aligned with market numbers is crucial to develop countermeasures. Media news reports have highlighted safety concerns with colours used in liquid laundry capsules, implying that these are attractive to toddlers and hence may contribute to unintentional interaction that could lead to poisoning incidents in the home , 2. On tPrevious studies on the early development of colour preference in children are inconsistent and only limited data are available that explain colour preference in toddlers , 6. OldeThis research set out to formally explore if differences in colours and contrasting colour designs used in mono and multi-coloured capsules result in different levels of attractiveness to toddlers and to explore the impact of a more ecologically valid study design. Using a controlled lab set-up in combination with a more ecologically valid approach will allow us to explore whether previously observed effects occur under typical conditions for the population at large rather than being limited to a laboratory set-up . The curThe research was comprised of four studies–study 1 to 4. Studies 1 and 2 were carried out in the first week of field work and studies 3 and 4 the second following week. Participants taking part in study 1 and 2 also took part in study 3 and 4 together with additional toddlers, required to increase the statistical robustness of studies 3 and 4 whose design divided the toddlers into two independent groups. Participants were recruited from the local community in Frankfurt, Germany representing a cross section of local race and demographics. This study was reviewed and approved in advance by Procter & Gamble’s Independent Human Safety Compliance Department  ref number ELN EXP-16-BI0420, and informed consent was provided by all parents of panellists in writing. In equivalence to university ethics committees, these procedures ensure consistency in approach as well as the protection of the rights, safety, and well-being of all test subjects as well as assuring that P&G’s human testing research program is in compliance with the regulatory requirements, ESOMAR world research codes, Company business and ethical standards, and the Declaration of Helsinki. An overview of the study designs can be found in Study 1: Out of context eye tracking study–examining differences in toddlers’ visual attention to a high-resolution image of different coloured liquid laundry capsules when set in a circle alongside one another on a neutral background Study 2: A more ecologically valid eye tracking study–examining differences in toddlers’ visual attention to a high-resolution image of different coloured liquid laundry capsules when presented in their open packaging (box) in an “under the sink” kitchen cupboard set-up, alongside other household cleaning items. This was designed to mimic that of typical storage in European households Figs  and 4Study 3: Out of context behavioural observation study–examining differences in toddlers’ grasping choice when presented with 3D plastic replicas of differently coloured liquid laundry capsules set in a line alongside one another on a neutral background Study 4: A more ecologically valid behavioural observation study–examining differences in toddlers’ grasping choice when presented with 3D plastic replicas of differently coloured liquid laundry capsules in their open packaging (box) in an “under the sink” kitchen cupboard set-up alongside other household cleaning items. This was designed to mimic that of typical storage in a European household, as per Study 2 Method: On a television monitor, each toddler viewed a single high-resolution image of six different colour capsules arranged in a circle . The imaParticipants: 134 toddlers (mean age in months and standard deviation (M ± SD), 21.59 ± 6.41; 63 boys, 71 girls; range (R), 12–36) were assigned to the task. A total of 23 toddlers were excluded from the study for not meeting the inclusion criteria for sufficient eye tracking data (see below for a more detailed description of these criteria). The data were collected from the remaining 111 toddlers .Stimulus materials: Replica plastic 3D printed capsules of equal size, weight and shape were made. These were painted and varnished with one finish to visually represent, as closely as possible, common capsule colours . These colours were mono-colour green, blue, white, purple and multi-coloured low and high contrast (light blue/blue/dark blue and green/blue/white). One capsule replica representing each of the six colour/colour combinations were arranged in a circle on a neutral grey background. The ordering and sequence of the capsules in the circle was randomised using SAS9.4 software to ensure that each individual colour was in a different position. High resolution images for each position were taken and a single shot was projected individually to each participant on a television monitor following the randomisation pattern.Experimental set-up: A remote infrared eye tracker was used . This equipment registers the focal gaze  every 12 milliseconds (sampling frequency: 120 Hz), and can distinguish spatial changes when the gaze angle differs by 0.3 degrees. The data were used to calculate the direct notice rate of capsules and fixation duration, using Tobii Studio version 3.4.5 software. Looking at a specific capsule (direct noticing) was inferred when the output indicated that the focal gaze of the toddler fell directly on the capsule in the image. Fixation duration was defined as the time spent looking at any given capsule within the 4 second stimulus display. The quality of the recording was checked for every image, and data was excluded for toddlers who did not have any gaze data or who had a large offset (i.e. consistently looking below/above or beside the products). Toddlers with a total fixation duration below 0.75 seconds were excluded from the analyses.Procedure: All eye tracking sessions were conducted in the presence of the accompanying parent, with the toddler sitting on their parent’s lap during the session to minimise stress and maintain a relatively fixed position in relation to the eye tracker, at eye level and 75–100 cm from the screen of the television monitor. The toddler’s gaze was initially calibrated using a picture of a kitten, shown at five specific points on the screen . Study 2 was completed first and all toddlers went on to immediately complete this study and within the same research conditions.Statistical Analysis: A mixed model was used for statistical analysis of fixation durations. Within this model the capsule colour, order within the circle and sequence of the six different coloured capsule stimuli were taken as fixed effects, the participant as a random effect and fixation duration as a response variable. Tukey Honest Significant Difference (HSD) was then used for post-hoc analysis pairwise comparisons across the six coloured capsules –with 15 paired comparisons in total. JMP 13.1 was used for this analysis. The same statistical analysis procedure was followed for the notice rate using a Glimmix model (SAS 9.4). For all comparisons, significance was assessed at the 95% confidence level.Results: For the fixation duration results, the mixed model showed that the fixed effects of colour  = 5.73, p<0.0001) and order  = 3.02, p = 0.01) were statistically significant. This means that colour significantly impacted the mean value of fixation duration as did the position of the capsule. The mixed model showed that the fixed effect of sequence  = 1.06, p = 0.39) was not significant.For the notice rate results the Glimmix model showed that the fixed effects of colour  = 7.09, p < .0001) and order  = 2.83, p = 0.02) were also statistically significant. Consistent with fixation duration it showed that the fixed effect of sequence  = 1.06, p = 0.39) was not statistically significant.The paired comparison results for fixation duration and notice rates between colours are summarised in For the mono-coloured capsules, the findings showed no statistically significant differences in the fixation duration that the toddlers spent looking at the mono-coloured capsules (p>0.07) or in the notice rates (p>0.06).Confidence interval calculations for the paired comparisons of the six colours were completed. The range for the confidence intervals of fixation duration was between -0.49 to 0.35 and the range for the confidence intervals for the % notice rate was from -53 to 26. These ranges demonstrate relatively large variation in the fixation duration and notice rates between toddlers, compared to the minimum significant differences (0.19 seconds and 20%).Findings: The fixation duration and notice rate were significantly greater for the high contrast capsule versus the blue and the purple. For the low contrast capsule a statistically significant difference in greater notice rates and fixation duration was shown versus the blue capsule. There were no statistically significant differences between the mono-coloured capsules.Method: On the same television monitor, each toddler viewed six high resolution images of the six different colour capsules used in study 1. This time the capsules were shown in their open packaging (i.e. a box) along with capsules of the same colour placed in the kitchen cupboard set-up. Each of the six images was projected for four seconds, one after another in a pre-assigned sequence of six. This study measured visual attention using the fixation duration (seconds) and the % notice rate of the opening of the box displaying the capsules as the defined area of interest see Figs  and 4.Participants: The same toddlers participated in this study as in Study 1. However, the number of participants varied between the different images, as toddlers whose gaze was not focused on the screen were excluded from the analysis.Stimulus materials: Six high resolution images of a kitchen cupboard set-up containing cleaning product facsimiles were used. Each cupboard image contained nine identical control household cleaning items  and an open box displaying one of the six colours of capsules used in Study 1 see . The onlProcedure: In this study following calibration of the equipment, the six images were displayed on the screen, interspersed with a video of a circus cartoon of which the length was randomly varied from two to nine seconds. The ordering of the projected images was randomised using SAS9.4 software to ensure that each capsule colour shown was balanced and that it occurred at each possible position of the image sequence.Statistical Analysis: A mixed model was used for statistical analysis of fixation duration. Within this model the colour, order and sequence of the six different images were taken as fixed effects; the participant as a random effect and fixation duration as a response variable. The order and sequence that the images were shown was randomised and balanced. JMP 13.1 was used for this analysis. The same statistical analysis procedure was followed for the notice rate using a Glimmix model (SAS 9.4). For all comparisons, significance was assessed at the 95% confidence level.Results: For the fixation duration results, the mixed model yielded no significant effects for any of the fixed factors  = 0.73, p = 0.60; sequence, F = 1.66, p = 0.15, or colour, F = 1.23, p = 0.29). Likewise, for the notice rate results, all of the fixed effects in the Glimmix model were not significant  = 1.11, p = 0.35; sequence, F = 0.87, p = 0.50; colour, F = 0.58, p = 0.72). The mean values and standard deviations for the fixation durations and notice rates between colours are summarised in Confidence intervals for the paired comparison of the six different images were completed. The range for the confidence intervals of fixation duration was -0.22–0.23 and the range for the confidence intervals for the notice rate was -19%–22%. These ranges demonstrate relatively small variations in the fixation duration and notice rates between toddlers, compared to the minimum significant differences (0.19 seconds and 20%) and those found in Study 1.Findings: No statistically significant differences were found in the toddlers’ fixation duration or notice rates for any of the different coloured capsules displayed in their open box in the cupboard set-up.Method: After completing study 4 and following a 20–30 minute break, Toddlers were sat on a stool at a toddler’s table for a controlled environment grasping choice task.Participants: A total of 188 toddlers  participated in this behavioural task. These toddlers included the 134 toddlers used earlier in studies 1 and 2, plus 54 additional toddlers who were recruited into study 3 and 4 to ensure the base size was robust.Stimulus materials: This task involved six different arrangements (subsets) of three different coloured capsule replicas drawing from the six colours used in studies 1 and 2  using a randomisation pattern. Each time the capsule replicas were placed on set markers on a grey card attached to the table top in front of the seated toddler, to form a standardised line on a neutral background see .Procedure: A MaxDiff procedure was used . Each toStatistical Analysis: SSI web software version 9.1 , 15 was Results: The ANOVA yielded a significant effect of the factor Colour, F = 17.78, p<0.0001. Tukey HSD as post-hoc tests showed the pair comparison results in Findings: In an out-of-context set-up, the white, purple, and both contrast capsules were chosen with significantly higher probability than the green or blue capsules.Method: This behavioural study assessed toddler grasping choices from the cupboard set-up used in study 2 for two of the six colour capsule replicas, by means of a between-subjects design. Green mono-colour and multi-coloured high contrast capsule replicas were used as the most differentiated in visual appearance.Participants: The participant sample included 240 toddlers, 134 of which had also taken part in studies 1 and 2. A total of 118 toddlers  were assigned to the task using the green capsule, and 122 toddlers  to the task using the high contrast capsule.Stimulus materials: The study employed two identical rooms  each with identical cupboard set-ups containing the nine cleaning product facsimiles as described in Study 2 . The cleProcedure: Toddlers were allocated to one of the rooms and allowed up to 10 minutes to settle, so that they were at ease and able to play, during which time the open cupboard was concealed behind a screen. When the toddler had settled, the researcher moved closer to the cupboard and invited the toddler to “take one thing from the cupboard that you would most like to play with”. The position in front of the cupboard was the same for all toddlers and all items were reachable in principle. After the result was recorded, the toddler was asked to give the item to their caregiver, and the item was put to one side. The grasping choice task was repeated three times in total. The frequency with which either a single capsule or the complete box containing the capsules was chosen, was calculated for first, second and third choice. The sum of the three choices was then additionally calculated.Statistical Analysis: The results of the two differently coloured capsules from the cupboards were compared using a Chi-square test (Fisher’s exact test) in JMP13 software. A Monte Carlo simulation was used to confirm the repeatability of the statistical results . For allResults: The percentages of toddlers who chose the green capsules and the multi-coloured high contrast capsules among their first three chosen items from the cupboard are summarised in The Monte Carlo simulation was conducted with the observed probabilities from Findings: No statistically significant differences were found in the percentage of toddlers grasping the green or multi coloured high contrast capsules from the cupboard.This research set out to formally explore if differences in colours and contrasting colour designs used in mono and multi-coloured liquid laundry capsules result in different levels of toddler attractiveness to better understand market accidentology data which, contrary to opinion expressed in the media , 2, showEye tracking was used to measure variations in visual attention and notice rate of toddler’s when looking at different coloured capsules to determine if any colours were particularly preferred. Behaviour observation tasks then measured the degree in which different coloured capsules drove the act of grasping, which examined if particular colours also manifested in different levels of a toddler’s desire to physically interact with them . These tasks were completed in both a neutral out of context set-up and in a more ecologically valid setting to help mimic a realistic home laundry environment.The studies completed in the out of context set-up, showed that the colour of the capsule had a significant impact on both the visual attention and the grasping choice, but that visual attention may not necessarily be predictive of the act of grasping. These results were at odds from those in a more ecologically valid setting which demonstrated both that the colour of the capsule did not have a significant impact, and that the visual attention may be a good predictor of grasping. Importantly, the results from the more ecologically valid setting were consistent with market accidentology data which also show no impact of colour to incident rates.In the out of context eye tracking study the capsules with the multi-coloured high and low contrast designs were noticed by a higher percentage of toddlers and elicited longer looking times versus some of the mono-colour capsules . No differences were found between the mono-coloured capsules, i.e. green, blue, purple and white. In the out of context behavioural study examining grasping choice, the multi-coloured high and low contrast designs were chosen with a higher probability than the mono-coloured blue or green. The mono-coloured white and purple were also chosen with higher probabilities than the others, along with the high and low contrast designs. These results suggest that whereas colour may play a role in visual attention and grasping choice, results achieved in visual attention experiments may not necessarily be predictive of grasping choice.Taylor et al. highlight the increased noticing by toddlers may not necessarily indicate liking, but may instead reflect other factors, such as complexity, salience or novelty–in other words, their increased noticing may have more to do with objects being different or unusual, and so ‘catching their eye’ . This maIn the more ecologically valid eye tracking study no statistically significant differences between the six different coloured capsules were found in the fixation duration or % notice rates of the toddlers. Also, in the more ecologically valid grasping choice task no differences in the probability of choice were observed between the two capsules studied, that is the mono-coloured green and multi-coloured high contrast capsules. We are confident that these results reflect the real situation given that the mean differences between the different coloured capsules found in the studies are small, confidence intervals are narrow and that the results were achieved within the context of large sample sizes. Additionally within the grasping choice exercise where a Monte Carlo simulation was completed, results confirmed high repeatability.The more ecologically valid study findings were aligned with real-world poisoning incident numbers, where the trends in unintentional interaction by toddlers leading to reported incidents, show no consistent differences between the most prevalent colours  were looking at 2D images of liquid laundry capsules which may lose certain characteristics of the actual 3D object, such as distinctive texture, lustre, etc. that could be interesting to a toddler.The out of context findings of a statistical preference for multi-coloured capsules in Study 1, and in the case of Study 3, of a statistical preference for mono-coloured white and purple as well as multi-colours over green and blue, showed that colour may have an impact on the level to which toddlers may be attracted to capsules but that visual attention may not necessarily correlate with the act of grasping. These colour preference results are not aligned with real-world data. In comparison, no statistical differences in levels of attractiveness when looking at mono-coloured or multi-coloured capsules were found when investigated within a more ecologically valid setting (Studies 2 and 4). Within this context visual attention was predictive of grasping and importantly matched in-market, real-world data. Overall, the data from the more ecologically designed studies do not support a link between the colour of capsules and differences that colours may elicit in either visual attention or grasping–with its associated risk of poisoning incidents. The findings suggest that caution should be exercised in extrapolating outcomes from research conducted in an out of context setting to predict the risk of poisoning incidents in the home, and recommends a more ecological study design with a valid real-world setting to do this.This work was completed to contribute to a broader study designed to help uncover what drives toddler interaction with laundry capsules that may lead to poisoning incidents in the home to help develop effective counter-measures. Findings support the importance of child-safe packaging and parental awareness as strategies to prevent unintentional interactions , rather than changes in capsule colour design.S1 Fig(XLSX)Click here for additional data file.S2 Fig(XLSX)Click here for additional data file.S3 Fig(XLSX)Click here for additional data file.S4 Fig(XLSX)Click here for additional data file.S5 FigSupporting statistical data from all studies.(XLSX)Click here for additional data file.