Methacholine bronchial provocation test for assessment of bronchial hyperresponsiveness in preschool children

Introduction

Asthma is a chronic airway inflammation disease resulting from interactions among genetic factors, environmental pollution, and allergen sensitization (1-3). The prevalence of asthma is estimated to be 300 million globally (1) and still increasing (1,2). There has been a rapid increase in the prevalence of asthma among children in China, possibly because of severe air pollution, and altered environmental exposures and dietary patterns (2-5). The cumulative prevalence of asthma in children under 14 years of age has increased from 1.90% in 1990 (4) and 1.09% in 2000 (5) to 3.02% in 2010 (6). Moreover, approximately 30% of asthmatic children have been underdiagnosed in urban areas of China (5,6). In clinical, the diagnosis of asthma relies heavily on clinical history, however, the measurement of bronchial hyperresponsiveness (BHR), a cardinal pathophysiologic feature (7), with practical approaches such as bronchial provocation test (BPT) (8), is helpful for the diagnosis and differential diagnosis of asthma, especially for those with uncertain history.

Forced expiratory volume in one second (FEV₁) has been the “gold standard” for determining BHR in children aged five years or greater (1). Nevertheless, BPT with spirometry may be challenging among preschool children because of their developmental characteristics (limited ability to comprehend and cooperate), which reduced the reliability of results. Understanding the feasibility of BPT in preschool children (with uncertain history, particularly those with chronic cough and wheezing) may be of practical value (e.g., for differential diagnosis of asthma) (9). Despite the publication of European Respiratory Society/American Thoracic Society recommendations of spirometry (7), no guidelines for BPT specifically targeting at preschool children have been drafted.

We hypothesized that methacholine bronchial provocation test (MCh-BPT) would be safe and feasible in preschool children. To this end, we conducted MCh-BPT in healthy children and preschool children who previously had wheezing, we also evaluated the optimal parameter for determining the positive response for diagnosing BHR.

Methods

Subjects

Children aged 4–6 years were recruited from a kindergarten located in Guangzhou and the pediatric outpatient department of The First Affiliated Hospital of Guangzhou Medical University. All subjects were categorized into healthy children or wheezing children, according to the history of recurrent wheezing. All subjects had normal height and weight (±2 standard deviations) (10), had no history of other severe cardiopulmonary diseases, and had no acute airway infection within 4 weeks. Healthy children had no recurrent wheezing diseases, no prior exposure to noxious gases or dust; had normal chest physical examination findings. Children with wheezing had either a history two or more episodes of wheezing (without upper or lower airway infection), or a clinical diagnosis of asthma (1,11). We excluded subjects with baseline FEV₁ <60% predicted, and those who failed to complete MCh-BPT. Subjects that under allergen immunology were also excluded. Inhaled short-acting and long-acting β2 receptor agonist were withheld for at least 8 and 24 hours respectively, before any bronchial challenge test. Antihistamines and leukotrienes receptor antagonists were withheld for at least 3 days prior to the study.

The study protocol was approved by the ethics committee of The First Affiliated Hospital of Guangzhou Medical University, and parents signed informed consent.

Spirometry

Spirometry was performed with MasterScope spirometer (Carefusion Co. Ltd., Hoechberg, Germany) by experienced technicians according to international guidelines (7). Baseline spirometry was performed for 3–8 times (at 1-min intervals) to obtain at least two technically acceptable curves. Reference values were derived from the equation by Zhang et al. (12).

MCh-BPT

MCh-BPT with doubling (for children with wheezing) and quadrupling (for healthy children) provocative concentrations of methacholine was performed with Masterscreen system (DeVilbiss 646 nebulizer, power: 160 µL/min, Carefusion Co. Ltd., Hoechberg, Germany) by using Yan’s and Zhong’s approach of deep inhalation (13,14). The initial and final dose of MCh (Sigma-Aldrich Co, St Louis, Mo) was 0.39 and 12.80 µmol, respectively (Table S1). Repeated inhalation challenges were performed at 1-minute intervals. These procedures were terminated when FEV₁ reduced by 20% or the final dosing was accomplished (Figure S1). 200–400 mcg salbutamol was administered via a spacer (Volumatic, Allen & Hanbury’s, UK) to relieve discomfort among subjects whose FEV₁ fall exceeded 20%. The cumulative doses of MCh causing FEV_0.5, FEV_0.75 and FEV₁ to decrease by 15% (PD₁₅FEV_0.5, PD₁₅FEV_0.75, PD₁₅FEV₁) and 20% (PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁), causing peak expiratory flow (PEF) to decrease by 20% (PD₂₀PEF), and causing maximal mid-expiratory flow (MMEF) to decrease by 35% (PD₃₅MMEF) were calculated.

Table S1 Work flow of MCh-BPT
Full table

Figure S1 Procedure of MCh-BPT. Mch-BPT, methacholine bronchial provocation test. FEV₁, forced expiratory volumes in one second.

For children with wheezing, failure of test was defined as inability to complete spirometry. MCh-BPT would be rescheduled one week thereafter in case the initial MCh-BPT (but not spirometry) was unsuccessful. No further attempts were made in case two consecutive MCh-BPT could not be completed successfully. For healthy children who tested positive to the initial MCh-BPT, they were invited for repeated MCh-BPT at one-month interval. BHR could be confirmed among healthy children who tested positive (FEV₁ fall exceeded 20%) on both occasions. However, among children with wheezing, BHR was diagnosed if they tested positive at either occasion.

Statistical analysis

Statistical analysis was performed by using the SPSS 16.0 software package (SPSS Inc., Chicago, Illinois, USA). Data were expressed as mean ± standard deviation for normal distribution or otherwise median (interquartile range) for non-normal distribution. Independent t-test or Mann-Whitney U test was used to compare the difference between the two groups. The chi-square test was used to compare the categorical variables. P<0.05 was considered as statistically significant.

Results

Baseline characteristics

A total of 252 (190 healthy and 62 wheezing) children aged 4–6 years were enrolled. The recruitment flow chart is shown in Figure 1. There is no significantly different of height and weight between healthy children and those with wheezing (P>0.05). The demographic characteristics and history of atopy are shown in Table 1.

Figure 1 Subject enrollment. Mch-BPT, methacholine bronchial provocation test.

Table 1 Characteristics in different age groups
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Success rate of spirometry

The success rate of performing spirometry in healthy children was 73.2% (139/190) for the initial visit and 82.1% (156/190) for two visits. The success rate of the 4-year-old group was significantly lower than that of 5-year-old group and 6-year-old group (68.0% vs. 86.7% vs. 96.4%, both P<0.05). The mean duration of expiration in healthy children was 1.74 s, with seven (4.5%) children whose expiration lasted for no more than one second. In children with wheezing, the success rate of spirometry and MCh-BPT was 85.5% (73.7% vs. 88.0% vs. 94.4% in 4-, 5- and 6-year-old groups, respectively), without significant differences between healthy and wheezing groups (P>0.05) (Table 2).

Table 2 Rate of children who successfully completed spirometry and MCh-BPT in different age groups
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Comparison of BHR

Of the 156 healthy children who successfully performed spirometry and MCh-BPT, 11 had an FEV₁ decrease for greater than 20% during the initial visit. At reassessment one month thereafter, 10 children (6.4%) had an FEV₁ decrease for greater than 20%. There was no significant difference of the values of PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁of the 10 BHR positive children in healthy subject group. Of the 10 children who had an FEV₁ decrease for greater than 20%, eight had a history of allergy, frequent upper respiratory tract infection, eczema, allergic rhinitis, and family asthma history.

Of the 53 children with wheezing who had successfully completed spirometry and MCh-BPT, 49 (92.5%) tested positive to MCh-BPT. The positive rate of MCh-BPT did not differ significantly between different age groups (85.7% in 4-year-old group vs. 95.5% in 5-year-old group vs. 94.1% in 6-year-old group, P>0.05) (Table 2). Although there was no significant difference in the positive rate of MCh-BPT [97.4% (37/38) vs. 80.0% (12/15), P>0.05], however, with significant lower PD₂₀FEV₁ in children with clinically diagnosed asthma than those with a history of wheezing only children (4.40±3.67 vs. 7.87±3.78 µmol, P=0.007).

Diagnostic value of different parameters

Of the 49 children with wheezing that tested positive to MCh-BPT, 46 subjects had both an expiration of more than one second and an FEV₁ decrease of greater than 20%. The positive rates of FEV_0.5, FEV_0.75, PEF and MMEF are shown in Table 3. Comparison of the provocative doses revealed that both FEV_0.75 and FEV_0.5, but not PEF and MMEF, could be the surrogates of FEV₁ in children with an expiration of less than one second.

Table 3 Diagnostic values of FEV_0.5, FEV_0.75, PEF, and MMEF, according to FEV₁
Full table

The PD₁₅FEV_0.5, PD₁₅FEV_0.75 and PD₁₅FEV₁ were significantly lower in children with wheezing than those tested positive to MCh-BPT in healthy children [2.35 (2.99) vs. 5.01±2.55 µmol for PD₁₅FEV_0.5, 2.48 (3.19) vs. 5.18±2.38 µmol for PD₁₅FEV_0.75, 2.78 (5.02) vs. 5.49±2.62 µmol for PD₁₅FEV₁, all P<0.05, Table 4]. The area under the curve (AUC) of receiver operating characteristic curves (ROC) of PD₁₅FEV_0.5, PD₁₅FEV_0.75, PD₁₅FEV₁ and PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁ for the diagnosis of BHR was 0.944 (95% CI: 0.900, 0.988), 0.935 (95% CI: 0.887, 0.984), 0.914 (95% CI: 0.858, 0.970) and 0.932 (95% CI: 0.883, 0.981), 0.922 (95% CI: 0.869, 0.975), 0.914 (95% CI: 0.858, 0.970), respectively (Figure 2).

Table 4 Comparison of different cumulative doses of MCh for diagnosing BHR
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Figure 2 Receive operation characteristic curve of PD₁₅FEV_0.5, PD₁₅FEV_0.75, PD₁₅FEV₁ (A) and PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁ (B) for diagnosing asthma. The area under the curve (AUC) of receiver operating characteristic curves (ROC) of PD₁₅FEV_0.5, PD₁₅FEV_0.75, PD₁₅FEV₁ and PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁ were 0.944 (95% CI: 0.900, 0.988), 0.935 (95% CI: 0.887, 0.984), 0.914 (95% CI: 0.858, 0.970) and 0.932 (95% CI: 0.883, 0.981), 0.922 (95% CI: 0.869, 0.975), 0.914 (95% CI: 0.858, 0.970), respectively. FEV_0.5, forced expiratory volumes in 0.5; FEV_0.75, forced expiratory volumes in 0.75; FEV₁, forced expiratory volumes in one second.

The positive rate of MCh-BPT, determined with PD₂₀FEV₁, was 6.4% in healthy children and 92.5% in children with wheezing. PD₂₀FEV₁ in children with wheezing was significantly lower than that of healthy children [4.17 (6.54) vs. 7.97±3.67 µmol, P<0.05], whereas PD₂₀FEV_0.5 [3.89 (5.02) vs. 6.73±3.57 µmol, P>0.05] and PD₂₀FEV_0.75 [3.75 (6.09) vs. 6.90±3.46 µmol, P>0.05] were comparable between the two groups. Among children with wheezing, PD₂₀FEV_0.5, PD₂₀FEV_0.75 and PD₂₀FEV₁ were significantly lower in children with a clinical diagnosis of asthma than in those who had a history of wheezing but no clinically diagnosed asthma [3.77±3.21 vs. 6.31±3.0 µmol, 3.50 (6.01) vs. 6.30±2.91 µmol, 4.50±3.67 vs. 7.86±4.0 µmol, P<0.05].

Adverse effects

Adverse events happened during the MCh-BPT included transient wheezing, cough, pharyngeal itching, hoarseness, sore throat, shortness of breath, chest tightness, which were mild and comparable in different age groups (Table 5). Mild wheezing and cough were significantly more common in children with wheezing than in healthy children (41.5% vs. 2.6%, 13.2% vs. 1.3%, P<0.01), whereas the incidence of sore throat, hoarseness and chest tightness was comparable between the two groups (all P>0.05). After challenge, 200–400 mcg inhaled salbutamol had been given through a spacer to the children whose FEV₁ decreased more than 20% from baseline, or self-reported chest-tightness wheezing, or wheezing by auscultation. Ten minutes after the inhalation of salbutamol, FEV₁ recovered to the baseline and the symptoms relieved in all of the children with adverse events. There was no serious adverse event requiring continuous nebulization with bronchodilators and/or corticosteroids.

Table 5 Adverse events in different age groups
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Discussion

This study revealed that preschool children who were able to perform spirometry could successfully complete MCh-BPT, with tolerable and minor adverse events. PD₂₀FEV₁ remains to be an optimal parameter for assessing BHR with MCh-BPT in preschool children. For children with expiration for less than one second, both PD₂₀FEV_0.5 and PD₂₀FEV_0.75 can be surrogates of PD₂₀FEV₁.

Currently, PD₂₀FEV₁ is extensively recommended as a positive indicator of bronchial provocation tests (including MCh-BPT) in elderly children and adults (15,16). However, some studies have reported that the quality control standards for adults might not be suitable to directly extrapolate to preschool children (6,17,18). The diagnosis of asthma among children aged 5 years or younger has been challenging because of the risks of misdiagnosis. For instance, children without asthma may often experience intermittent respiratory symptoms such as wheezing and cough (19,20). Therefore, ascertainment of the feasibility of spirometry and MCh-BPT for assessment of BHR, and the safety and diagnostic value of MCh-BPT among preschool children may provide valuable insights to clinical management of diseases with BHR (particularly asthma).

We found that the sensitivity and specificity were both 90.0% and 93.6% for PD₂₀FEV_0.5 and PD₂₀FEV_0.75, which did not differ significantly from that of PD₂₀FEV₁. However, the sensitivity and the specificity were only 71.9% and 66.7% for PD₂₀PEF, and 77.97% and 66.67% for PD₃₅MMEF. Our findings indicated that in preschool children, for those with an expiratory duration of less than one second, both PD₂₀FEV_0.5 and PD₂₀FEV_0.75, but not PD₂₀PEF or PD₃₅MMEF, could serve as the surrogates for PD₂₀FEV₁ for assessment of BHR. Bentur et al. (21) reported that when PD₂₀FEV₁ was applied as the main parameter for reflecting BHR, it would be more feasible, safer, and require less provocative agents to be inhaled than assessment with the symptoms or the breath sounds of wheezing in preschool children. PD₂₀FEV₁ was significantly lower in children with wheezing than in healthy children, whereas PD₂₀FEV_0.5 and PD₂₀FEV_0.75 were comparable between the two groups, indicating that PD₂₀FEV₁ might be preferable to PD₂₀FEV_0.5 and PD₂₀FEV_0.75 for evaluation of BHR in preschool children.

Noteworthily, healthy children who tested positive to MCh-BPT displayed a significantly higher incidence of exposure or sensitive to allergens, frequent upper airway infections, and allergic rhinitis than those with negative results (P<0.01). It is likely that drugs, food allergens, frequent upper airway infection history, and allergic rhinitis may have contributed to the development of, or exacerbated, BHR. A family history of asthma has been identified as a major risk factor for asthma in children (22,23), whereas asymptomatic BHR may predispose to asthma (24). The mechanisms for the BHR among some of the healthy children warranted further studies.

Methacholine has been the most common provocative agent for BPT in clinical practice. There are international guidelines that offered detailed instructions to perform MCh-BPT (25). However, the safety of MCh-BPT in preschool children is unclear. We have previously reported that (26) the use of histamine BPT for diagnosing BHR in children aged 11–14 years was safe, with no serious adverse events having been reported. There are also literature reports (27) documenting the use of MCh-BPT in infants and toddlers with tidal breathing method, the results of which showed minor safety concerns. Kivastik et al. (28) have confirmed the safety of MCh-BPT with tidal breathing methods and tripling dose increment, by using air flow blocking technology to measure respiratory resistance in children aged 3 to 6 years old. These pieces of evidence have reaffirmed the safety of MCh-BPT to be applied among preschool children.

Some children developed pharyngeal itching, sore throat, hoarse cough, cough, chest tightness, and mild wheezing. However, all these symptoms were ameliorated through throat gargling, water ingestion, having a rest, or inhalation of salbutamol for 200 µg within 10–30 minutes. No serious adverse event was reported, indicating that MCh-BPT in preschool children was safe. The frequency of cough and mild wheezing was significantly higher in children with wheezing than in healthy children. In children with wheezing, the incidence of transient wheezing during MCh-BPT was higher in those with abnormal lung function.

This study assessed BHR in preschool children and confirmed that the methacholine bronchial provocation test was safe and applicable in children aged 4–6 years. Similar with toddlers and adults, some preschool children may have asymptomatic BHR. Whether this would require further medical evaluation or dynamic follow-up merits further investigations. However, some limitations should be considered. First, the sample size and single-center study design might have limited the validity of our conclusion. Moreover, we did not include 3-year-old children, some of whom might also be capable of performing the MCh-BPT.

In summary, although most preschool children in this study can successfully and safely complete MCh-BPT for the diagnosis of BHR in preschool children, however higher success rate was found in larger age groups. PD₂₀FEV₁ remains an optimal indicator of BHR for MCh-BPT, both PD₂₀FEV_0.5 and PD₂₀FEV_0.75 could be surrogate endpoints in preschool children, particularly those with expiration of less than one second. PD₁₅ has a good diagnostic value as PD₂₀ for diagnosing of BHR in preschool children.

Acknowledgments

Thanks all of the subjects participated in this study.

Funding: This study was supported by The National Key Scientific & Technology Support Program (No. 2015BAI12B10).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study protocol was approved by the ethics committee of The First Affiliated Hospital of Guangzhou Medical University, and parents signed informed consent.

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