The bronchial provocation test that uses an aerosol of hypertonic (4.5%) saline as an indirect stimulus was developed over a number of years by Anderson and others in Australia 1 2 3 4 . The test subsequently became known as the hypertonic saline (HS) test. In epidemiology it was first used in a study in children with exercise as a comparator challenge 5 . The HS test has now been used safely in both adults and children for a wide variety of studies for 20 years 6 7 8 9 . A 15% reduction in forced expiratory volume in one second (FEV₁) is used to define abnormality 10 . The dose of 4.5% saline to provoke this fall in FEV₁ (PD₁₅) is the index used to express the airway sensitivity to this stimulus or the airway hyperresponsiveness (AHR) 10 . There are some technical difficulties with the HS test that preclude it from being a common operating standard for investigators 4 .

In order to overcome some of these problems a dry powder preparation of mannitol was developed to provide a more simple form of osmotic challenge 11 . The studies using dry powder mannitol demonstrated that it had a very similar profile to HS 12 . Further those subjects responsive to other challenge tests that acted 'indirectly' to cause airways to narrow e.g. exercise, eucapnic voluntary hyperpnea 13 , and adenosine monophosphate 14 , were also responsive to mannitol. The major advantage in using provoking stimuli that act 'indirectly' to cause airways to narrow is the high specificity for identifying the type of AHR that is altered by drugs used in the treatment of asthma 15 16 , and inhaled corticosteroids in particular. Sensitivity to mannitol is reduced or even totally inhibited following chronic administration of inhaled corticosteroids (ICS) 17 , therefore allowing monitoring of therapy 18 . Further, in subjects whose asthma is well controlled with ICS, response to mannitol has been used to predict exacerbations following back titration of steroid dose 19 . In these respects challenge with mannitol is different from challenge with methacholine. Whilst responsiveness to methacholine can be reduced 1 to 2 doubling doses in response to treatment with inhaled steroids, most patients still record a positive response 20 21 22 and this agent has not been useful to guide for titration of steroid dose 23 .

We aimed to determine the efficacy and safety profile of a commercial preparation of dry powder mannitol in subjects with and without signs and symptoms of asthma. A phase III, multi-centre, open label, operator-blinded, crossover design, randomised trial, with follow-up was performed comparing the airway response to mannitol with the HS test and a standard clinical assessment of asthma.

All the subjects classed as asthmatics at entry had an established history of asthma, with most having symptoms before 19 years of age. Asthmatic subjects had normal FEV₁ values and there was no difference compared to the non-asthmatics (Table 1). A total of 646 subjects completed at least one challenge with 627 exposed to mannitol and 636 to HS. The mean dose of mannitol administered was 433.6 ± 237.2 mg, with the mean for subjects with a positive mannitol test being 239.0 ± 185.0 mg. As expected, the mean ± SD cumulative dose for a negative mannitol test was 635 ± 0.9 mg. For the HS challenge, the mean dose ± SD for all subjects was 21.6 ± 13.5 ml, 12.9 ± 11.3 ml in subjects with a positive HS challenge and 32.4 ± 6.3 ml for subjects with a negative challenge. The output of the ultrasonic nebuliser ranged from 0.24 to 4.4 ml/min, with a mean of 2.1 ± 0.5 ml/min.

In the efficacy population (n = 592) that included both asthmatics and non-asthmatics, the severity of symptoms using the questionnaire specific to the week prior to commencement of the study revealed that there were 39.0% (n = 231) with wheeze, 32.8% (n = 194) with trouble breathing, and 11.3% (n = 67) who had respiratory symptoms that interfered with normal activities. There were 45.2% (n = 292) who had daytime cough that lasted for a mean ± SD of 4.0 ± 2.5 days, with 82.5% (n = 241) stating that it was only occasional. Cough at night time was reported by 31.6% (n = 187) and 49.5% (n = 293) reported using a reliever puffer in the past week. Only 6.3% (n = 37) reported having a cold or flu in the past week. There were 78.4% taking at least one medication for asthma. Of those assessed as asthmatics at entry 27.7 % were taking ICS alone and 38.5% in combination with a LABA, 15.6% were taking only a SABA and 3 subjects taking only a LABA. Only 1.7% were taking leukotriene antagonists, 4.6% were taking an anti-cholinergic and 2.2% were taking sodium cromoglycate.

As designated by the clinical assessment, 82.3% (n = 487) of the efficacy population (n = 592) were asthmatic and 17.7% (n = 105) were non-asthmatic. Of the 378 adult asthmatic subjects, 50.5% (n = 191) were classed as mild, 39.9% (n = 151) were classed as moderate, and 9.5% (n = 36) as severe (Table 2). For the 109 paediatric asthmatic subjects, 22.0% (n = 24) were classed as infrequent episodic, 17.4% (n = 19) as frequent episodic, and 60.6% (n = 66) were classed as persistent.

Of the 592 subjects 50.0% (n = 296) were positive to the mannitol challenge and 54.4% (n = 322) positive to HS. For those who were positive the maximum mean % fall in FEV1 was similar for both mannitol (21.0% ± 5.7 (range: 15.0–45.5)) and for HS (21.3% ± 5.9 (range:15.0–48.62)) and there was a relationship between maximum % fall in FEV₁ to both challenges (r_p = 0.62, p < 0.001). The maximum mean % fall in FEV₁ in children less than 12 years of age was 21.1% ± 6.0 (range:15.85 × 40.30) for mannitol and 22.0% ± 6.4 (range:16.34–48.62) for HS. Only 14 subjects recorded a maximum fall in FEV₁ greater than 30% and the majority had a fall between 15% and 25% (Figure 2). For hypertonic saline 18 subjects recorded a fall of 30% or more.

The geometric mean (95% CI) for PD₁₅ was 112.20 mg (97.72 mg, 128.82 mg) for mannitol and 5.01 ml, (4.27 ml, 5.75 ml) for HS. The median PD₁₅ for mannitol was 148.1 mg and for HS it was 6.2 ml. There was a moderate, though statistically significant, correlation between log transformed PD₁₅ between mannitol and HS (r = 0.53, p < 0.0001). Those adults with a classification of mild asthma had a PD₁₅ for mannitol that was significantly different from those classed as having moderate and severe asthma (Table 2). There was a trend for those children with episodic asthma to have higher PD₁₅ values however there was no significant difference between the responses and their clinical severity classification.

The maximum mean % fall in FEV₁ from baseline for subjects who were clinically assessed as non-asthmatic was 5.5% ± 4.8 after mannitol and 5.4% ± 4.9 after HS. The median (range) RDR for those with a negative challenge was 0.0075 (0.0–0.0317) for mannitol and 0.15 (0.0–0.70) for HS. For the group who were non-asthmatic the RDR geometric mean (95% CI) for mannitol was 0.0029 (0.002, 0.004) and for the HS was 0.11 (0.085, 0.139).

The highest proportion (28.7%) of subjects achieving a positive mannitol challenge result did so in the cumulative dose range of >155 × 315 mg, 18.2% were positive in the >75 × 155 dose range and 16.2% in the >35 × 75 dose range (Figure 3). Eighty percent of those who achieved a fall of 15% or more did so in a dose = 315 mg (10 capsules).

Nineteen (3.0%) subjects had repeat mannitol doses, 89.5% (n = 17) of whom went on to achieve a PD₁₅, the remaining two subjects having maximum falls in FEV₁ of 14.55% and 14.85%. Thirty-six recorded a PD₁₅ to mannitol but did not achieve a PD₁₅ to HS, while 62 achieved a PD₁₅ to HS but not to mannitol. There was no significant sequence effect (p= 0.2) and no significant difference between the treatment order or the study site for mannitol and HS responses.

The mean ± SD time taken for a positive mannitol challenge was 17.3 ± 7.09 minutes and for HS was 15.0 ± 9.1 minutes. For a negative challenge the mean ± SD for mannitol was 26.4 ± 6.1 minutes and for HS it was 27.3 ± 3.6 minutes. It should be noted that this included the time taken to complete the post challenge measurement of the vital signs.

For subjects with a positive challenge test, the time to recover to 95% baseline FEV₁ was similar for both mannitol and HS, being 19.4 ± 8.8 minutes and 19.3 ± 8.7 minutes respectively. The maximum recovery time was 65 minutes for mannitol and 79 minutes for HS. There were 344 (54.9%) subjects who received 200 mcg of salbutamol after the mannitol challenge, and 370 (58.2%) after HS. A second salbutamol dose of 200 mcg was given to 46 (7.3%) subjects after mannitol and 38 (6.0%) after HS, with additional bronchodilator given in 6 (1.0%) subjects following the mannitol challenge and 2 (0.3%) following HS.

There were 535 (85.3%) subjects who experienced cough during the mannitol challenge and 468 (73.5%) during the HS challenge. The mean cumulative dose at which cough was first reported was 62.7 mg for mannitol and 5.07 ml for HS. The majority of subjects, 70.8% for mannitol and 64.8% for HS, experienced occasional cough causing no delay in administering the next dose. Frequent cough causing a delay in the challenge was noted in 83 (13.2%) subjects during the mannitol challenge and 51 (8%) subjects during HS. There were 8 (1.3%) subjects during mannitol and 5 (0.8%) during HS who were noted to have a cough severe enough to stop the challenge although 2 and 4 respectively did record a 15% fall in FEV₁ at the time they were stopped.

There were 14 (2.2%) subjects who commenced but did not complete the mannitol challenge and 9 (1.4%) subjects who did not complete the HS challenge. The most frequent causes of an incomplete challenge were incorrect calculation of the target 15% reduction in FEV₁ (which resulted in premature termination of the challenge) and cough (see above).

The sensitivity and specificity for mannitol to identify responsiveness to HS and for the clinical diagnosis are given in Table 3. As expected the sensitivity for mannitol to identify a clinical diagnosis of asthma was lower due to many asthmatics taking ICS, however the specificity remained high. The sensitivity in children (<12 yrs) was 85.5% (95% CI: 76.1, 94.8) and the specificity was 68.4% (95% CI: 47.5, 79.0).

Subjects diagnosed with asthma who were using ICS (either alone or in combination with a beta₂ agonist) were divided into mannitol positive and negative. Of the 291 subjects mannitol positive, 204 (70.1 %) had been using ICS prior to the first challenge; of the 196 subjects negative to mannitol, 159 (81.1%) had been using ICS prior to the first challenge. As an exploratory analysis, the subjects who were mannitol negative and taking ICS before the study were removed from the analysis of sensitivity and specificity for mannitol with respect to the clinical assessment and there was an improvement in sensitivity (88.7%) and specificity (95.0%).

All asthmatics had an established history of asthma with good lung function that was similar to that seen with the non-asthmatics. Further, reliever bronchodilator was taken in 51.3% in the week prior to commencement of the study, however only 11.9% reported symptoms interfering with normal activity. The majority of adult asthmatic subjects, classified using GINA guidelines, had mild disease and most probably represent the population to be assessed using a bronchial provocation test, i.e. those with good lung function and mild symptoms.

The median doses of mannitol (148 mg) and HS (6.2 ml) to cause a 15% fall in FEV₁ were similar to those that have previously been identified as the cut-off point between moderate and mild AHR to mannitol (155 mg) and HS (6.1 ml) 10 . Thus 50% of the asthmatic subjects were in the mild range of AHR to these agents and this was consistent with the cohort's treatment and symptom profile, i.e. symptoms less than once per week.

These results demonstrate the safety of the inhaled mannitol challenge to identify AHR. There were no serious adverse events recorded. There was no difference in the overall safety profile demonstrated by a similar adverse event profile between mannitol and HS. The low incidence of headache, the most frequent complaint after both challenges, was likely to be a direct result of the challenge procedure. Headache was more prevalent in those with a negative mannitol challenge and likely due to the larger number of forced expiratory manoeuvres required compared to those with a positive challenge.

The mean maximum fall in FEV₁ was similar for both the mannitol and HS challenges and close to the target FEV₁ of 15%. Relative to the potential fall in FEV₁ that can occur following challenges such as exercise, this is a moderate decrease in FEV₁. Few subjects had a fall in FEV₁ greater than 30%, a value that is frequently attained when testing with exercise or with dry air as the stimulus 28 29 . Only two subjects responded positively to the initial mannitol dose of 5 mg and the falls in FEV₁ were 15% and 26%. Twelve subjects had a PD₁₅ to hypertonic saline = 1.0 ml which is equivalent to a 30 sec exposure to the aerosol.

Only two of the 19 subjects who had a between dose fall in FEV₁ of >10% failed to achieve a PD₁₅ and for both these subjects the maximum fall achieved was greater than 14.5%. Thus a fall in FEV₁ of 10% between doses of mannitol should also be considered as a positive challenge and signal an end to the challenge procedure. The PD₁₅ can then be extrapolated from the recorded response at the 10% fall.

Bronchoconstriction provoked by other challenge tests may be accompanied by significant reductions in SpO₂, e.g. exercise challenge 28 . However the reduction in SpO₂ during the mannitol challenge was small and was probably of clinical significance in only 3 subjects (i.e., >3%). There were only small and clinically insignificant changes in blood pressure, heart rate, and breathing frequency after challenge. This is in contrast to a challenge with exercise that can be associated with substantial changes in blood pressure, heart rate, and breathing frequency 28 . The insignificant changes in vital signs are relevant when choosing a test for the assessment of airway responsiveness in older subjects.

Cough is a recognised aspect of both the mannitol and HS challenges 30 . The majority of subjects (>85%) had either an occasional cough or no cough in response to the mannitol challenge. This finding is consistent with the mild severity of disease in the majority of asthmatic subjects and the high proportion taking ICS. Severe cough was infrequent, occurring in only 1.3% of subjects during the mannitol challenge. For 8 subjects the cough was too severe to continue the challenge.

The sensitivity of mannitol to identify subjects with AHR to HS was lower than expected, given the results of an earlier study 11 . The majority of subjects (52 of 62) who were negative to mannitol and positive to HS had AHR to HS within the mild range (PD₁₅>6.1 ml). The reason for the discrepancy is not clear from the results but several explanations can be postulated. One explanation is that the HS is administered with tidal breathing whereas mannitol is given with an inhalation to total lung capacity. Cockcroft et al 31 have reported a similar discrepancy between methacholine responsiveness in mild subjects when the dosimeter technique that uses a deep inspiration is compared with the tidal breathing technique. Alternatively, it may relate to a higher than usual output of the nebulisers delivering the 4.5% saline. It would be expected that if too much fluid deposits in the airways it could amplify the airway narrowing effect of a small amount of smooth muscle contraction, thereby causing a false positive response 32 . Other possible reasons include subjects becoming more compliant with their medication after their initial challenge, and that the period between last medication and the challenge was variable, although a minimum of 12 hr was required.

It is likely that the relatively high number of negative challenges in subjects with asthma is largely due to the fact that such a high proportion of the subjects were using ICS and therefore their airway inflammation was well controlled. Airway sensitivity to mannitol is reduced or inhibited by treatment with ICS 17 18 , a feature that allows it to be used for monitoring therapy 33 . The use of ICS largely explains the discrepancy between the sensitivity to both challenge tests and the clinical assessment, which was made on information that included medications taken by the subject. Of the total group of diagnosed asthmatic subjects, 75% were taking ICS.

Based on earlier studies, the sensitivity of the mannitol challenge would have been expected to be higher and, up to 95% in asthmatics not taking ICS 12 . Of the 196 subjects classified as asthmatic by a clinician and who were negative to mannitol, 81% (n = 159) were taking ICS. These subjects represented 33% of the 487 diagnosed asthmatic subjects. Re-analysis of the data after exclusion of this group showed a sensitivity of 88.7% for mannitol to identify a clinical diagnosis. This value is more consistent with findings in earlier studies. It should be noted however that use of the mannitol challenge for diagnostic purposes in subjects taking ICS warrants caution in the interpretation of a negative test result.

Koskela et al 18 have suggested that "if mannitol is used to monitor effect of ICS in asthma, the goal of treatment should be unresponsiveness". Further, Leuppi et al have used a negative test as a starting point for back titration of steroids 19 . Thus for those 70.1% of asthmatics taking ICS with a positive response to mannitol, the dose of ICS may have been inadequate and/or the compliance with its use poor so that the airway inflammation was still active.

In conclusion the mannitol challenge was generally safe and well tolerated. No serious adverse events were recorded. Mannitol PD₁₅ had a sensitivity of 81% and specificity of 87% with respect to PD₁₅ for 4.5% saline. A positive mannitol test cut off of a 15% fall in FEV₁ (PD₁₅) provided appropriate sensitivity and specificity with respect to clinical diagnosis of asthma even when the patient's baseline FEV₁ was within the normal range. Based on an analysis of patients with a clinical diagnosis of asthma, and excluding those with a negative test result and on current corticosteroid therapy, mannitol PD₁₅ had a sensitivity of up to 89% to detect the presence of asthma and specificity of 95% for clinical diagnosis of asthma.

All the authors contributed to the design of the protocol for this study. The data management and statistical analysis of the results was contracted out by Pharmaxis Ltd to Datapharm Australia Pty Ltd. This paper is a summary of their report and all the authors made a contribution to this summary.