Heterogeneity of surrogate outcome measures used in critical care studies: A systematic review.

BACKGROUND: The choice of outcome measure is a critical decision in the design of any clinical trial, but many Phase III clinical trials in critical care fail to detect a difference between the interventions being compared. This may be because the surrogate outcomes used to show beneficial effects in early phase trials (which informed the design of the subsequent Phase III trials) are not valid guides to the differences between the interventions for the main outcomes of the Phase III trials. We undertook a systematic review (1) to generate a list of outcome measures used in critical care trials, (2) to determine the variability in the outcome reporting in the respiratory subgroup and (3) to create a smaller list of potential early phase endpoints in the respiratory subgroup. METHODS: Data related to outcomes were extracted from studies published in the six top-ranked critical care journals between 2010 and 2020. Outcomes were classified into subcategories and categories. A subset of early phase endpoints relevant to the respiratory subgroup was selected for further investigation. The variability of the outcomes and the variability in reporting was investigated. RESULTS: A total of 6905 references were retrieved and a total of 294 separate outcomes were identified from 58 studies. The outcomes were then classified into 11 categories and 66 subcategories. A subset of 22 outcomes relevant for the respiratory group were identified as potential early phase outcomes. The summary statistics, time points and definitions show the outcomes are analysed and reported in different ways. CONCLUSION: The outcome measures were defined, analysed and reported in a variety of ways. This creates difficulties for synthesising data in systematic reviews and planning definitive trials. This review once again highlights an urgent need for standardisation and validation of surrogate outcomes reported in critical care trials. Future work should aim to validate and develop a core outcome set for surrogate outcomes in critical care trials.

Re-analysis of ventilator-free days (VFD) in acute respiratory distress syndrome (ARDS) studies.

BACKGROUND: Over recent decades, improvements in healthcare have reduced mortality and morbidity rates in many conditions. This has resulted, in part, from the identification of effective interventions in randomised trials, and in conducting such trials, a composite outcome measure (COM) with multiple components will increase event rates, which allows study completion with a smaller sample size. In critical care research, the COM "ventilator-free days" (VFD) combines mortality and duration of mechanical ventilation (MV) into a single continuous measure, which can be analysed in a variety of ways. This study investigates the usefulness of Poisson and two-part Poisson models compared to t-distribution for the analysis of VFD. METHODS: Data from four studies (ALbuterol for the Treatment of ALI (ALTA), Early vs. Delayed Enteral Nutrition (EDEN), Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute Lung Injury (ALI) to reduce pulmonary dysfunction (HARP-2), Statins for Acutely Injured Lungs from Sepsis (SAILS)) were used for analysis, with the VFD results summarised using mean, standard deviation (SD), median, interquartile range (25th and 75th percentiles) and minimum and maximum values. The statistical analyses that are compared used the t-test, Poisson, zero-inflated Poisson (ZIP) and two-part Logit-Poisson hurdle models. The analyses were exploratory in nature, and the significance level for differences in the estimates was set to 0.05. RESULTS: In HARP-2, which compared simvastatin and placebo, the mean (SD) VFD for all patients was 12.0 (10.2), but this mean value did not represent the data distribution as it falls in a zone between two peaks, with the lowest frequency of occurrence. The mean (SD) VFD after excluding patients who died before day 28 and patients who did not achieve unassisted breathing were 15.9 (8.7) and 18.2 (6.6), respectively. The mean difference (95% CI) between the two groups was 1.1 (95% CI: 0.7 to 2.8; p = 0.20) based on an independent t-test. However, when the two-part hurdle model was used, the simvastatin arm had a significantly higher number of non-zero values compared to the placebo group, which indicated that more patients were alive and free of mechanical ventilation in the simvastatin group. Similarly, in ALTA, this model found that significantly more patients were alive and free of MV in the control group. In EDEN and SAILS, there was no significant difference between the control and intervention groups. CONCLUSION: Our analyses show that the t-test and Poisson model are not appropriate for bi-modal data (such as VFD) where there is a large number of zero events. The two-part hurdle model was the most promising approach. There is a need for future research to investigate other analysis techniques, such as two-part quantile regression and to determine the impact on sample size requirements for comparative effectiveness trials.

Aspirin as a Treatment for ARDS: A Randomized, Placebo-Controlled Clinical Trial.

BACKGROUND: There is no pharmacologic treatment for ARDS. Platelets play an important role in the pathophysiology of ARDS. Preclinical, observational, and clinically relevant models of ARDS indicate aspirin as a potential therapeutic option. RESEARCH QUESTION: Is enteral aspirin (75 mg, once daily) safe and effective in improving surrogate outcomes in adult patients with ARDS? STUDY DESIGN AND METHODS: This randomized, double-blind (patient and investigator), allocation-concealed, placebo-controlled phase 2 trial was conducted in five UK ICUs. Patients fulfilling the Berlin definition of ARDS were randomly assigned at a 1:1 ratio to receive enteral aspirin (75 mg) or placebo, for a maximum of 14 days, using a computer-generated randomization schedule, with variable block size, stratified by vasopressor requirement. The primary end point was oxygenation index (OI) on day 7. Secondary outcomes included safety parameters and other respiratory physiological markers. Analyses were by intention to treat. RESULTS: The trial was stopped early, due to slow recruitment, after 49 of a planned 60 patients were recruited. Twenty-four patients were allocated to aspirin and 25 to placebo. There was no significant difference in day 7 OI [aspirin group: unadjusted mean, 54.4 (SD 26.8); placebo group: 42.4 (SD 25); mean difference, 12.0; 95% CI, -6.1 to 30.1; P = .19]. Aspirin did not significantly impact the secondary outcomes. There was no difference in the number of adverse events between the groups (13 in each; OR, 1.04; 95% CI, 0.56-1.94; P = .56). INTERPRETATION: Aspirin was well tolerated but did not improve OI or other physiological outcomes; a larger trial is not feasible in its current design. TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT02326350; URL: www. CLINICALTRIALS: gov.

Transepithelial nasal potential difference in patients with, and at risk of acute respiratory distress syndrome.

BACKGROUND: Impaired alveolar fluid clearance, determined in part by alveolar sodium transport, is associated with acute respiratory distress syndrome (ARDS). Nasal sodium transport may reflect alveolar transport. The primary objective of this prospective, observational study was to determine if reduced nasal sodium transport, as measured by nasal potential difference (NPD), was predictive of the development of and outcome from ARDS. METHODS: NPD was measured in 15 healthy controls and in 88 patients: 40 mechanically ventilated patients defined as 'at-risk' for ARDS, 61 mechanically ventilated patients with ARDS (13 who were previously included in the 'at-risk' group) and 8 ARDS survivors on the ward. RESULTS: In at-risk subjects, maximum NPD (mNPD) was greater in those who developed ARDS (difference -8.4 mV; 95% CI -13.8 to -3.7; p=0.005) and increased mNPD predicted the development of ARDS before its onset (area under the curve (AUC) 0.75; 95% CI 0.59 to 0.89). In the ARDS group, mNPD was not significantly different for survivors and non-survivors (p=0.076), and mNPD was a modest predictor of death (AUC 0.60; 95% CI 0.45 to 0.75). mNPD was greater in subjects with ARDS (-30.8 mV) than in at-risk subjects (-24.2 mV) and controls (-19.9 mV) (p<0.001). NPD values were not significantly different for survivors and controls (p=0.18). CONCLUSIONS: Increased NPD predicts the development of ARDS in at-risk subjects but does not predict mortality. NPD increases before ARDS develops, is greater during ARDS, but is not significantly different for controls and survivors. These results may reflect the upregulated sodium transport necessary for alveolar fluid clearance in ARDS. NPD may be useful as a biomarker of endogenous mechanisms to stimulate sodium transport. Larger studies are now needed to confirm these associations and predictive performance.

The Effectiveness of Individual or Group Physiotherapy in the Management of Sub-Acromial Impingement: A Randomised Controlled Trial and Health Economic Analysis.

Background: Shoulder pain is common in primary care. The management of subacromial impingement (SAI) can include corticosteroid injections and physiotherapy. Physiotherapy can be on an individual or group basis. Aim: To examine the clinical effectiveness and make an economic analysis of individual versus group physiotherapy, following corticosteroid injection for SAI. Design and Setting: A single-blind, open-label, randomised equivalence study comparing group and individual physiotherapy. Patients referred by local general practitioners and physiotherapists were considered for inclusion. Method: Patients were randomised to individual or group physiotherapy groups, and all received corticosteroid injection before physiotherapy. The primary outcome measure was shoulder pain and disability index (SPADI) at 26 weeks. An economic analysis was conducted. Results and Conclusion: 136 patients were recruited, 68 randomised to each group. Recruitment was 68% of the target 200 participants. SPADI (from baseline to 26 weeks) demonstrated a difference (SE) in mean change between groups of -0.43 (5.7) (p-value = 0.050001), and the TOST (two-one-sided test for equivalence) 90% CI for this difference was (-10.0 to 9.14). This was borderline. In a secondary analysis using inputted data, patients without SPADI at week 26 were analysed by carrying forward scores at week 12 (mean difference (95% CI) = -0.14 (-7.5 to 7.3), p-value = 0.014). There is little difference in outcome at 26 weeks. Group physiotherapy was cheaper to deliver per patient (£252 versus £84). Group physiotherapy for SAI produces similar clinical outcomes to individual physiotherapy with potential cost savings. Due to low recruitment to our study, firm conclusions are difficult and further research is required to give a definitive answer to this research question. (NCT Clinical Trial Registration Number NCT04058522).

Low-dose (0.01%) atropine eye-drops to reduce progression of myopia in children: a multicentre placebo-controlled randomised trial in the UK (CHAMP-UK)-study protocol.

BACKGROUND/AIMS: To report the protocol of a trial designed to evaluate the efficacy, safety and mechanism of action of low-dose atropine (0.01%) eye-drops for reducing progression of myopia in UK children. METHODS: Multicentre, double-masked, superiority, placebo-controlled, randomised trial. We will enrol children aged 6-12 years with myopia of -0.50 dioptres or worse in both eyes.We will recruit 289 participants with an allocation ratio of 2:1 (193 atropine; 96 placebo) from five centres. Participants will instil one drop in each eye every day for 2 years and attend a research centre every 6 months. The vehicle and preservative will be the same in both study arms.The primary outcome is SER of both eyes measured by autorefractor under cycloplegia at 2 years (adjusted for baseline). Secondary outcomes include axial length, best corrected distance visual acuity, near visual acuity, reading speed, pupil diameter, accommodation, adverse event rates and allergic reactions, quality of life (EQ-5D-Y) and tolerability at 2 years. Mechanistic evaluations will include: peripheral axial length, peripheral retinal defocus, anterior chamber depth, iris colour, height and weight, activities questionnaire, ciliary body biometry and chorioretinal thickness. Endpoints from both eyes will be pooled in combined analysis using generalised estimating equations to allow for the correlation between eyes within participant. Three years after cessation of treatment, we will also evaluate refractive error and adverse events. CONCLUSIONS: The Childhood Atropine for Myopia Progression in the UK study will be the first randomised trial reporting outcomes of low-dose atropine eye-drops for children with myopia in a UK population. TRIAL REGISTRATION NUMBER: ISRCTN99883695, NCT03690089.

The prevention and treatment of glucocorticoid-induced osteopaenia in juvenile rheumatic disease: A randomised double-blind controlled trial.

BACKGROUND: Children and young people (CYP) with chronic rheumatic conditions; Juvenile Idiopathic Arthritis, Juvenile Systemic Lupus Erythematosus, Juvenile Dermatomyositis and Juvenile Vasculitis, treated with steroids, have low bone density, increased fracture risk and are likely to have suboptimal peak bone mass. There is currently no evidence base for the management of steroid-induced bone loss in children with rheumatic diseases. METHODS: We undertook a multi-centre double dummy double-blind randomised placebo controlled trial to investigate whether the bisphosphonate risedronate was superior to alfacalcidol or calcium and vitamin D supplementation in the prevention and treatment of steroid-induced osteopaenia in these children. Patients were stratified and randomised in a 1:1 ratio, into: placebo; alfacalcidol; risedronate. The primary outcome was the change in lumbar spine bone mineral density z score (LSaBMDz) measured by dual energy x-ray absorptiometry at one year. Secondary outcome was fracture rate. RESULTS: Two hundred and seventeen patients were recruited to the study. Seventy seven placebo, 71 alfacalcidol, and 69 risedronate. Highly statistically significant differences were observed in the change in LSaBMDz between the placebo and risedronate groups; 0.274, 95% CI (0.061, 0.487) (p < 0.001) and between the risedronate and the alfacalcidol groups; 0.326 95% CI (0.109, 0.543) (p < 0.001). The difference observed between the alfacalcidol and placebo group was not statistically significant.Highly statistically significant differences were seen in the change in Total Body Less Head aBMD-Z Score between the placebo and risedronate groups (p < 0.01) but not between the alfacalcidol and risedronate groups. No significant differences in fracture frequency, adverse or serious adverse reactions were observed between the groups. CONCLUSIONS: Children and adolescents receiving steroids for rheumatic diseases benefit from prophylactic treatment with bisphosphonates to increase LSaBMD. Alfacalcidol is ineffective.

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9.

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50µg/mouse) was evaluated. RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells. CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.

The effectiveness of non-pharmacological interventions in reducing the incidence and duration of delirium in critically ill patients: a systematic review and meta-analysis.

PURPOSE: To evaluate the effect of non-pharmacological interventions versus standard care on incidence and duration of delirium in critically ill patients. METHODS: We searched electronic and grey literature for randomised clinical trials up to March 2018. Two reviewers independently screened, selected and extracted data. Meta-analysis was undertaken using random effects modelling. RESULTS: We identified 15 trials (2812 participants). Eleven trials reported incidence of delirium. Pooled data from four trials of bright light therapy showed no significant effect between groups (n = 829 participants, RR 0.45, 99% CI 0.10-2.13, P = 0.19, very low quality evidence). Seven trials of various individual interventions also failed to report any significant effects. A total of eight trials reported duration of delirium. Pooled data from two trials of multicomponent physical therapy showed no significant effect [n = 404 participants, MD (days) - 0.65, 99% CI - 2.73 to 1.44, P = 0.42, low quality of evidence]. Four trials of various individual interventions also reported no significant effects. A trial of family voice reorientation showed a beneficial effect [n = 30, MD (days) - 1.30, 99% CI - 2.41 to - 0.19, P = 0.003, very low quality evidence]. CONCLUSIONS: Current evidence does not support the use of non-pharmacological interventions in reducing incidence and duration of delirium in critically ill patients. Future research should consider well-designed and well-described multicomponent interventions and include adequately defined outcome measures.

Oral health behaviours of parents and young children in a practice-based caries prevention trial in Northern Ireland.

OBJECTIVES: The NICPIP trial evaluated the costs and effects of a caries prevention intervention delivered to 2- to 3-year-old children attending dental practices in Northern Ireland. This supplementary study explored the oral health behaviours of children and their parents to help understand the reasons for the trial's findings. METHODS: A mixed methods study that included a questionnaire completed by all parents (n = 1058) at the time they brought their child for the NICPIP final clinical assessment. The questionnaire collected data on frequency of toothbrushing and sugar consumption. Questionnaire data were analysed by trial group and caries status. Parents of trial participants (n = 42) were invited to take part in telephone interviews. Parents were purposively sampled according to trial group and whether or not their child developed caries. The interviews explored how and why oral health behaviours happened. Interview data were audio-recorded, transcribed verbatim and analysed thematically. RESULTS: The questionnaire data indicated that toothbrushing and between-meal sugar snacking were common in the majority of children. The children of parents who automatically reminded their child to brush their teeth were more likely to remain caries-free (Odds Ratio 1.24; 95% CI 1.08, 1.41; P = .002). Frequency of sweet drink consumption was associated with the child developing caries (Odds Ratio 0.88; 95% CI 0.79, 0.98; P = .021). The interview data showed that parents had positive attitudes towards brushing both in terms of perceived importance and expected outcomes. Attitudes towards sugar snacking were more complex, with parents reporting difficulties in controlling this behaviour. Sugar was described as being something that was "ever present" in children's lives. CONCLUSIONS: Toothbrushing was widely adopted from a young age, but between-meal sugar consumption was highly prevalent. The results suggest that effective family-level and population-level interventions are needed to reduce sugar consumption if substantial improvements in caries prevention are to be achieved.

A randomised controlled trial to measure the effects and costs of a dental caries prevention regime for young children attending primary care dental services: the Northern Ireland Caries Prevention In Practice (NIC-PIP) trial.

BACKGROUND: Dental caries is the most common disease of childhood. The NHS guidelines promote preventative care in dental practices, particularly for young children. However, the cost-effectiveness of this policy has not been established. OBJECTIVE: To measure the effects and costs of a composite fluoride intervention designed to prevent caries in young children attending dental services. DESIGN: The study was a two-arm, parallel-group, randomised controlled trial, with an allocation ratio of 1 : 1. Randomisation was by clinical trials unit, using randomised permuted blocks. Children/families were not blinded; however, outcome assessment was blinded to group assessment. SETTING: The study took place in 22 NHS dental practices in Northern Ireland, UK. PARTICIPANTS: The study participants were children aged 2-3 years, who were caries free at baseline. INTERVENTIONS: The intervention was composite in nature, comprising a varnish containing 22,600 parts per million (p.p.m.) fluoride, a toothbrush and a 50-ml tube of toothpaste containing 1450 p.p.m. fluoride; plus standardised, evidence-based prevention advice provided at 6-monthly intervals over 3 years. The control group received the prevention advice alone. MAIN OUTCOME MEASURES: The primary outcome measure was conversion from caries-free to caries-active states. Secondary outcome measures were the number of decayed, missing or filled tooth surfaces in primary dentition (dmfs) in caries-active children, the number of episodes of pain, the number of extracted teeth and the costs of care. Adverse reactions (ARs) were recorded. RESULTS: A total of 1248 children (624 randomised to each group) were recruited and 1096 (549 in the intervention group and 547 in the control group) were included in the final analyses. A total of 87% of the intervention children and 85% of control children attended every 6-month visit (p = 0.77). In total, 187 (34%) children in the intervention group converted to caries active, compared with 213 (39%) in the control group [odds ratio (OR) 0.81, 95% confidence interval (CI) 0.64 to 1.04; p = 0.11]. The mean number of tooth surfaces affected by caries was 7.2 in the intervention group, compared with 9.6 in the control group (p = 0.007). There was no significant difference in the number of episodes of pain between groups (p = 0.81). However, 164 out of the total of 400 (41%) children who converted to caries active reported toothache, compared with 62 out of 696 (9%) caries-free children (OR 7.1 95% CI 5.1 to 9.9; p < 0.001). There was no statistically significant difference in the number of teeth extracted in caries-active children (p = 0.95). Ten children in the intervention group had ARs of a minor nature. The average direct dental care cost was £155.74 for the intervention group and £48.21 for the control group over 3 years (p < 0.05). The mean cost per carious surface avoided over the 3 years was estimated at £251.00. LIMITATIONS: The usual limitations of a trial such as generalisability and understanding the underlying reasons for the outcomes apply. There is no mean willingness-to-pay threshold available to enable assessment of value for money. CONCLUSIONS: A statistically significant effect could not be demonstrated for the primary outcome. Once caries develop, pain is likely. There was a statistically significant difference in dmfs in caries-active children in favour of the intervention. Although adequately powered, the effect size of the intervention was small and of questionable clinical and economic benefit. FUTURE WORK: Future work should assess the caries prevention effects of interventions to reduce sugar consumption at the population and individual levels. Interventions designed to arrest the disease once it is established need to be developed and tested in practice. TRIAL REGISTRATION: Current Controlled Trials ISRCTN36180119 and EudraCT 2009-010725-39. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 20, No. 71. See the NIHR Journals Library website for further project information.

Sector analysis of 125I permanent prostate brachytherapy provides a rapid and effective method of evaluating and comparing pre- and post-implant dosimetry.

PURPOSE: To evaluate a sector analysis program in the assessment and comparison of pre- and post-implant dosimetric parameters during the development of an (125)I permanent prostate brachytherapy service. METHODS AND MATERIALS: A total of 50 consecutive men being treated with permanent prostate brachytherapy had dose-volume analysis in 12 sectors of their pre-implant ultrasound (USpre) and post-implant CT (CTpost) studies. Individual sectors were created by dividing prostate into three equal lengths, namely base, midgland, and apex. Each of these volumes was then divided into four axial sectors. Dosimetric parameters were compared in adjoining sectors within each study and between studies. RESULTS: There were statistically significant differences between individual sectors on USpre and CTpost volumes with CTpost higher than USpre (p=0.001). Statistically significant differences were found in corresponding sectors on USpre and CTpost for all dosimetric parameters. The dosimetric parameters were significantly lower on CTpost in the anterior base and midgland (p=0.001) and significantly higher at the posterior apex and midgland (p=0.05). Dose homogeneity was demonstrated in adjoining sectors in all USpre and most adjoining sectors on CTpost. CONCLUSIONS: Sector analysis allows rapid assessment of USpre and CTpost dosimetry. It offers a scientific method of identifying areas of increased and reduced dosing on CTpost when compared with USpre, providing a learning tool to refine dosimetric analysis and highlight sectors where implant quality could be improved.

Transmission dynamics of methicillin-resistant Staphylococcus aureus in a medical intensive care unit in India.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a global pathogen and an important but seldom investigated cause of morbidity and mortality in lower and middle-income countries where it can place a major burden on limited resources. Quantifying nosocomial transmission in resource-poor settings is difficult because molecular typing methods are prohibitively expensive. Mechanistic statistical models can overcome this problem with minimal cost. We analyse the transmission dynamics of MRSA in a hospital in south India using one such approach and provide conservative estimates of the organism's economic burden. METHODS AND FINDINGS: Fifty months of MRSA infection data were collected retrospectively from a Medical Intensive Care Unit (MICU) in a tertiary hospital in Vellore, south India. Data were analysed using a previously described structured hidden Markov model. Seventy-two patients developed MRSA infections and, of these, 49 (68%) died in the MICU. We estimated that 4.2% (95%CI 1.0, 19.0) of patients were MRSA-positive when admitted, that there were 0.39 MRSA infections per colonized patient month (0.06, 0.73), and that the ward-level reproduction number for MRSA was 0.42 (0.08, 2.04). Anti-MRSA antibiotic treatment costs alone averaged $124/patient, over three times the monthly income of more than 40% of the Indian population. CONCLUSIONS: Our analysis of routine data provides the first estimate of the nosocomial transmission potential of MRSA in India. The high levels of transmission estimated underline the need for cost-effective interventions to reduce MRSA transmission in hospital settings in low and middle income countries.