Efficacy and tolerability of SEDIFLÙ in treating dry or productive cough in the pediatric population (SEPEDIA): A pilot,randomized,double-blind,placebo-controlled,multicenter clinical trial

Abstract

The aim of this trial was to assess the effectiveness of Sediflù, a medical device containing active herbal ingredients, on nocturnal and diurnal persistent coughs in children, with a duration of 3 to 7 days. Children with a dry and/or productive cough were enrolled in this prospective, interventional, multicenter, placebo-controlled, double-blind, randomized clinical study. Clinical efficacy was assessed through the evaluation of the soothing action of Sediflù against dry and/or productive coughing, both at night and during the day, and other effects of coughing associated with quality of sleep: frequency, child’s quality of sleep, parental quality of sleep and severity. Treatment with Sediflù improved both night-time and day-time cough scores from day 2. The diurnal score also improved significantly in the Sediflù group at days 3 and 7. Sediflù syrup can be considered a valid treatment for cough management in younger children with upper respiratory tract infections, shortening the cough duration.

Background

Coughing and irritation of the mucosa are mainly associated with upper respiratory tract infections, such as the common cold. In young children, respiratory tract infections are extremely common and frequently recurrent. ¹ Pathogens often colonize the upper respiratory tract (nose or mouth) prior to causing lower respiratory infections or invasive diseases. The oral mucosa and epithelium play a protective role against the entry of microorganisms into the body. ² This mucosa is the main target tissue of microorganisms, such as viruses. Normally, primary infection results in viral replication at mucosal surfaces.

Upper respiratory tract infection is very frequent. Adult subjects experience 2 to 5 episodes per year, whereas school children suffer 7 to 10 episodes per year, coughing up 140 days per year. Children are almost 4 times more likely to experience upper respiratory infection-associated acute coughs than adults. ³ In this context, coughing is the most common symptom of many inflammatory diseases.

Coughing is a physiological response to airway irritation. It has 2 main functions: (1) to prevent the entry of food and fluids into the lower airways and (2) to favor the removal of material that exceeds the transport capacity of the mucociliary system. Coughing can be initiated by pharyngeal stimulation or mucosal dehydration. A cough may be classified according to its duration and characteristics. It is considered to be occasional when it lasts less than 3 days. Persistent coughs that last more than 7 days are usually treated with medicinal products. However, persistent coughs lasting more than 3 days but less than 7 can be particularly bothersome and their appropriate management might help to improve symptoms, without compromising the patient’s natural cough recovery mechanisms. Coughs may be wet, such as a productive cough associated with secretion, or dry, such as an irritative cough. ³

An acute cough consequent to an upper respiratory tract infection may persist in children for many days. A cough, mainly when prolonged, can impact quality of life, cause anxiety and distress, and affect the sleep of children and their parents or cohabitants.^3,4 Coughing, especially during the night, can be particularly troubling to children and to their parents, as it often results in discomfort to the child and loss of sleep for both the child and their parents. In addition, there have been very few assessments of the associated sensations: irritation, the urge to cough, and even pain, yet these may be more important symptoms for patients with laryngitis or a post-viral cough than the actual coughing. ⁵ In addition, a sore throat represents inflammation or irritation. Throat irritation also dehydrates the pharyngeal mucosa and is commonly accompanied by a dry cough, aggravating the irritative state.

No treatment option has been associated with clear benefits in terms of cough recovery. ⁶ Given that some therapeutic options exhibit low effectiveness and also produce undesirable adverse effects, they are not optimal treatments. ⁷ Non-pharmacological interventions, such as throat and pharynx demulcents and emollients, are an alternative for cough management and mucosa protection and do not have side effects.

A suitable strategy to treat coughing and irritation is to form a protective barrier by a formulation able to adhere to and reside on the mucosal surface, in the oral cavity as far as the pharynx. This protective barrier can be achieved with a demulcent and/or emollient effect. A demulcent is a substance that forms a soothing film over a mucous membrane to relieve minor pain and inflammation. Emollients are the ingredients responsible for the smooth feel of the skin or mucosa.

The demulcent capacity is a physical action that confers a protective effect on the mucous membranes. This action can lead to decreased coughing, and its efficacy has been demonstrated in clinical studies elsewhere. ⁸ Furthermore, the formation of this layer on the epithelial surface or the oral cavity allows rehydration, increasing saliva production, desensitization, and a decrease in local inflammation and the cough reflex. ⁸ Demulcent, emollient, and lubricant effects are reinforced by a mucoadhesive effect.

The ideal physical and chemical characteristics for a cough syrup can be obtained by a complex mixture of natural substances exerting lubricant, demulcent, and protective barrier effects. ³ The optimal combination of ingredients to produce a humectant action is natural sweetness, such as honey, which stimulates salivation, and herbal extracts associated with complementary actions. Up to 85% of the benefit of cough syrups may be due to the physical and chemical effects of the syrup, which contribute to its demulcent action.

The syrup tested in this study is a class IIa medical device containing different active herbal ingredients: eucalyptus honey and dry extracts of horehound flowers (Marrubium vulgare L.), sundew flowers (Drosera rotundifolia L.), ivy leaves (Hedera helix L.), and plantain leaves (Plantago lanceolata L.).

The ingredients of this syrup involve a combination of actions with a potential benefit for cold symptoms. Sediflù uses 2 main mechanisms to produce the desired effects: (a) mucolytic and expectorant actions (moisturizing and fluidifying action of mucus that favors its physiological elimination) and (b) demulcent effect (soothing and protective effects against external irritants by forming a protective barrier and increasing saliva production to reduce the cough reflex). ⁸ Due to this double action, it is recommended both for productive coughs (due to its mucolytic and expectorant effect, as well as its demulcent action) and for dry coughs (due to its demulcent effect).

The aim of this randomized trial was to assess the effectiveness of this medical device, as compared with a placebo, on nocturnal and diurnal persistent coughs in children, with a duration of 3 to 7 days.

Methodology

Study Treatment

The product administered to the treatment group is a vegetable extracts-based medical device manufactured by Sakura Italia Srl and marketed in different EU territories with their corresponding brand names (Seditus tos, Sediflù tosse, Actirub toux, and Complexe expert Sirop toux séche et grasse). It is a syrup containing eucalyptus honey, dry extract of horehound flowers (Marrubium vulgare L.), dry extract of sundew flowers (Drosera rotundifolia L.), dry extract of ivy leaves (Hedera helix L., 10% hederacoside C), and dry extract of plantain leaves (Plantago lanceolata L., 5% total phenols and 1% verbascoside). The placebo product was an inactive syrup, without the active components listed above, with the same appearance, density, brix and flavor as Seditus.

Either the treatment or the placebo was administered in 2 doses per day (morning and evening), 10 ml each, for 7 days, as recommended in the instructions for use of the marketed product.

Design

The study was conducted as a prospective, interventional, multicenter, placebo-controlled, double-blind, randomized clinical study to evaluate the clinical response and tolerance of the medical device in patients aged 5 to 11, with a dry and/or productive cough.

Study Population

Children were recruited, between November 2020 and March 2021, in 2 primary care centers in Madrid (Consultorio local de Brunete, Brunete, Madrid, Spain and Pediatría Dr Carlos Núñez de Prado Aparicio, Majadahonda, Madrid). The criteria for inclusion were the signature of informed consent by their parents, having an age ranging from 5 to 11 and suffering at the time of study initiation from a dry and/or productive cough, with a duration of 3 to 7 days.

Patients were excluded if they were under therapies that could interfere with the evaluation of the product under test, especially other antitussives or cough relief products, or if the children suffered from serious lung, kidney and/or liver diseases, neoplasms of any kind, allergy, hypersensitivity or any other type of incompatibility with some of the components of the product under study. Other exclusion criteria were participation in another clinical study in the last month, any surgery scheduled during their participation in the study or any other cause that, in the opinion of the investigator, could compromise compliance with the protocol.

A simple balanced randomization (1:1) was carried out, in which the random distribution of each group (either treatment or placebo) was ensured. The purpose of randomization was to ensure that each patient had an equal chance of being assigned to either of the 2 treatment groups.

Ethical Considerations

The study was conducted in compliance with the ICH-GCP (International Committee on Harmonization of Good Clinical Practice) Guideline and appropriate procedures were always followed to ensure compliance with Regulation (EU) 2016/679. It was approved by the local Ethics Committee of Madrid (CEIm). The participants’ parents gave their written informed consent before the study was carried out.

Primary and Secondary Endpoints

The primary endpoint of the study was to assess the clinical efficacy of Seditus through the evaluation of its soothing action against dry and/or productive coughing, both at night and during the day.

The following clinical findings were documented during visits in accordance with both the Cough Clinical Score (CCS) (modified from Chung, 2002) ⁹ and Paul’s Night Cough Questionnaire. ¹⁰

Cough Clinical Score provides the primary endpoint, and it is composed of 2 variables: night-time cough score and day-time cough score. This severity perception method includes not only severity criteria, but also intensity and impact of cough as a whole (Table 1).

Table 1.

Day-Time and Night-Time Cough Clinical Scores (CCS) (Chung Modification, 2002). ⁹

Cough score	Symptom for day-time	Symptom for night-time
0	Absent	Absent
1	For a short period (approximately a few minutes)	Only at awakening/only before falling asleep
2	For 2 short periods (approximately 10 minutes)	Awaken once/early awaken due to cough
3	Frequent cough that does not interfere with normal activities	Frequently awaken due to cough
4	Frequent cough that interferes with normal activities	Frequent cough for the most part of the night
5	Disturbing cough for the most part of the day	Disturbing cough

The secondary endpoints were the evaluation of the tolerability of Seditus in accordance with Paul’s Night Cough Questionnaire, a survey that specifically assesses the effects of coughing on several variables associated with quality of sleep: frequency, child’s quality of sleep, parental quality of sleep, severity, and bothersomeness (Table 2).

Table 2.

Evaluation of Tolerability According to Paul’s Night Cough Questionnaire. ¹⁰

Question	Score
1. How frequent was your child’s coughing last night?	0: Not at all 1: Not much 2: A little 3: Somewhat 4: A lot 5: Very much 6: Extremely
2. How severe was your child’s cough last night?
3. How bothersome was last night’s cough to your child?
4. How much did last night’s cough affect your child’s ability to sleep?
5. How much did last night’s cough affect your (parental) ability to sleep?

The clinical safety was evaluated through the registration of adverse effects in patients and the identification of those that are device-related adverse effects.

The duration of the study was 14 days per patient, with a screening visit (or visit 1), where either Seditus or a placebo was provided to the patients, 1 week of treatment (visit 2 at day 7) and 1 follow-up week (through a phone call to monitor the product’s safety) (Figure 1). The assessment was continuous (from days 0 to 7) through the subject’s diary. At the end of the treatment period (visit 2), the bottles of the administered products (both treatment and placebo) were returned to the principal investigators so that they could assess compliance with the posology described in the protocol.

Figure 1.

Study schedule.

Statistical Analysis

A descriptive statistical analysis of the results of quantitative biometric variables at different experimental times was carried out, including basic descriptive parameters (central tendency and variation) that reliably expose the distribution of the main variable at each time. Night-time and day-time cough scores are considered qualitative variables, ranging from 0 (absent) to 5 (the most disturbing cough). A subgroup analysis was performed for each variable, according to data ≥ 3 and < 3, to assess the effects in children with more intense symptoms.

Cumulative logit mixed-effects models (CLMMs) were used, adjusting them to the data distributions in each response variable (day-time cough score, night-time cough score, frequency score, child’s sleep score, parental sleep score, severity score, and bothersomeness score), with the aim of evaluating the clinical response of the product at each experimental time (D0, D1, D2, D3, D4, D5, D6, and D7). The effect of the product on the values of the main variables in the statistical analyses is interpreted with reference to the initial time (D0), which corresponds to the screening and first administration of the treatment, and between treatment groups at each experimental time. All the models used in the study for data analysis are present in the “clmm” function of the “ordinal” package of the R software.

The performance of multiple biometric measurements over time (volunteers evaluated during different time points), and therefore correlated, was taken into account by including random effects at the level of each individual, allowing the interception of the models to vary randomly between individuals in the trial. The value of significance established for all the statistical tests of the study is P < .05.

Results

Baseline Characteristics

Sixty children with a dry and/or productive cough were recruited in the study. Of them, 56 were enrolled, randomized to either the treatment (n = 27) or placebo (n = 29) group and completed the study. The mean age of the children was 8.65 ± a standard deviation (SD) of 2.14, with similar baseline characteristics for the 2 treatment groups (Table 3). Boys and girls accounted for 53% and 47%, respectively, of the total sample of enrolled children.

Table 3.

Baseline Characteristics of Children Enrolled for the Study.

Group	Parameter	Mean	Standard deviation	Minimum	Maximum
Sediflù (n = 27)	Age (years)	8.82	2.44	5	11
	Weight (kg)	33.64	12.94	17	67
	Height (cm)	135.17	16.77	107	172
Placebo (n = 29)	Age (years)	8.47	1.82	6	11
	Weight (kg)	31.66	12.93	17	65
	Height (cm)	131.02	14.43	111	163
Total (n = 56)	Age (years)	8.65	2.14	5	11
	Weight (kg)	32.65	12.87	17	67
	Height (cm)	133.09	15.65	107	172

Efficacy of the Product: Cough Score

According to the primary endpoint of the study, the changes in night-time and day-time cough scores were evaluated between D0 (visit 1, basal time) and the other experimental times (D1-D7). There is a decrease in night-time and day-time cough scores in the Sediflù group between the experimental times and the baseline from the second day (D2) to the last day (D7) (Figure 2). The difference between D2 and D0 is statistically significant, and it is maintained throughout the rest of days (D3-D7) (P < .01).

Figure 2.

Evolution of the day-time and night-time cough scores ± standard error over time of Sediflù.

Significant differences were also detected favoring the Sediflù group, in the inter-group analysis, for both scores. The improvement of the night-time cough score is statistically significantly higher in the Sediflù group (28% lower) at D4, in comparison with the placebo group (P < .05). Similarly, at D5, D6, and D7, patients in the Sediflù group also reduced the score by 36%, 44%, and 71%, respectively, as compared with the placebo group (P < .05) (Figure 3). In line with these findings, the diurnal score improved significantly in the Sediflù group (22% lower) at D3, in comparison with the placebo group (P < .05) as well as at D4, D5, D6, and D7, with a reduction of 36%, 43%, 58% and 71%, respectively, in comparison with the placebo group (P < .01).

Figure 3.

Average percentage change in treatment group relative to the placebo by the day-time and night-time cough score ± standard error over time.

As per the day-time and night-time cough score, the variables studied with Paul’s questionnaire rapidly improved after treatment with Sediflù. In fact, there was a significant decrease in the scores of the 5 nocturnal variables associated with the quality of sleep (frequency, severity, bothersomeness, and child’s quality of sleep and parental quality of sleep) from D2 (Figure 4). The difference between D2 and D0 is statistically significant, and it is maintained throughout the rest of the days (D3-D7).

Figure 4.

Evolution of the frequency, severity, bothersomeness, child’s sleep, and parental sleep scores ± standard error over time of Sediflù.

Considering the intra-group analysis, at D7, the Sediflù group causes a reduction in the frequency score of 89% in comparison with D0 (P < .001). For the severity score, the Sediflù group showed a decrease of 87% (P < .001) from D0. The bothersomeness score also suffered a significant decrease at D2 in the Sediflù group, going down to 87% at D7, as compared with the baseline values at D0.

In relation to the intra-group analysis of the child’s quality of sleep score, a significant improvement was observed from D2 in the Sediflù group, with a score reduction of 30% (P < .001), reaching an improvement of 89% (as compared with the baseline values) at D7. Similarly, the parental sleep score showed a significant reduction (P < .01) of 30% in the Sediflù group at D2. This score reduction reached 92% at D7, having the original values as the reference.

To further evaluate the effect of Sediflù in children with a more severe cough (ie, children with cough scores ≥ 3), a subgroup analysis was conducted, dividing all the participants into 2 subgroups (≥ 3 and < 3). The intra-group analysis showed a significant reduction of both frequency and severity scores from first day (D1) of treatment in children who received Sediflù (P < .05).

Considering the inter-group analysis for frequency, severity, bothersomeness, child’s sleep and parental sleep, Sediflù showed a significant improvement, for all variables (70%, 51%, 52%, 58%, 66% lower, respectively) at D7, in comparison with the placebo group (P < .01). These percentages are particularly relevant taking into consideration that the baseline scores of all the variables in the Sediflù group were higher than those present in the placebo group, thus reinforcing the effect of the treatment with Sediflù syrup, in comparison with placebo (Figure 5).

Figure 5.

Average percentage change in the treatment group relative to the placebo across various factors, including the frequency, severity, bothersomeness, child’s sleep, and parental sleep scores ± standard error over time.

Safety

No adverse effects were observed either in the treatment or placebo group.

Discussion

This study corresponds to a prospective, interventional, multicenter, randomized, placebo-controlled, double-blind clinical investigation designed to evaluate the potential benefits of Sediflù syrup for children with a dry or productive cough that persists for 7 days. Children without known severe underlying diseases commonly suffer, particularly during the cold months, from several respiratory infections involving mainly the upper respiratory tract. Therefore, effective and safe therapeutics are needed. There is no scientific evidence to justify the use of over-the-counter cough medicinal products (antitussives, mucolytic, and/or antihistamines), as they could have potentially serious side effects, and thus should not be prescribed to children on a regular basis. Products with honey and herbal extracts, such as Sediflù syrup, are an effective and safe alternative treatment to drugs. In these upper respiratory tract infections, coughing is a functional disorder frequent in children and it is one of the most frequent reasons for consultation in daily pediatric practice. ¹¹ - ¹³

Sediflù syrup is a medical device containing eucalyptus honey and dry extracts of plantain leaves (Plantago lanceolata L.), horehound flowers (Marrubium vulgare L.), sundew flowers (Drosera rotundifolia L.), and ivy leaves (Hedera helix L.). The aerial part of plantain or ribwort plantain (Plantago lanceolata L.) has been used for centuries by traditional medicine in the treatment of infectious disorders of the respiratory tract. Its beneficial properties are due to the significant content of mucilaginous polysaccharides, mainly rhamnogalacturonans, galactans, arabinogalactans, and xylogalacturonans. ¹⁴ Horehound flowers (Marrubium vulgare L.) have also been traditionally used in Europe as well as in Southern and Eastern Mediterranean countries, and one of their therapeutic uses is treatment of respiratory diseases. ¹⁵ Ivy leaves (Hedera helix L.) showed effectiveness for treating symptoms of respiratory tract infections and improving the respiratory function, mainly for pediatric coughs, by reducing their frequency and intensity, with high tolerability. ¹⁶ - ²⁰ Finally, sundew flowers (Drosera rotundifolia L.) have a long history as a remedy for coughs, being probably one of the most widely distributed carnivorous plant species. ²¹

The results of this clinical trial show a significant reduction in night-time and day-time cough symptoms in children who received Sediflù syrup, as measured using a specific and validated evaluation scale. Antitussive effects, in night and day times, improved significantly at day 2 and they continued ameliorating during the 7 days of treatment, with a significant difference favoring the Sediflù treatment, in comparison with the placebo.

Similar findings were observed in cough frequency, severity, and bothersomeness, as well as in the child’s quality of sleep and parental quality of sleep, which are variables specifically designed to assess nocturnal coughing. Indeed, the reduction of disturbances in night-time coughing is a very important topic in cough management, as it causes considerable discomfort and sleep disturbances both in children and their parents.

Sediflù syrup reduces the frequency and severity of coughing from the first day of treatment in a subset of children who had more severe coughs (ie, CCS ≥ 3). Rapid recovery, 24 hours after the first administration of Sediflù, is of extreme interest, provided that these variables are recognized as the 2 main disturbances associated with coughing. Concerning bothersomeness and the quality of sleep of children and their parents, significant improvements were also observed for these variables in the Sediflù group.

The effectiveness Sediflù is a consequence of the combined action of its ingredients. This syrup treats coughing through a mechanical mode of action related to demulcent, bioadhesive, protective, and expectorant characteristics of the combination of honey with these herbal extracts, without an action similar to antitussive drugs that acts either on the cough center of central nervous system that controls coughing or on the peripheral cough receptor, mainly located in the trachea and pharynx.

The active components of Sediflù syrup, in addition to honey, include mainly mucilaginous polysaccharides from plantain, lactones and phenolic compounds from horehound, saponins from ivy and naphthoquinones from sundew.

The ingredients of Sediflù syrup create a protective film on the mucosa, calming coughing and protecting the upper respiratory tract. In an inflammatory process of the throat and pharynx, the function of the epithelial surface is altered causing subsequent physical irritation of the membranes. Adhesion to the mucosa limits contact with external irritating agents, promotes hydration and reduces damage caused by the mucosal contact of microorganisms and other irritant agents. ²² - ²⁴

Honey is used to treat upper respiratory tract symptoms and it is generally believed to be safe outside of the pediatric population. Honey is a lay remedy for upper respiratory tract infections and has an emerging evidence base for its use. ²⁵ The World Health Organization (WHO) in 2001 considered the use of honey of interest in the treatment of coughs, due to its demulcent properties ²⁶ . The WHO reports that demulcents may soothe the throat and can be recommended to provide some relief from coughing in children. As evidenced by some previous studies, honey products may have a beneficial effect for symptomatic relief of coughing, even nocturnal coughing, associated with upper respiratory tract infections.^4,25,27,28

Mucilaginous polysaccharide-containing plants are widely used for therapeutic treatment of irritations of mucous membranes in the pharynx regions. In addition to the demulcent activity of honey, these polysaccharides adhere to the oral and pharynx mucosa and exert a mechanical barrier effect. In fact, polysaccharides are related to a protective effect on irritation of the mucosa and with a cough-reduction effect mediated by chemical or mechanical changes.^22,23 It is a protective effect produced mechanically but not pharmacologically, due to the demulcent effect of polysaccharides. Moreover, the bioadhesive effect allows the formation of a protective layer of polysaccharides due to the mucoadhesive effect of their hydrophilic macromolecular structures. Polysaccharides have a high number of hydroxyl groups, allowing a high binding capacity with water and causing hydration.^22,24 The formation of this layer on the epithelial surface allows rehydration, desensitization, a decrease in local inflammation, and a reduction in dry coughing. Hydration is one of the recognized non-pharmacological interventions in the cough approach and is causally related to the reduction of the sensitivity of the cough reflex. ²⁹ Furthermore, the effect on the reduction of local inflammation is produced by a mechanical barrier effect related to mucoadhesive capacity.

Extracts obtained from Plantago sp. are characterized by their mucilaginous polysaccharide content. ¹⁴ Their effect is complemented with lactones from horehound, mainly marrubiin, a labdane diterpene, which is characteristic for the genus, and it acts as a good antioxidant agent in conjunction with various phenolic compounds. ³⁰ Ivy leaves contain saponins (hederacosides) and produce a secretolytic effect, inducing type II alveolar epithelial cells to generate more surfactant yielding for reduction of mucus viscosity, a physicochemical effect. ¹⁷

Moreover, the sweet taste and palatability provided by honey, and its physical and chemical properties, are responsible for modulating cough sensitivity. ³¹ - ³³ It is an effect related to the stimulation of sensory pathways that influence the normal response of the body, contributing to reflex processes, such as salivation. These sensory pathways are also associated with those that participate in the cough process, being able to inhibit their reflex, without pharmacological mechanisms.

The study was conducted between November 2020 and March 2021, when the use of masks was compulsory in Spain as a result of the COVID-19 pandemic. Due to this fact, cough episodes have been milder and less frequent than in previous years. This is the main limitation of this study, since the cough-associated disturbances of the children enrolled were lower than expected. In addition, children in the Sediflù group had baseline scores for all the variables studies slightly higher than those present in the placebo group. Finally, although the inclusion criteria only allowed for the enrollment of children with a cough duration between 3 and 7 days, specific information on the duration of the cough in each comparison group was not collected. Children with COVID-19 were not included in the study; however, no other tests were performed. Despite these limitations, Sediflù syrup proved to be effective in reducing all the variables associated with both diurnal and nocturnal coughing in children and confirmed its safety profile as well as its tolerability. Further studies should be conducted with a larger sample size and a wider age range of the participants.

Conclusion

Sediflù syrup exerts positive effects by reducing the main disturbances associated with day-time and night-time cough scores, shortening the duration of coughs in children. Cough severity and frequency are significantly reduced from day 1 of treatment with Sediflù, which showed a very good safety profile. Based on these findings, Sediflù syrup can be considered a valid treatment for cough management, especially in younger children with upper respiratory tract infections, thanks to the creation of a protective mechanical barrier on oropharyngeal mucosa and its complementary physicochemical effects.

Author Contributions

CN: Contributed to conception, contributed to acquisition, drafted manuscript, critically revised manuscript, gave final approval, agrees to be accountable for all aspects of work ensuring integrity and accuracy. MCC: Contributed to conception, contributed to acquisition, drafted manuscript, critically revised manuscript, gave final approval, agrees to be accountable for all aspects of work ensuring integrity and accuracy. FT: Contributed to conception, contributed to interpretation, drafted manuscript, critically revised manuscript, gave final approval, agrees to be accountable for all aspects of work ensuring integrity and accuracy. CC-R: Contributed to conception, contributed to interpretation, drafted manuscript, critically revised manuscript, gave final approval, agrees to be accountable for all aspects of work ensuring integrity and accuracy. ER: Contributed to design, contributed to analysis and interpretation, drafted manuscript, critically revised manuscript, gave final approval, agrees to be accountable for all aspects of work ensuring integrity and accuracy.