Bronchoprovocation with hypertonic saline solution in asthmatic children

Instituição: Departamento de Pediatria da Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brasil 1Mestre em Pediatria e Ciências Aplicadas à Pediatria pela Unifesp; Fisioterapeuta; Docente do Centro Universitário de Ensino Superior de Maceió (Cesmac), Maceió, AL, Brasil 2Doutora em Pediatria e Ciências Aplicadas à Pediatria pela Unifesp; Fisioterapeuta; Docente da Universidade Nove de Julho (Uninove), São Paulo, SP, Brasil 3Professor Titular e Livre-Docente da Disciplina de Alergia, Reumatologia e Imunologia do Departamento de Pediatria da Escola Paulista de Medicina da Unifesp, São Paulo, SP, Brasil  ABSTRACT


Introduction
In Brazil, asthma is responsible for 350,000 hospitalizations annually, and it is the fourth leading cause of hospitalization in the Brazilian public Unified Health System (SUS), and the third among children and young adults.It is defined as a chronic inflammatory disease, characterized by hyperreactivity of the lower airways and airflow limitation, with spontaneous reversion or by treatment.It is classified regarding severity, according to clinic and spirometric parameters, in: intermittent, mild persistent, moderate persistent, and severe persistent (1) .
The assessment of pulmonary function is the method of choice to determine the restriction of airflow and establishing the diagnosis of asthma.In symptomatic individuals with normal spirometry, the diagnosis of asthma can be confirmed by demonstrating the presence of bronchial hyperreactivity (BH) through bronchoprovocation tests (BPTs), which reflect the sensitivity or the ease of airway response to external stimuli or a marked response to β-2 agonist agents (1) .
The BPTs considered gold standard are those using pharmacological agents such as histamine and methacholine (2)(3)(4) .However, the difficulty of access to these agents has motivated the search for alternative provocative agents.In 1981, there was the first report of the use of hypertonic saline solution (HSS) (4) .Inhalation on HSS increases water loss via airways to the outside world.After such osmolar stimulus, the release of mediators from bronchial mucosa cells occurs, causing the contraction of the smooth muscle and then bronchospasm (4)(5)(6) .
BPT with HSS has good sensibility and specificity in the assessment of BH in children and adolescents.Although BH occurs in normal asymptomatic patients, patients with asthma have it more often (6)(7)(8)(9)(10) .In asthmatic patients, BPT with methacholine, although not universally, require gradient in the concentration of drug needed to induce the decrease of 20% or more in the forced expiratory volume in the first second (FEV 1 ) in relation to asthma severity.In most cases, more severe cases of asthma need lower doses of methacholine to induce the same level of bronchoconstriction (2) .
In this context, the objective of this study was to verify if a BPT with HSS at 4.5% allows detecting gradient of response according to disease severity and the presence of atopy in asthmatic children and adolescents.

Method
This was a cross-sectional study, in which the sample size calculation was based on a pilot study with 14 individuals.These patients presented: intermittent asthma (three), with mean of 11.6 years and reduction of 20% of FEV 1 in mean time of 5 minutes (30 seconds to 9 minutes and 30 seconds); persistent mild asthma (six), with mean of 8.1 years and mean time of decrease of 3 minutes and 54 seconds (1 minute and 30 seconds to 5 minutes and 30 seconds); moderate persistent asthma (three), with mean of 9.6 years and mean time of decrease in 2 minutes and 10 seconds (1 minute and 30 seconds to 3 minutes and 30 seconds); and severe persistent asthma (two), both with 11 years and mean time of decrease of 5 minutes and 30 seconds.Using as variable the time of FEV 1 decline (in seconds), considering the maximum error acceptable of 30 seconds, significance level of 5% and power of 80%, it would be necessary to include 75 patients.In this pilot study, it was found that children aged 6 or older had an adequate cognitive development to participate in the test.
The study included 75 patients aged between 6 and 18 years, diagnosed with asthma for at least 1 year and able to perform spirometry appropriately.These patients were referred by doctors from SUS outpatient services or from services linked to the Health Department of the state of Alagoas, which is a reference for the treatment and medical monitoring of children and adolescents with asthma.
Patients were non-smokers, had no other associated disease (pulmonary, cardiac or neurological) that could interfere in the evaluations nor had had upper respiratory tract infection in the last 30 days and/or exacerbation of asthma in the last week.In addition to clinical data obtained on admission, patients were classified according to severity of asthma in: intermittent, mild persistent, moderate persistent and severe persistent (1) .Then, patients and their parents were informed about the discontinuation of medications that could affect the test results: systemic corticosteroids (3 weeks), antihistamines and inhaled corticosteroids (48 hours), and theophylline and/or β-2 adrenergic agents (24 hours).
Patients underwent immediate hypersensitivity puncture skin tests to aeroallergens: Dermatophagoides pteronyssinus, D. farinae e Blomia tropicalis (FDA Allergenics ® ), taking the solution of histamine (1 mg/mL) as positive control and the excipient solution, as negative.We considered as a positive result the test that induced papular induration, whose diameter was higher than or equal to 3 mm (11) .Patients with at least one positive test were defined as atopic and those in which all results were negative, as non-atopic.
After acclimatization for 5 minutes in a special room at 22º C, children performed the measurement of their pulmonary function while seated, with nose clip and Easy One spirometer (pm) (Cradle Medical Technology ® , model 2010, USA).All were previously trained for the proper execution of spirometry (12) .The values obtained (FEV 1 , forced vital capacity -FVC -and the relation between FEV 1 /FVC) were compared with normal values (Polgar and Promadhat (12) ) and expressed in a percentage of the predicted value, being considered reference for the other evaluations.
Patients were, then, submitted to the BPT with HSS at 4.5%, which is the most widely used because it combines high sensitivity, specificity, reproducibility and it is standardized (5) , with an ultrasonic nebulizer (Pulmossonic Star Luxo II, Soniclear ® ) with 10 mL volume, flow rate around 1.25 mL/ minute (steady), 80% aerosol and diameter less than 4 microns.
During the BPT, children inhaled HSS at 4.5% for increasing periods (0.5; 1; 2; 4 and 8 minutes) at intervals of 2 minutes, totaling 15 minutes and 30 seconds, depending on a positive response or not.FEV 1 was measured 1 minute after the end of each inhalation.Drops of FEV 1 equal or higher than 20% in relation to the basal value authorized the interruption of BPT.Patients who did not have a drop after 15 minutes and 30 seconds of inhalation were categorized as having negative bronchoprovocation (4,5) .
At the end of BPT, a new evaluation was performed: vital signs, PA and SpO 2 .Patients also received inhalation with β-2 agonist agent of short duration (salbutamol aerosol via metered dose inhaler -400 mcg/dose) to relieve possible bronchospasm.
For statistical analysis, initially, we performed the comparison between the different degrees of severity of asthma.The absence of differences made the patients with intermittent and persistent mild asthma comprise the IM Group while those with persistent moderate or severe asthma composed the MS Group.The IM Group worked as a control group, once the Research Ethics Committee of Universidade de Ciências da Saúde de Alagoas (Uncisal) did not authorize the submission of non-asthmatic individuals to BPT with HSS at 4.5%.All variables were also analyzed considering atopic patients versus non-atopic ones.
Sample homogeneity was verified by the Kolmogorov-Smirnov test.The variables analyzed did not present normal distribution, and their values were described as median values.The Kruskal-Wallis non-paramedic test was employed for variables such as time and percentage of FEV 1 decrease, according to severity of asthma and the presence of atopic status.For the analysis of vital signs changes, we used the Mann-Whitney non-paramedic test.We considered as statistical significance when p≤0.05.
The study was approved by the Research Ethics of Universidade Federal de São Paulo (Unifesp), protocol 1039/08, and Universidade de Ciências da Saúde de Alagoas (Uncisal), protocol 817/2008.Patients and their guardians were informed about the research and signed an informed consent form.

Results
Out of all asthmatic patients referred to be submitted to BPT with HSS at 4.5% (n=96), eight patients were not admitted for having basal value of FEV 1 70% lower than the predicted value, other eight because they presented other associated diseases that could interfere with the evaluations and five because they could not perform acceptable and reproducible spirometry.Thus, 75 children and adolescents with asthma were included in the study.
Such patients, after the immediate hypersensitivity skin test, composed two groups: atopic (n=60) and non atopic (n=15) to household dust mites (Table 1).At the beginning of the outbreak, the two groups were similar in relation to age and predicted basal values of FEV 1 (Table 1).BPT with HSS was positive in 58 patients (77.3%), 9 (60.0%)non-atopic and 48 (80.0%), atopic.Although there was no difference between the percentage values of FEV 1 at the end of the test, the time needed for the drop to occur was significantly higher in the non-atopic.The percentage of FEV 1 reduction in relation to basal values was higher among the atopic, when compared to non-atopic patients (Figures 1 and 2).Considering individuals with negative test, the mean reduction of the FEV 1 was 2.6% (variation of <1 to 5%) among the non-atopic and 10.7% (variation of <1 to 17%) among atopics.Among the atopic patients, four patients (30%) presented reduction higher than 15%.
The initial analysis, considering the severity of asthma as variable, did not show statistically significant differences between patients with intermittent and mild persistent asthma, what allowed us to group them (intermittent + mild -IM Group).The same occurred in relation to patients with moderate persistent asthma and severe persistent asthma (moderate group + severe -MS).However, among the non-atopics, the  (1) , before starting treatment; n.: number of individuals per group comparison between patients with persistent moderate asthma and patients with persistent severe asthma was not possible, due to the fact that there were no patients with severe persistent asthma in this group.Among atopic and non-atopic patients, there were no significant differences regarding predicted basal values for the pulmonary parameters assessed according to asthma severity.Among the atopics, we verified significantly lower values in the ratio FEV 1 /FVC and in the peak of expiratory flux (PEF) in the MS patients after BPT with HSS, a fact that was not observed among non-atopics.
Regarding the FEV 1 , a significant reduction was observed among those with M asthma in the non-atopic group.In the atopic group, there was a reduction on the time needed for the 20% drop of FEV 1 to occur and higher percentage of drop in predicted FEV 1 among those with MS asthma after BPT, which was not observed among the non atopic.
The comparative analysis of the presence or absence of atopy among patients, divided according severity of asthma, showed significant changes only in Forced Expiratory Flow from 25 to 75% (FEF 25-75 ) in the IM Group, before and after bronchoprovocation, and it was higher among non-atopics.Variation of FVC was lower than 5% after bronchoprovocation.Comparison between MS subgroups showed that the predicted value of FEV 1 after bronchoprovocation was lower in the atopics, as was the time needed for the 20% drop in FEV 1 .The presence of asthma induced by exercise was not evaluated.
Vital signs represented by RR, HR, systolic BP (SBP), diastolic BP (DBP), AT and SpO 2 , both before and after bronchoprovocation, were similar between groups.Among atopics, there was an increase in HR and a decrease in SpO 2 , after bronchoprovocation in the IM subgroup.In this group there were no changes in other vital signs after the exam.In the MS subgroup, only the SpO 2 decreased.In the nonatopic group, there were no differences in vital signs before and after bronchoprovocation between subgroups.

Discussion
The BPT with saline solution proved to be safe, since its interruption was not necessary in any patient because of the emergence of adverse effects, by request of the patients or because they felt bad or uncomfortable during the procedure.
The presence of atopy is associated with increased BH, possibly because of inflammatory changes triggered by repeated exposure to the allergen.On the other hand, the resulting BH causes increased inflammation response and symptoms (1) .In the present study, patients that were sensitive to dust mites were considered atopic, because the latter are the main etiologic agents of respiratory allergies in tropical areas (14) .The frequency of positive BPT with HSS at 4.5% was higher in patients with moderate and severe asthma, who remained more symptomatic.A similar finding was observed by other authors, who reinforced the existence of a direct relation between the number of wheezing episodes and de degree of BH (15,16) .
The presence of gradient response to BPT, with HSS at 4.5%, depending on the severity of asthma was also documented.Although the two groups of patients were initially similar to FEV 1 , when submitted to BPT with HSS at 4.5%, those atopic with persistent or severe asthma presented a faster and more marked drop in FEV 1 than the group with intermittent or mild persistent asthma.In a way, these findings support the idea that individuals with more severe asthma have more pronounced BH.Other studies have observed the relation between severity and bronchial reactivity by different methods.Cassol et al found more profound decline in FEV 1 in individuals with severe asthma, followed by those with moderate and severe, with exercise induced methods (16) .Avital et al performed BPT with methacholine in children with mild or moderate asthma and confirmed the need for lower doses of methacholine to determine the drop of at least 20% in FEV 1 values, among those with moderate asthma (17) .
Another point to consider regarding the parameter used for the diagnosis of BPT as positive: 20% drop in baseline (5) FEV 1 or 15% drop (4,6) , which alters the sensibility to BPT.Because of this, we fixed the positivity rate for BPT with HSS at 4.5% in 20%.
The measurement of FVC may vary depending on bronchial obstruction, elastic recoil, and muscular effort; the PEF reflects the proximal airways caliber and is a dependent effort (12) .Thus, reduction of these values could mean bronchial obstruction caused by the test, as it could also suffer interference from the fatigue caused by the various spirometric tests performed.The median of FVC was reduced, in the IM Group, in 8.6% for non-atopic and 15.8% for atopic, and, in the MS Group, the reduction was of 1.4% for non-atopic and 18.6% for atopic.There was also a reduction in PEF, in which the median for the IM Group was of 19.6 (non-atopic) and 22.6% (atopic), as well as for the MS Group, 23.5 (non atopic) and 31.8%(atopic).
The FEF 25-75 is considered the most sensitive parameter for the detection of obstruction in the populations at risk, and represents the changes in the peripheral airways of medium and small caliber (12,18) .The reduction of this parameter was marked, taking as reference studies that considered as positive response the decrease in FEV 1 and/or in the FEF 25-75 of at least 25% (19) .In the present study, we observed a decrease in the median of the IM Group of 27.4% (non-atopics) and 34.5% (atopics), as well as in MS, of 36.1% (non-atopics) and 44.0%(atopics).As the variation of FVC was lower than 5% after bronchoprovocation, the alterations of FEF 25-75 may have resulted from the inhalation.
Considering the other clinical variables analyzed, there were no significant changes, except for the increase in HR in the IM atopic group.Even with this change, patients remained within normal limits, without health risks.The SpO 2 showed a slight decrease in the atopic group, but the reduction, although statistically significant, apparently had no clinical significance, which was reduced from 98 to 97%, and the decline was more pronounced among those characterized as atopic.A study performed by Solé et al found no relationship between the decrease in SpO 2 and provocative concentration, and found no relation between the decrease in SpO 2 and the percentage of FEV 1 variation in BPT (20) .
BPT with HSS at 4.5% proved to be safe, easy to perform and required little cooperation from participants.At the end, most patients complained about the unpleasant taste of the salt, the excessive production of saliva, the prolonged duration of the test, particularly among those in which there was no induction, besides fatigue for the repeated spirometry calculations, especially in younger individuals.
In the present study, it was verified that BPT with HSS at 4.5% determines the gradient of answer in children and adolescents with asthma, according to the severity of the disease.Severe forms are followed by greater and faster drops in FEV 1 .The frequency of positive BPTs was higher among atopic patients, as well as in those with more severe asthma.According to some authors (1,2) , the documentation of the presence of BH has been essential for the diagnosis of asthma, especially in patients with atypical forms.Thus, a BPT with SSF at 4.5%, which is safe, easy to perform, and allows documenting gradient intensity of asthma, should be an important instrument to be incorporated in the followup of patients with asthma, because it allows an objective assessment of the disease.

Aknowledgements
To Centro Universitário Cesmac for the support, to the physiotherapists Ana Lúcia de Gusmão Freire, Patrícia Nobre, Edjane de Biase and Jaciene Farias.To the doctors who cooperated in this research: Dirlene Brandão, Rosa Maranhão, Ana Mary Xavier and Ana Cláudia B. Dawsley.To nurse Rita and Doctor Rogério Barboza.

Figure 1 -Figure 2 -
Figure 1 -Time needed (seconds) for 20% reduction in FEV 1 , in the group of children and adolescents with asthma, according to presence of atopy

Table 1 -
Clinical and anthropometric characteristics of children (described as median, higher and lower limits) FEV 1 : forced expiratory volume in fist second; D. Dermatophagoides; BPT+vo: positive bronchoprovocation test; %BPT+vo: percentage of positive bronchoprovocation in relation to the group; *according to IV Brazilian Guidelines on Asthma Management 2006