Team:ULaval/Description
INSPIRATION
The air quality and the presence of airborne pathogens have become increasingly important in recent years. However, monitoring air quality is tedious; it requires specialized personnel and is generally lengthy and costly, meaning that a contaminant is often discovered too late. With the rise of new diseases, the resurgence of old ones, and the growing concerns of disease transmission in hospitals (Eames et al., 2009; Mirhoseini et al., 2016; Bing-Yuan et al., 2018; Khan et al., 2017), the need to have more efficient methods to monitor their presence is ever more critical. To assess the need for airborne pathogen detection, we distributed market study surveys to people working in the food production industry as well as doctors and researchers specializing in infectiology, fields within which air quality control is of the uttermost importance. The preliminary results confirmed the need for an airborne pathogen detection device to improve air quality monitoring in various environments. In accordance with the comments gathered, we aim at creating a tool that could be used on-site to rapidly identify the presence of airborne pathogens.
Project Description
Aerobiology
Aerobiology is the study of bioaerosol in the air. Bioaerosols can be composed of whole microorganisms or fragments of microorganisms or other living organisms. These microorganisms or fragments usually originate from a surface where the organism can live and multiply. The bioaerosols are then generated through many processes. They can be as simple as wind dragging the microorganisms from a surface into the air. They can also be produced by an infected person through many means such as speaking or sneezing which both expel droplets of various sizes at different speeds (Verreault, Moineau et al. 2008).
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Bioaerosols can be composed of solid or liquid particles. Bioaerosols can be dry (droplet nuclei) or watery (droplets), this factor is determined by the means of production of the bioaerosol. The size of bioaerosols can range from 0,002µm and 100µm, the size of bioaerosols affects the duration the particle can stay airborne. Thus, some particles can remain suspended in the air for very long periods of time, increasing the odds that someone would inhale them. The size also influences the location where the bioaerosols can deposit within the respiratory tract. (Verreault, Moineau et al. 2008).
Aerobiology is an important domain of research because many agents can be transmitted through the air and cause various infections. Indeed, it is possible for potentially pathogenic microorganisms, toxic products, and allergens to be disseminated into the air. In fact, bioaerosols can be inhaled by humans and cause allergic reactions or diseases such as tuberculosis, measles or many more (Maki, Puspitasari et al. 2014). However, microorganisms cannot grow in the air and can lose their infectious potential or viability because of their exposition to stress like drying, UV rays, and the temperature. To cause disease, a person needs to be exposed to a minimal dose of viable microorganisms which depends on the nature of the microorganism, this is called the infectious dose.
It is possible to detect those bioaerosols and identify their content through air sampling. To sample the air, many devices can be used, but most of these rely on a pump that will suck in great volumes of air and capture the bioaerosols (Walton and Vincent 1998). To be identified, the microorganisms found in samples need to be tested through different means, some of which will require culturing of the sample (Nehme, Letourneau et al. 2008). By sampling the air and identifying its content, it is possible to detect the presence of harmful pathogens and take measures to avoid exposition or to eliminate their presence from the air. Thus, it is important to sample the air and take measures to control the presence of microorganisms in the air so that infections can be prevented or so that the spread of pathogens can be put to a halt (Verreault, Moineau et al. 2008).
Figure 1.Aerosol deposition pattern in different regions of the human respiratory tree according to the aerodynamic diameter of the particles. This is the value averages for men and women obtained from a light breathing exercise through the nose (courtesy of Prof Duchaine)
Figure 2.Different aerosols of solid and liquid particles and their size range (courtesy of Prof Duchaine)
Why is air quality control important?
Aerosols containing microorganisms or biological fragments, also known as bioaerosols, can represent an important threat to human health (Mbareche et al., 2019). In fact, a wide variety of organisms, including bacteria, fungi, and viruses, can be transmitted or transported by the airborne route. They can induce nosocomial infections, infections acquired in hospitals and healthcare facilities, which can cause complications or be deathly (Department of Health et al., 2019b). A virus such as the measles virus, Poxvirus (chickenpox) and the Norovirus (enterogastritis) have been known to be transmitted or transported in the air.
From January 1st, 2019 to April 15th, 2019, 170 countries reported 112 163 measles infection cases, nearly 300% more than the previous year (WHO, 2019). It can lead to major respiratory and neurological complications that can be fatal (Centers for Disease Controls and Preventions, 2018a). Measles caused overall close to 110 000 deaths in 2017 (WHO, 2019).
Even though an effective vaccine is available against the measles virus (93% with one dose, 97% with two doses [Centers for Disease Control and Prevention, 2019]), the lack of vaccination coverage led to the resurgence of this highly contagious virus in recent years (Paules et al., 2019). Furthermore, the administration of the live viral vaccine for measles is also not always possible, for example, in the event of immunosuppressed patients (Arvas, 2014), leaving the non-immunized population at high risk of developing the disease.
The number of cases of measles being contracted in healthcare settings through the air is unknown. However, no air monitoring is currently in place (Department of Health et al., 2019a). Since patient afflicted by measles may transit by theses environment to seek treatment, they could contaminate the air and easily lead to the transmission of the disease to other patients.
Chickenpox is caused by the Varicella zoster virus (VZV). A vaccine is also available for the VZV (94% effective [U.S. Department of Health & Human Services, 2018]), but the vaccination coverage is highly variable worldwide and many countries opted for only one dose of the vaccine, which can still lead to the development of symptoms and transmission (Wutzler et al., 2017). Nosocomial transmission of the VZV is a well recognized medical and health care problem (Seward and Marin, 2014). It is associated with significant morbidity and mortality in high-risk populations, such as neonates, pregnant women and immunocompromised patients (Kim et al., 2018). Even in otherwise healthy patients, the VZV can cause deadly complications including encephalitis, pneumonia, sepsis (Centers for Disease Controls and Preventions, 2018c). VZV can be contracted can direct contact with an infected person, but also through the inhalation of contaminated droplets (Kim et al., 2018). As for other human pathogenic viruses, there is currently no air monitoring procedures in place for VZV.
The norovirus is the principal cause of gastroenteritis, both in adults and children (Rosa et al., 2013). Even though it is considered to be an enteral virus, it has recently been described that it can remain infectious in the air and be transported on long distances, which could promote infections through the fecal-oral route (Bonifait et al., 2015). It is estimated to cause 60 billion dollars lost worldwide due to healthcare costs and lost productivity (Centers for Disease Control and Prevention, 2018b). Outbreaks of Norovirus infections are also frequently recorded in healthcare settings (Maccannell et al., 2011). There is currently no vaccine or antiviral medication available for this infection (Rocha-Pereira et al., 2016) and the infectious dose required to contract the disease is very low (<100 viral particles) (Atmar et al., 2008). Hence, infection prevention methods are the only way to limit the occurrence of a Norovirus outbreak. Constant monitoring of the virus in the air, combined with already in place protocols (e.i. surface cleaning), could contribute to the establishment of tight infection prevention procedure by ensuring patients are not exposed to the virus.
Increased surveillance of viral pathogens seems mandatory to get better control over the propagation of those potentially deadly diseases. Constant monitoring could also increase the knowledge of transmission and the epidemiological importance of the airborne route. The development of an easy-to-use device could lead to the accumulation of a much greater number of epidemiologic data related to the air transmission route that could further our knowledge of these diseases and lead to better protection of the population.
Current state of microbial air quality monitoring
Although air quality control and airborne pathogen controls seems critical, the tools currently used to achieve it are complex, require trained personnel, well-equipped facilities, and are time consuming (Department of Health et al., 2019b). Consequently, microbial air quality control is mostly performed by research groups or commercial experts.
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Due to this high complexity of air sampling and characterization, no systematic microbial air quality management protocol is established in healthcare facilities, even though ensuring the air quality in these environments is fundamental due to the vulnerable nature of the population it holds (Settimo et al., 2017).
Microbial air quality monitoring involves multiples steps, including air sampling, samples transportation, sample treatment, and detection, that requires the manipulation and transportation of samples and liquids. The lack of automatization of these processes renders them inadequate to rapidly detect pathogens in the environment such as healthcare facilities. In the context of our project and considering the possibilities of synthetic biology, we focused on the automatization and detection systems.
Detection methods and sample treatments
Most of the current detection methods can be divided into two groups: culture or molecular techniques. Sample treatment also is highly dependent on the detection method selected since samples can rarely be analyses directly (e.i. presence of contaminants, requires dilution or concentration, the genomics material is located inside living cells, etc.). Theses may currently require extensive manipulations by specialized scientists.
Culture methods imply the impaction or plating of microorganisms directly on solid culture media (e.i. Tryptic Soy Agar, Bengal Rose Agar, etc. ) and incubation which will enable the multiplication of viable microorganisms and the formation of colonies. Those can be counted and identified to estimate the concentration of microorganisms per volume of air. These techniques have the advantages of differencing between still infectious microorganisms and inactivated (“death”) ones. However, it requires extensive working force (e.i. Plating, incubation, counting) and infrastructure (i.e media preparation and sterilization, incubators). Furthermore, these techniques cannot be easily applied to the detection of viral microbes, since their multiplication depends on the presence of a suitable host. Hence, the detection of viral particles requires the cultivation of the host (bacterial, human cells, etc.), which can be time-consuming or even impossible in cases where hosts cell cannot be cultured independently. These renders culture methods impracticable for fast and easy microbial air quality monitoring on the field.
The second method detects or quantifies microorganisms in air samples by the analysis of genomic material (DNA or RNA). Therefore, it requires a step of nucleic acid extraction and purification of the air samples to ensure proper efficiency of detection. The two most used molecular techniques are sequencing and Quantitative Polymerase Chain Reaction (qPCR). The sequencing method uses nucleic acids amplification methods to obtain the nucleotide sequence in part or as a whole. It is highly informative but is expensive, lengthy and inadequate for field detection. qPCR allows the quantification of microorganisms based on a correlation between a standard curve in a number of genomic copies. The primers selected let you target specific microorganisms or groups of organisms, Bacteria for example (16S). Although highly versatile, this method requires trained personnel for sample preparation and the use of a thermal cycler, which fluctuate between specific temperature to allow the polymerase chain reaction. Therefore, it is not an appropriate choice for quantification of field.
In short, no detection method currently used is appropriate to create an all-in-one and easy-to-use air-monitoring device for healthcare environments.
Our solution
Team ULaval intends to create a device, called A.D.N. (Airborne Detector for Nucleic acids, DNA in French) to rapidly detect minute concentrations of airborne pathogens. The device would rely on novel, microfluidics-based DNA extraction, and detection of target DNA sequences by molecular sensors.
Our molecular sensors are based on the riboregulator system, more specifically on the toehold switches technology (Green et al., 2014).
We are currently working to develop the microfluidic circuit that will be used to extract the genetic material from the samples, as well as to host the cell-free reaction chamber.
An air particle collector, such as the SASS 2300 will be linked to the microfluidic chip within which the genetic material of the collected particles will be mixed with the designed molecular sensor.
Binding of the molecular sensor to the target DNA sequence of a specific airborne pathogen will allow for the production of fluorescent or colored protein that can be detected.
Knowing that airborne pathogens are often found in low concentration in the air, we intend to use an isothermal amplifying system (Notomi et al., 2000) which will allow for the detection of smaller amounts of the airborne pathogens.
We aim to develop a tool that is easy to use, meaning that anyone could operate it and interpret the output, therefore removing the need for specialized personnel. By using a cartridge-based system that relies on microfluidics for DNA extraction of air samples, alongside a reusable sampling and detection apparatus, we think that the device could be fully automated, very sensitive and cheap of use. With the help of a researcher specializing in air biology and airborne pathogens, we intend to conduct real-life tests of our apparatus, as well as developing an extensive simulation model for our nucleic acid detector.
We are also working on the development of a software allowing for rapid design of toehold switches including both automated decisions and guidance on decisions to be made by the user in order to maximise the efficiency of the design process and hence obtain the desired toehold switches.
Who could use our device?
Our device was first intended to be used in healthcares setting, including hospitals and long-term care facilities....
Our device could be put to use in two different ways. First, directly on levels or rooms that host high-risk patients and that are susceptible to outbreaks or following an outbreak to ensure proper decontamination. Following discussions with experts, this might be difficult to implement due to a shortage of healthcare personnel and their already heavy workload. Therefore, the second option might be more realistic. It consists in implementing our device to the recirculating air system already in place in those environments. The task of initiating air monitoring, even if fairly simple, could then be the responsibility of the maintenance personnel and the infection prevention team. The combination of our detection method with corrective measures (increase air change rate, UV, ozone, etc.) could create a solid air quality management system that would ensure the safety of the patients. In addition, air treatment units of healthcare facilities usually treat high volumes of air coming from multiple rooms. The pathogenic contaminants are therefore more likely to be detected than by sampling levels or rooms individually.
On the other hand, the possible applications of our device go far beyond the healthcare system or even air quality monitoring. In fact, the large versatility of our device enables us to consider its utilization for the detection of any organism that arbor DNA or RNA genomes. It can analyze any liquid samples, meaning water, surfaces (e.i. With the use of a calcium alginate swabs) or solid sample (may need preprocessing) could by treated for microbial detection
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