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A multi-strain Synbiotic may reduce viral respiratory infections in asthmatic children: a randomized controlled trial

Background and objective Asthma is a growing problem worldwide. Acute exacerbations impose considerable morbidity, mortality, and increased cost. Viral respiratory infections are the most common cause (80–85%) of pediatric asthma exacerbations and admissions to the hospital. The aim of this study was to determine the effect of a new synbiotic Lactocare® on viral respiratory infections and asthma exacerbations in asthmatic children. Methods In this double blind, placebo-controlled, randomized clinical trial, 72 children with mild persistent asthma, aged between 6 and 12 years, were randomized to receive either Lactocare®, a Synbiotic containing 1 billion CFU/Capsule of Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium infantis, Lactobacillus bulgaricus, and Fructooligosacharide (Zist Takhmir, Tehran, Iran) or placebo daily for 60 days. The primary outcome was the number of viral respiratory infections, and secondary outcomes were school absence, salbutamol and prednisolone usage, outpatient visits, and hospital admission for asthma. The outcomes were compared among study groups using the SPSS 11.5 program and the Mann Whitney and Fisher exact tests. Results Of the 72 children who were enrolled with mild persistent asthma, 36 were assigned randomly to be treated with synbiotic and 36 with placebo. The number of viral respiratory infections was significantly higher in placebo group than the synbiotic group during the first month of intervention (0.74 ± 0.12 vs. 0.44 ± 0.1, p < 0.007) but not during the second month (0.5 ± 0.8 vs. 0.5 ± 0.8, p < 0.641). Considering the total duration of the study (two months), infection episodes also were significantly lower in the synbiotic group (0.92 ± 0.15 vs. 0.69 ± 0.11, p < 0.046). Salbutamol consumption was significantly lower in the synbiotic group, but there were no significant differences in school absenteeism, oral prednisolone use, outpatient visits, or hospital admissions. Conclusion This new synbiotic (a mixture of seven probiotic strains plus fructooligosacharide may reduce episodes of viral infection in asthmatic children. Trial registration This study is registered in Iranian Registry of Clinical Trials with registration number of IRCT201509234976N3. Funding This research was supported financially by the Research Council of Mashhad University of Medical Sciences (Grant Number: 911048).


Introduction
Asthma is an increasing problem worldwide. It is the most common chronic disease of childhood, and it is a common cause of childhood morbidity, including school absences, emergency visits, and hospitalizations (1). The acute exacerbations that continue to occur constitute a major burden on health care resources and have been associated with decreased lung growth (2,3). Viral respiratory infections are the most common cause (80 -85%) of pediatric asthma exacerbations and admissions to the hospital (4). Recent studies have shown that the airway's epithelial cells in asthmatics can produce less anti-viral inteferons IFN-β and IFN-λ when infected with Human Rhinoviruses (HRVs), making them potentially more susceptible to viral infections (5). Current medications for the prevention of virus-induced exacerbation of asthma's symptoms are not effective, and novel therapies should be sought (6). Hand hygiene techniques, a balanced diet, probiotics, and the immunostimulant OM-85 are considered to be the most effective methods for preventing the common cold in asthmatic children (6). Many clinical trials have used probiotics for the prevention of the common cold in healthy children and adults with promising results (7)(8)(9)(10)(11)(12)(13)(14). Synbiotics, defined as a combination of a probiotic and a prebiotic, the aim of which is to increase the survival and activity of probiotics, can reduce respiratory viral infection by modulation of the Th1/Th2 responses in the lungs (15)(16)(17). Some studies have shown that synbiotics may reduce the duration of viral infections in healthy school children and reduce Th2 cytokine in asthmatic children (18,19). The aim of this study was to determine efficacy of synbiotic Lactocare®, a mixture of seven probiotic strains plus fructooligosacharide (FOS) in reducing seasonal viral upper respiratory infection in asthmatic children.

Research design and setting
This double-blind, placebo-controlled, randomized clinical trial was conducted at Sarvar Teaching Hospital in Mashhad, Iran, with the sample population recruited from the outpatient pediatric allergy clinic from September 2012 to September 2014.

Participants
Children in the age range of 6 to 12 years with mild persistent asthma and a history of recurrence (at least two viral upper respiratory infections in past autumn) were enrolled. We excluded those with with congenital abnormalities or syndromes, antibiotic or probiotic consumption during the two weeks before the study, and any underlying disease. During the first visit, demographic information was collected, including gender, age, birth weight, antibiotic consumption, family history of allergies, exposure to allergens and irritants, history of recurrent infection, and severity of asthma.

Sample size
According to previous studies that showed a 20-30% reduction (14) in common colds with a probiotic, we estimated a sample size of 35 in each group using an α value of 0.05 with a power of 90% and possible 20% follow-up loss. N = 7.8(S1 2 +S2 2 )/(M2-M1) 2 = 28.38

Randomization
Computer-generated randomization was conducted by a university statistician.

Blinding
In this double-blinded study, the treatment plan of each group was prepared by the hospital pharmacist and delivered to the investigator. The patient was not aware of the type of solution that was being used. Interventions were concealed from the study's investigators, the pharmacist, and the parents to minimize treatment bias.

Interventions
Following randomization of the patients, they received daily for two months either Lactocare®, a synbiotic containing 1 billion CFU/Capsule of Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium infantis, Lactobacillus bulgaricus, and Fructooligosacharide (Zist Takhmir, Tehran, Iran) or a placebo that was matched with the color, size, and shape of the medication. Before enrolment in the study, an education session was conducted focusing on recommended hygienic measures, including frequent hand washing, use of disposable tissues, having sick family members wear a face mask and keep a distance of a meter away from the patient, and avoiding any kind of smoking. Patients were assessed by a single physician at enrolment, at the end of the first month, at the end of second month, and every time they had respiratory tract symptoms when a viral respiratory infection was suspected. A complete history of any new Page 2835 symptom, drug usage (salbutamol, prednisolone), school absenteeism, outpatient visits, and hospital admission was taken, and physical examinations were done to rule out any presumed new signs, such as wheezing, otitis media, and pneumonia.

Outcomes
The total number of respiratory viral infection episodes over two months of follow-up was considered as the primary outcome. Acute viral upper respiratory tract infection episodes (acute nasopharyngitis or common cold) were diagnosed clinically by a single pediatrician in the presence of diagnostic symptoms (fever, sneezing, cough, rhinorhea, and sore throat) that lasted at least 48 hours. Multiple episodes were counted only if the patient was without symptoms for at least 72 hours between episodes. School absenteeism, salbutamol usage, prednisolone usage, outpatient visits, and hospital admissions were considered as secondary outcomes.

Research ethics
The protocol for this study was approved by Ethics Committee of Mashhad University of Medical Sciences.

Statistical methods
The SPSS 11.5 program (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. For normally distributed variables, values were reported as mean ± SD. The t-test, chi-squared test, and/or the Fisher exact test were used as appropriate to compare baseline demographics and the clinical characteristics among the study groups. Normality was assessed using the 'Kolmogorov-Smirnov' test.

Baseline characteristics
Of the 75 children enrolled with mild, persistent asthma, 37 were assigned randomly to be treated with Synbiotic and 38 with placebo. One patient in the synbiotic group was excluded from the analysis due to non-compliance and two patients in placebo group were excluded for the following reasons: lost to follow up (n = 1) and abdominal pain (n = 1). Seventy-two children completed the study, 36 patients in each groups (Figure 1). There were no significant differences in baseline characteristics between the synbiotic and placebo groups (Table 1).

Viral respiratory infection
There was a significant difference for the number of viral respiratory infections between the intervention and placebo groups during the first month of treatment (p < 0.007); this was not the case during the second month (p < 0.641) ( Table 2). Considering the total duration of the study (two months), infection episodes also were significantly lower in the synbiotic group (p < 0.046).

Salbutamol consumption
Salbutamol consumption was significantly lower in the synbiotic group than in the placebo group (p < 0.017), but there were no significant differences between the two groups in school absenteeism oral prednisolone, outpatient visits, or hospital admissions (Table 3).

Side effects
There were no side effects in either group.

Discussion
In the present study, a synbiotic containing a mixture of seven probiotic strains and the prebiotic fructooligosacharide was associated with fewer episodes of viral respiratory infection in children with mild persistent asthma. These results are promising as the common cold occurs each fall and winter, and current drugs for the prevention of virus-induced exacerbation of asthma are not very effective. In this study, we focused on patients with a history of recurrent infection in the previous season, as among inner-city children with asthma. A history of exacerbation in the previous season is the strongest predictor of asthma exacerbation in fall and winter (20). Several studies of healthy children and adults have shown the benefits of probiotics in respiratory infections. In 2015, a Cochrane review written by Hao et al. concluded that probiotics were better than placebo in reducing the number of participants who experienced episodes of acute upper respiratory infection, reducing the mean duration of an episode, reducing the use of antibiotics, and reducing cold-related absences from school (14). The therapeutic role of probiotics in asthma has been tested in a few clinical trials considering various outcomes and study designs. The earlier studies failed to show any significant benefits. In 1997, Wheeler concluded that yogurt that contained L. acidophilus produced borderline elevated interferon gamma levels in asthmatic adults, but it did not improve lung function or quality of life (21). In 2007, Giovannini et al. showed that consuming fermented milk containing a Lactobacillus casei over a 12-month period did not change the episodes of fever or diarrhea in asthmatic children whose ages ranged from two to five (22). These results may be explained by the fact that the effects of probiotics depend on dose, duration, and the strains used in trials, and only specific strains with accurate dose can reduce the episodes of upper respiratory infections. Some researchers confirmed the benefits of probiotics by using different study protocols. In Poland, Gutkowski and colleagues evaluated the effects of the treatment of asthmatic children with a 12-week regimen of Trilac capsules, which contained a mixture of Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus delbrueckii subsp. Bulgaricus (23). Trilac significantly improved lung function and reduced episodes of asthma exacerbations. Previous studies have shown that a mixture of multiple probiotic strains might be more effective than a single strain, although the components of the mixture should be chosen carefully based on their proven efficacy for treating that special disease, because the efficacy of the mixtures may be decreased by any inhibitory effects that exist between the different probiotic strains (24). We used a mixture of seven probiotic strains plus the prebiotic fructooligosacharide (FOS). Many of these strains have proven efficacy in reducing viral respiratory infections. The prebiotic in the symbiotic mixture improved the survival of the probiotics and the host endogenous good bacteria, and it also stimulated induction of regulatory T lymphocytes and immune tolerance. In 2011, Van de Pol et al. showed that treatment with synbiotics (short-chain galactooligosaccharides, long-chain fructo-oligosaccharide and Bifidobacterium breve) in adults with allergic asthma significantly reduced the production of Th2-cytokines after allergen challenge and improved peak expiratory flow rates (19). In 2013, a systematic review of the effects of probiotics in allergic airway diseases was conducted by Dos et al. and they concluded that probiotics did not improve quality of life scores in asthmatics but could improve the following parameters, i.e., longer time free from episodes of asthma and decrease in the number of episodes of rhinitis per year. They also concluded that routine use of probiotics as an additive therapy in subjects with allergic airway diseases should not be recommended (25). Our study also showed the effects of synbiotics on reduction of number of viral respiratory episodes in asthmatic children. In another study in Austria, Stocker and colleagues concluded that in asthmatic children in the age range of 6 to 12, laser acupuncture for 10 weeks plus probiotic Enterococcus faecalis for seven weeks had fewer days of acute febrile infections than the control group (26).
In our study, although prednisolone usage, outpatient visits, school absences, and hospital admissions were lower in the synbiotic group, the difference was not statistically significant. This was the same as the results of another study in which Lactobacillus rhamnosus (ATCC 53103) made no significant change in asthma medications during Birch pollen season (27). In fact, some strains of probiotics may even worsen the asthma-like symptom and increase the need for rescue medication (28). An extensively hydrolyzed formula with Bifidobacterium breve M-16V and a galacto / fructooligosaccharide mixture prevents asthma-like symptoms, including 'frequent wheezing' and 'wheezing and/or noisy breathing' in infants with atopic dermatitis (29). These different results emphasize the importance of accurately selecting the proper probiotic strain in treating allergic disorders. In our study, the use of synbiotics significantly reduced Salbutamol usage during the period of viral respiratory infections. One other study also showed that probiotics may reduce bronchodilator usage in asthmatic patients (23). The advantages of our study were the use of a new synbiotic that contained seven probiotics, the study population (children), and careful followup of the patients several times during the study. The limitations of this study were that we did not perform pulmonary function tests or laboratory evaluation of immunologic effects.

Conclusions
A synbiotic that contains a mixture of seven probiotic strains plus fructooligosacharide may reduce episodes of viral respiratory infection in asthmatic children. For clear recommendations, well-designed RCTs with longer follow-up, larger sample sizes, and different probiotic mixtures are needed to determine the ideal, effective product, dosage, and duration for asthmatic patients.

Acknowledgments:
This manuscript was derived from a Paediatric thesis by Dr. Toktam Ganji, and the research was supported financially by the Vice Chancellor for Research, Mashhad University of Medical Sciences. The authors wish to acknowledge Professor Peter Sly from The University of Queensland for his scientific support and reviewing the manuscript.