The emphasis of this study was on the development and refinement of a cough aerosol model in healthy human volunteers, detected and verified with a laser diffraction system.

Major findings in this study include: a) the respiratory system generates droplets of many different sizes during coughing; b) droplets smaller than ten-microns account for up to 99% of the total number of droplets that are expelled as a bioaerosol during coughing; c) due to its size distribution and amount, the cough bioaerosol has the potential to contribute directly, indirectly and/or through airborne route to the transmission of respiratory infections, including Influenza A caused by the H1N1 virus; d) age, sex, weight, height and corporal mass have no effect on the size and number of emitted droplets; e) our approach has the potential to identify high emitters and/or outliers; f) these results create a foundation for the development of a standardized human cough aerosol model; g) the acquired data creates a foundation for the development of tools in airway hygiene for secretion management, as well as in prevention of droplet-spread illnesses.

During the preparatory phase of this study, our research group was concerned that healthy non-smokers would find it difficult to voluntarily perform a reproducible "near-real-cough aerosol". We considered requesting participants to undergo respiratory challenges such as inhaling hypertonic saline to induce augmented airway secretion and/or capsaicin to elicit a "real cough". The concern was due to technical facts: the thickness of the airway mucus layer in healthy non-smokers is 5 to 10 microns [39, 40].

The International Organization for Standardization (ISO) has stated "the laser diffraction technique has evolved such that it is now a dominant method for determination of droplet size distribution". (ISO 13320:2009 (E) 2009) [41]. While testing and tuning the laser diffraction system we found that our initial concerns were inaccurate: Healthy non-smokers are excellent models to characterize cough aerosol droplets. Thus, we avoided using any challenging intervention that could alter the physical properties of the airway mucus layer.

The first limitation of this study was that the laser diffraction system, according to the manufacturer, was not intended to assess aerosolized mucus from the airways when coughing. This created an uncertainty as to whether we could capture any droplets at all. However, thanks to some unique technical expertise in our group, our lab was able to use the machine for our intended purposes, if slightly limited.

Other limitations were related to the performance of the laser diffraction system with a fluid that has optical properties different than water. Berge and Pearce report that the Refraction Index of Air is 1:0 and of Water is: 1.3, and Reid reports that the refractive index of mucus is very similar to that of pure water [42, 43].

Properties of mucus are different in health than during disease. Mucus in healthy individuals is more opaque than water. In diseased state, mucus opaqueness could be more pronounced further affecting optical properties. During bacterial infection there is formation of pus or mucus could become bloody, altering most physical and optical properties.

However, during acute viral respiratory infections such as flu-like diseases, respiratory secretions are watery and clear. Therefore our assumption is valid for influenza, SARS-CoV, and avian influenza, which remain our priority. To assess cough aerosol in diseases like Tuberculosis, Cystic Fibrosis or others caused by bacteria, we may need to determine the optical properties of diseased mucus first.

Any droplets travelling in the periphery of the plume and outside of the measurement zone are unaccounted for. Our experiment was designed to capture a representative section of the cough plume crossing the path length and the measurement zone, including droplets from the lateral periphery that cross the measurement section. We estimate that we captured a sample of 15% of cough droplets that are representative of the cough plume but we have no definitive way or method to accurately determine this yet.

There is a confounder we have not deal with yet, and is the contribution of saliva to the number and size of droplets detected. This is a topic for the next trials.

Linear correlation indicates a very weak association between height, weight and BMI with the size and number of cough droplets expelled. These findings lend further support to the concept that cough droplet diameter/number distribution is mainly determined by the physical properties of the layer of mucus, such as elasticity, cohesiveness. A mucus layer with low elasticity and poor cohesiveness due to infections (i.e. the watery mucus layer during a flu-like disease) or mucus exposed to respiratory agents that disrupt bondings tends to break apart with more ease. Consequently, this will form a larger number of droplets of different size.

A mucus layer with strengthened elasticity and high cohesiveness will be more resistant to break, and will form less number of droplets and/or produce fewer droplets of a larger size. This is a concept we described in previous publications [44, 45].

In healthy non-smoker individuals there is an optimum range among their mucus physical properties that allow it to behave in a balanced manner even at different frequencies. This enabled us to reach a milestone: enhance our understanding of the cough aerosol role in droplet-spread, pandemic-prone IRD.

Nevertheless, several factors observed in our design suggested that the "best effort" cough requires improvements. Specifically, the distance from the mouth to the laser beam and the position of the face, were identified as factors to further assess and improve in order to minimize the variability of the acquired data.

The open bench design was selected since we were interested in characterizing the cough bioaerosol in an indoor environment that could simulate and explain what would happen in a real-life emergency room, triage site, school, home, or any enclosed location where people gather. This approach would facilitate assessing the characterization of the bioaerosol coming out the respiratory system and dispersed into the surrounding environment.

Indoor conditions in the study site were maintained at similar room temperature, humidity, and atmospheric pressure as in the reception site of a hospital, with the exception of a lower rate of air exchange. Hence, the open bench format will not require a translation into real-life situations, unlike enclosed formats. It took our group a great deal of effort to overcome the limitation of the system and extract the number of droplets in the cough bioaerosol, since the laser diffraction system does not explicitly provide it. We have not encountered any information of any research team assessing cough droplets using an open bench format.

Researchers from various disciplines around the globe have dedicated a large number of studies to the investigation of cough aerosol droplets, using a variety of study designs, as well as multiple quantitative and qualitative methods and techniques. During our brief literature review, we found several key differences between those studies and our methods: they all used closed systems of various designs to assess the respiratory droplets; the majority of them used equipment with much lower resolution, limited range of sizes and biased droplet collection to characterize the size and number of droplets; and almost all of them used equipment with much lower data acquisition speeds than the one used in our study [27–37]. Without a doubt, these differences played a critical role in explaining why our data differs from the majority of data reported in the literature.

Furthermore, our research group considers that it is fundamental to reach a consensus in defining the components of the cough aerosol. Current terminology linked to cough aerosol and IRD transmission/dispersion (e.g. particles), are similar to terms used in air quality studies, where pollutants or "particles" are mostly composed of solid materials and gases generated during combustion process.

In this article our research group consistently uses the term droplets instead of particles to define the components of the cough aerosol, since water content dominates the composition of the airway mucus (~ 95%), with solid content filling out the remaining percentage. A consensus will enhance our effectiveness in IRD management and protection by reducing confounding terms. A consensus is also needed to clarify how the influenza virus is transmitted.

The high proportion of droplets smaller than one micron (97%), expelled as aerosol in a single cough, are susceptible to rapid evaporation when released to an environment with different humidity and temperature than inside the airways. This supports the probability that an airborne route of transmission could be a dominant force in the transmission of droplet-spread IRD. Interestingly, a group of researchers led by Palesi [46, 47], have published several studies indicating that, using a small mammalian model, a viral infection was transmitted to animals in different cages connected only by a tube with no direct contact involved. Airborne droplets, emitted by the infected group of animals, are the most likely mode of transmission in such a model. Hence, the contribution of droplets smaller than one micron in viral transmission merits further investigation.

Data from this study allow us to not only characterize the cough aerosol, but also to identify outstanding emitters. 10 individuals were categorized as high emitters of cough droplets. One of the high emitters was beyond two standard deviations greater than the average number of droplets expelled when coughing and was considered as an outlier, and the other nine only one standard deviation apart from the mean.

Data from our participants indicate that age, sex, weight, height or corporal mass have no statistically significant effect on the aerosol composition in terms of size and number of droplets, as confirmed by linear correlation assessment (Table 2) and ANOVA tests (Table 5). Results of the ANOVA test including all participants showed a tendency that did not reach a statistically significant difference in the following all droplet size categories. Excluding the outlier removed the tendency towards a difference that is statistically significant.

These results coincide with previous findings by Zayas (MSc Thesis, 1989) that viscoelastic properties, determined by rheology, from tracheal mucus do not differ in young and old healthy male/female adults who are non-smokers, including those mature non-smokers with pulmonary restrictive diseases [48].

The high emitter outlier was identified as a very fit athlete who practices high intensity sports. Such physical activities would very likely have a positive effect on lung mechanics, hence, allowing for a better lung capacity. However, it is tempting to speculate that if such a person happens to develop influenza they could become a "super-spreader" due to the high number of droplets expelled when coughing. Figure 2 illustrates the enormous difference between this outlier and the other participants. The same figure also highlights that droplets smaller than one micron (< 1 μm) clearly dominate over the rest of the droplets sizes.

Regarding the tri-modal size distribution, the third size mode at ~251 μm (between 215 - 464 μm size) is very small in our study. This third mode is of such small magnitude when compared with the other two modes that it is only evident in annexed Figure 3.4. Johnson in a recent publication, using a different methodology and technique, also reported a third mode peaking at around 200 μm [49]. We are preparing another article where we will discuss in more detail similarities and differences of our method and results from other teams of researchers working on the same topic. Our team of researchers is fully convinced that only a multi and trans-disciplinary collaboration will provide the optimal strategy to best manage, reduce infection, disease, and death due to IRD in rich and poor countries alike.

Another high emitter of cough droplets is a self-identified long-term (+ 30 years) ex-smoker. Previous studies found that viscoelastic properties of tracheal mucus from subjects exposed to tobacco smoke determined by rheological analysis are different (p < 0.05) with respect to the non-smoker population [50–53]. The remaining eight high emitters identified themselves as non-smokers. There was no investigation to verify if they have had any type of voluntary or involuntary exposure to tobacco smoke, or to other airborne insults.

Pharmacological and non-pharmacological factors are capable of disrupting the optimum balance of the physical properties found in mucus. Compounds that break-up and lyse the cross-linking binding sites at different levels of the mucin glycoprotein gel network can subsequently affect the natural balance of viscosity and elasticity of the airway mucus. Thus, transforming it in a less cohesive or more watery fluid, facilitating airway mucus droplet formation. This is an unexplored area that needs to be addressed.

A literature search yielded no scientific or empiric information regarding the effect that natural and/or non-natural compounds may exert on aerosolization of mucus during respiratory condition treatment. This is an area that also merits detailed research due to the wide and common use of these compounds and in light of recent epidemic-prone and pandemic-prone droplet-spread IRD. Therefore, it is critical to determine and grade the effect of both pharmacological and/or non-pharmacological factors on airway mucus aerosolization.

Seven participants were identified as Low emitters. Initially, we interpreted this result as a technical issue: that during the study these participants for some reason directed the cough airflow jet in a direction that prevented it to cross the measurement zone of the laser beam. The other possibility we are contemplating is that these participants may have inherent physical properties in their airway mucus that makes it more resistant to break up into droplets when coughing. There may be other explanations and we are preparing to explore these in future studies.

Based on the results obtained in this study we are confident that we have achieved a strategic and critical step towards optimizing management of droplet-spread epidemic-prone infectious diseases in the form of a detailed real-time characterization of the cough aerosol regarding the size and, more importantly, the number of droplets expelled when coughing.

The ERS definition of cough does not mention anything about droplets formed and expelled as a direct result of the interaction of the high-speed airflow with the layer of airway mucus during the expulsive phase. A consensus panel report on "managing cough as a defense mechanism and as a symptom" [54], endorsed by the American College of Chest Physician, the American Thoracic Society, and the Canadian Thoracic Society, made a detailed, highly clinical description of coughing. However, it does not discuss anything regarding cough aerosol droplets as the vehicle of transmission of IRD.

The fact that several of the most prestigious international professional organizations dealing with lung health and/or respiratory diseases, including the Council of Canadian Academies, are not discussing droplet formation and expulsion during coughing as a critical factor in IRD transmission and dispersion indicates that there is a knowledge gap and lack of consensus that require immediate attention [55–59].

This study provides both scientific support as well as encouragement to design evidence-based preventative measures and alternatives in existing technologies to optimize public health practices and personal protection barriers in bioaerosol control to prevent the spread of IRD. The human cough aerosol model could serve as the foundation for the development of an in-vivo, innovative and robust bioaerosol assessment tool. This tool could be quite useful during an IRD outbreak as a point of care diagnostic test for screening, detecting, and monitoring, individuals with an acute respiratory infectious medical condition. Our method yield results in less than five minutes, hence would reduce time inconveniences in scenarios where large amounts of individuals continuously gather; such as emergency entrances in hospitals, airports, bus/train stations, etc. By using an evidence-based preventative screening method, staff working in these scenarios will feel reassured that they have reliable protection.

This technology will complement and enhance protection to first responders and frontline health care workers caring for, and transporting, patients to and from health care institutions. Furthermore, it will also protect the general public during IRD outbreak transmitted by droplets via direct, indirect and airborne route.

Despite the fact that Canada has one of the most advanced healthcare systems in the world, Canadians' health remains at risk from droplet-spread IRD. The success in handling the SARS crisis was questioned, and the lesson to be taken from this experience is that Canada has room to improve in its ability to manage IRD outbreaks, not unlike most countries worldwide [60].

Our research group is aware that our findings clearly differ from other studies, but we also are aware that our findings still need further evaluation before confirmation of our data and findings. For the human cough aerosol model to achieve its full potential, a much larger sample of participants is required; hence ongoing recruitment of subjects is needed for the foreseeable future to confirm its value.

In addition, our research group at the Mucophysiology Laboratory, University of Alberta has been developing a novel non-vaccine strategy, mucomodulation [44, 45], that has shown the potential to slow or stop the spread of IRD, potentially saving lives as well as reducing the burden on resources for healthcare systems in rich and poor countries alike.

Currently the mucomodulation strategy has evolved into an ongoing comprehensive program known as The Edmonton Platform, and will serve as a complement to the WHO Pandemic Preparedness Plan. This program will be further detailed in a forthcoming article, however, it is conceived as the launching ground for methods and tools of innovative technologies, products, interventions, and strategies suitable to integrate into a country's health system, policies and strategies for IRD outbreak protection.

Data acquired in this study allowed us to achieve our main objective by establishing the fundamental basis of a standard human cough aerosol model. This would enable us all to acquire better knowledge and understanding of the human bioaerosol pattern by best characterizing the number and/or size of droplets production contained in the cough aerosol that might open new avenues in epidemic-prone and pandemic-prone IRD outbreaks preparedness.