Aerosol system for testing means for inactivation of airborne pathogens

Abstract

The present invention relates to an aerosol system for testing the effectiveness of means for inactivating airborne pathogens.

Description

[0001] AEROSOL SYSTEM

[0002] FOR TESTING MEANS FOR INACTIVATION OF AIRBORNE PATHOGENS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to an apparatus for testing the effectiveness of means for inactivating airborne pathogens comprising an aerosol system and at least one pathogen inactivating means.

[0005] BACKGROUND

[0006] Airborne disorders are caused by airborne pathogens, i.e., pathogens, such as bacteria, viruses and fungi, that are transmitted through the air, causing infections.

[0007] Airborne pathogens may be spread through sneezing, coughing, spraying of liquids, spread of dust, or any activity that results in the generation of aerosolized particles. The aerosolized particles may be generated from any source of infection, such as an infected subject, or biological waste products. A single human sneeze generates droplets with a size (geometric mean) ranging from 74 to 360 pm that can be spread for several meter. Other respiratory emission events include coughing and talking. It has been shown that 82% of particles detected during tidal breathing have a size of 0.300-0.499 pm.

[0008] Recently there has been strong concern for airborne pathogens, due to COVID-19 pandemic. COVID-19 is a highly contagious viral illness that has had a catastrophic effect on the world population with well over 750 million cases and almost 7 million deaths worldwide as of June 5, 2023. Virus-laden airway fluid (greater than 5 pm in diameter), or aerosol (smaller than 5 pm) can remain for hours. Therefore, a grand challenge is to effectively control air transmission of COVID-19 as well of other airborne pathogens to prevent infections.

[0009] To this aim, disinfecting means against airborne pathogens have been developed and still are. However, their effectiveness in blocking airborne transmission needs to be verified. In fact, typically, effectiveness of said disinfecting means is tested using droplets laid on surfaces.

[0010] At the same time, aerosol systems have been created to evaluate infectivity and stability of a virus aerosol, testing droplets size and virus persistence over time, but these systems are not suitable and have not been employed for testing disinfecting means. Moreover, currently available technologies for testing pathogens typically employ un- infectious, surrogate “avatars” of viruses, rather than real viruses, thus leading to unreliable results.

[0011] There is therefore a need for providing an apparatus capable of testing the effectiveness of disinfecting means against airborne pathogens, especially against respiratory viruses, preferably in their wild-type form.

[0012] BRIEF DESCRIPTION OF THE INVENTION

[0013] The limitations of the prior art are overcome by the present invention, providing a system capable of mimicking a natural airborne pathogen transmission and of collecting the pathogen after treatment by disinfecting means, for testing the effectiveness of disinfection.

[0014] The present invention is then directed to an aerosol system (100, 100’) for treating an aerosolized sample, comprising, in sequence:

[0015] - an aerosol generator (1, 1’), configured to aerosolize a sample,

[0016] - at least one aerosol treatment chamber (2), configured to contain an aerosolized sample (11, 11’), and

[0017] - at least one sample collector (3), configured to collect the aerosolized sample (21, 21’) that exits from the aerosol treatment chamber (2), wherein the aerosol generator (1, 1’), the aerosol treatment chamber (2) and the at least one sample collector (3) are in fluid communication.

[0018] The invention is also directed to an apparatus (200’, 300, 300’, 400’) for testing aerosol treating means, comprising: a) the aerosol system (100, 100’) described above, and b) at least one aerosol treating means (10, 10’, 30, 30’) configured to interact with the aerosol treatment chamber (2).

[0019] The aerosolized sample (21, 21’) that exits from the aerosol treatment chamber (2) of the apparatus (200’, 300, 300’, 400’) is a treated sample, that is collected in the sample collector.

[0020] Preferably, the apparatus of the invention is an apparatus (200’) for testing disinfecting means for inactivating an airborne pathogen, wherein the aerosol system (100’) comprises a bioaerosol generator (1’) for aerosolizing a sample comprising a pathogen, and wherein (b) the at least one aerosol treating means is at least one disinfecting means (10’) for inactivating pathogens.

[0021] The present invention is also directed to a method for testing disinfecting means for inactivating airborne pathogens, carried out by the apparatus (200’) for testing disinfecting means of the invention, and comprising the steps of:

[0022] - providing a sample comprising a pathogen; - aerosolizing said sample by means of the bioaerosol generator (L) of the aerosol system (100’) of the invention, obtaining an aerosolized sample (11’) comprising the pathogen;

[0023] - transferring the aerosolized sample (11’) into the at least one aerosol treatment chamber (2),

[0024] - disinfecting the aerosolized sample (11’) contained in the aerosol treatment chamber (2) by means of the at least one disinfecting means (10’), obtaining a disinfected aerosolized sample (21’),

[0025] - collecting the disinfected aerosolized sample (21’) by means of the at least one sample collector (3),

[0026] - recovering particles in the disinfected aerosolized sample (21’), optionally said particles comprising or consisting of the pathogen, and

[0027] - analysing the particles in an assay that is suitable to verify the viability of the pathogen and / or effectiveness of the disinfecting means.

[0028] The features and advantages of the present invention will become apparent from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the attached figures.

[0029] BRIEF DESCRIPTION OF FIGURES

[0030] Fig- 1 exhibits: (A) a scheme of the aerosol system (100) according to preferred aspects of the invention as described herein, and (B) a picture of an aerosol system under a class II biosafety cabinet. Here the sample collector (3) is an impinger connected to a vacuum pump (4); the arrows in A depict the direction of air suction operated by the vacuum pump. The aerosolized sample (11), generated by the aerosol generator (1), is depicted in A while being transferred into an aerosol treatment chamber (2); then the aerosolized sample (21) is depicted while exiting from the aerosol treatment chamber (2) to be collected by the impinger (3); the particles recovered from the collected aerosolized sample are depicted at the bottom of the impinger.

[0031] Fig- 2 exhibits: (A) a scheme of the apparatus (200’) for testing disinfecting means, according to preferred aspects of the invention, wherein the disinfecting means (10’) is a radio-frequency (RF) device emitting an electromagnetic signal, irradiating the aerosol treatment chamber (2), and (B) a picture of the apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3. The aerosolized sample comprising pathogens (11’), generated by the bioaerosol generator (1’) of a preferred embodiment of the aerosol system (100’) of the invention, is depicted in A while being transferred into the aerosol treatment chamber (2); then the disinfected aerosolized sample (21’) is depicted while exiting from the aerosol treatment chamber (2) to be collected by the impinger (3), connected to the vacuum pump (4); the pathogen’s particles recovered from the collected aerosolized sample are depicted in A as a grey box at the bottom of the impinger.

[0032] Fig- 3 exhibits (A) a scheme of the apparatus (200’) for testing disinfecting means, according to preferred aspects of the invention, and (B) a picture of the apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3, wherein the aerosol system comprises two treatment chambers (2’, 2”) in fluid communication and in series, and wherein the disinfecting means (10’) is a filter placed in a filter holder connecting the two treatment chambers (2’, 2”). The bioaerosol generator (L), the aerosolized samples, before (11’) and after disinfection (21’), the sample collector (3) and vacuum pump (4) are shown in A as in Fig. 2A.

[0033] Fig- 4 exhibits (A) a scheme of the apparatus (200’) for testing disinfecting means, according to preferred aspects of the invention, and (B) a picture of the apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3, wherein the disinfecting means (10’) is an ionizer, placed inside the aerosol treatment chamber (2). The aerosolized samples, before (11’) and after disinfection (21’) are shown in A as in Fig. 2A; the cruet of the bioaerosol generator (L) can be seen inside the aerosol treatment chamber (2), in B, while the sample collector (3) is not shown in B, being not yet connected.

[0034] Fig- 5 exhibits: (A) a scheme of the apparatus (200’) for testing disinfecting means, according to preferred aspects of the invention, and (B) a picture of the apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3, wherein the disinfecting means (10’) is a UV lamp, placed inside the aerosol treatment chamber (2); the sample collector (3) is shown in B in open configuration, not yet connected.

[0035] Fig. 6 exhibits: (A) a scheme of the apparatus (300) for testing sensors (30), according to preferred aspects of the invention, and (B) a picture of the same apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3, wherein the sensor (30) is an electrochemical sensor placed inside the aerosol treatment chamber (2), and connected to a computer (40) by means of an electric cable; the valve (6) downstream the treatment chamber is shown in closed configuration. The sample collector (3) is disconnected in B.

[0036] Fig. 7 exhibits: (A) a scheme of the apparatus (300’) for testing sensors (30’), according to preferred aspects of the invention, and (B) a picture of the same apparatus under a class II biosafety cabinet in a Biosafety Level 3 laboratory, BSL3, wherein the sensor (30’) is placed inside the aerosol treatment chamber (2). A device (40’) for reading the information of the biosensor (30’) is depicted in A.

[0037] Fig. 8 exhibits a scheme of the apparatus (400’) comprising at least one disinfecting means (10’) as in Fig. 6 and further comprising at least one sensor (30’) placed inside the aerosol treatment chamber (2), and connected to a computer (40); the valve (6) downstream the treatment chamber is shown in closed configuration.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention is then directed to an aerosol system (100) for treating an aerosolized sample, comprising, in sequence:

[0040] - an aerosol generator (1), configured to aerosolize a sample,

[0041] - at least one aerosol treatment chamber (2), configured to contain the aerosolized sample (11) generated by the aerosol generator (1),

[0042] - at least one sample collector (3), configured to collect an aerosolized sample (21) that exits from the aerosol treatment chamber (2), wherein the aerosol generator (1), the at least one aerosol treatment chamber (2) and the at least one sample collector (3) are in fluid communication.

[0043] The aerosol system according to preferred embodiments of the invention is illustrated in Fig. 1. The invention is also directed to an apparatus (200) for testing aerosol-treating means, comprising: a) the aerosol system (100) described previously, and b) at least one treating means (10) configured to interact with the aerosol treatment chamber (2).

[0044] Preferably, the apparatus of the invention is an apparatus (200’) for testing disinfecting means for inactivating an airborne pathogen, wherein (b) the at least one treating means (10) is at least one disinfecting means (10’) for inactivating airborne pathogens.

[0045] Therefore, the present invention is also directed to an apparatus (200’) for testing disinfecting means, comprising: a) an aerosol system (100’), comprising:

[0046] - a bioaerosol generator (1’), configured to aerosolize a sample comprising a pathogen,

[0047] - at least one aerosol treatment chamber (2, 2’, 2”), configured to contain the aerosolized sample (11’),

[0048] - at least one sample collector (3), configured to collect at least a portion of the aerosolized sample (21’) that exit from the at least one aerosol treatment chamber (2, 2’, 2”), wherein the aerosol generator (1’), the at least one aerosol treatment chamber (2, 2’, 2”) and the at least one sample collector (3) are in fluid communication; the apparatus (200’) further comprising: b) at least one aerosol disinfecting means (10’) for inactivating pathogens, configured to interact with the at least one aerosol treatment chamber (2, 2’, 2”).

[0049] The invention is further directed to a method for testing disinfecting means for inactivating airborne pathogens, carried out by the apparatus (200’) of the invention and comprising the steps of:

[0050] - providing a sample comprising a pathogen;

[0051] - aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11’) comprising the pathogen;

[0052] - transferring the aerosolized sample into the at least one aerosol treatment chamber (2),

[0053] - disinfecting the aerosolized sample (11’) contained in the aerosol treatment chamber (2) by means of the at least one disinfecting means (10’), obtaining a disinfected aerosolized sample (21’),

[0054] - collecting the disinfected aerosolized sample (21’) by means of the at least one sample collector (3),

[0055] - recovering particles in the disinfected aerosolized sample (21’), optionally said particles comprising or consisting of the pathogen, and

[0056] - analysing the particles in an assay suitable to verify the viability of the pathogen and / or effectiveness of the disinfecting means.

[0057] In further preferred embodiments, the at least one treating means configured to interact with the aerosol treatment chamber (2), comprises at least one sensor (30, 30’), preferably at least one biosensor (30’).

[0058] Therefore, the present invention is also directed to an apparatus (300, 300’) for testing sensors, comprising: a) an aerosol system (100, 100’) according to the invention and further comprising: b) at least one sensor (30, 30’), preferably placed inside the at least one aerosol treatment chamber (2, 2’, 2”), configured to detect particles in an aerosolized sample.

[0059] The invention is further directed to a method for testing sensors (30, 30’), carried out by the apparatus (300’) of the invention, and comprising the steps of:

[0060] - providing a sample optionally comprising a pathogen;

[0061] - aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11, 11’) optionally comprising the pathogen;

[0062] - transferring the aerosolized sample into the at least one aerosol treatment chamber (2, 2’, 2’ ’), said at least one aerosol treatment chamber containing at least one biosensor (30’) configured to detect airborne pathogens,

[0063] - operating the biosensor and obtaining information on the aerosolized sample (11, 11’). Advantageously, the information obtained from biosensors can be compared to the results of an assay analysing the collected sample. This allows to better test the sensor and / or to calibrate it.

[0064] The terms “sensor” is used herein to identify means capable of interacting with an aerosolized sample, and providing information on said sample. For instance, a sensor can detect the sample and provide information on its chemical and / or physical properties, such as chemical composition, concentration of certain elements, amount of sample, identifying the presence of a certain pathogen etc.; furthermore, it can detect variations of any of these properties over the observation period.

[0065] The term “biosensor” is used herein to identify means capable of interacting with an aerosolized sample comprising airborne pathogens, and providing information on said pathogen. Preferably, a biosensor is capable of providing information on the presence of pathogens and / or on the physical and / or chemical properties of the same, such as concentration, viability, amount of pathogen etc., and the variation of any of these elements over the observation period.

[0066] Preferably, the information provided by the sensor is transmitted to a receiving device, more preferably to a computer (40, 40’) comprising a computer processing unit (CPU), capable of elaborating said information, providing a readout.

[0067] In preferred embodiments, the receiving device and the sensor are not physically connected, but the device is capable of reading the information from the sensor and of elaborating it to provide a readout.

[0068] Exemplary sensors include sensors capable of transmitting the information through an electronic signal, e.g. to a receiving device that is electrically connected to the sensor, such as an electrochemical sensor. Exemplary sensors also include biosensors capable of detecting and measuring the concentration of a pathogen, e.g. through antibodies, aptamers, synthetic receptors, molecular imprinted polymers, etc.; said biosensors can be read by a specific device or assay to obtain the information. Further, non-limiting, exemplary sensors include optical sensors based on colorimetric and fluorescence techniques, surface plasmon resonance, chemical sensors, sensors based on electromagnetic waves or acoustic waves.

[0069] In further preferred embodiments, the apparatus comprises at least one sensor (30, 30’) placed inside the at least one aerosol treatment chamber (2, 2’, 2”), and at least one disinfecting means (10’), wherein the at least one sensor is configured to transmit a signal to the at least one disinfecting means, optionally wireless and / or by means of a computer, preferably wherein said signal activates the at least one disinfecting means.

[0070] According to preferred embodiments, such an apparatus can be used to test disinfecting means. Therefore, preferably, the invention is directed to a method for testing disinfecting means operated by an apparatus according to the invention, comprising at least one biosensor and at least one disinfecting means, wherein the at least one biosensor is configured to transmit a signal to the at least one disinfecting means, activating the at least one disinfecting means once received by the same, the method comprising the steps of:

[0071] - providing a sample comprising a pathogen, preferably in the form of a liquid suspension;

[0072] - aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11’) comprising the pathogen;

[0073] - transferring the aerosolized sample (11’) into the at least one aerosol treatment chamber (2, 2’, 2”), said at least one aerosol treatment chamber containing the at least one sensor (30, 30’) configured to detect airborne pathogens;

[0074] - providing a sample comprising a pathogen, preferably in the form of a liquid suspension;

[0075] - aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11’) comprising the pathogen;

[0076] - transferring the aerosolized sample (11’) into the at least one aerosol treatment chamber (2, 2’, 2”), said at least one aerosol treatment chamber containing at least one sensor (30’) configured to detect airborne pathogens; - operating the sensor and obtaining information on the aerosolized sample (11’);

[0077] - transmitting a signal to at least one disinfecting means (10’) activating the at least one disinfecting means (10’);

[0078] - disinfecting the aerosolized sample (11’) by means of the at least one disinfecting means (10’), and obtaining a disinfected aerosolized sample (21’),

[0079] - operating the sensor (30’) and obtaining information on the disinfected aerosolized sample (21’); optionally the method further comprising the steps of:

[0080] - collecting the disinfected aerosolized sample (21’) by means of the at least one sample collector (3),

[0081] - recovering particles in the disinfected aerosolized sample (21’), and

[0082] - analysing the particles in an assay that is suitable to verify the viability of the pathogen and / or effectiveness of the disinfecting means.

[0083] Preferably, the sensor is capable of detecting the variation of the concentration amount, and / orphysical and / or chemical properties of such pathogen after disinfection. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells, such as a population of cells.

[0084] The term “individual” or “subject” herein refers to a mammal, preferably human or non-human mammal, more preferably mouse, rat, other rodents, rabbit, dog, cat, pig, cow, horse or primate, most preferably human.

[0085] The terms “disinfecting / disinfection”, “neutralizing / neutralization” and “inactivating / inactivation” are herein used interchangeably to indicate the reduction or elimination of the pathogenicity of a pathogen. Said reduction or elimination of the pathogenicity can be achieved by destroying the pathogen and / or by reducing the concentration of the pathogen.

[0086] “Inactivating / disinfecting / neutralizing” means, as used interchangeably, are means capable of reducing or eliminating the pathogenicity of a pathogen. Said means can be mechanical, physical or chemical means.

[0087] In preferred aspects of the present invention said “disinfecting means” means emitting microwaves, light, or heat, chemical disinfectants, ionizers, or filters, such as polymeric filters having pores suitable to block the passage of the pathogen.

[0088] An “airborne” pathogen is a pathogen that can be transmitted through the air, such as bacteria, viruses and fungi / moulds.

[0089] In preferred aspects of the present invention the pathogen in the aerosolized sample is an airborne pathogen, more preferably a virus, most preferably a respiratory virus.

[0090] An aerosolized sample comprising a pathogen is also called “bioaerosol”.

[0091] Preferably the pathogen in the aerosolized sample is selected from: coronaviruses, rhinoviruses, Influenza viruses, Varicella viruses, Measles virus, Mumps virus, Hantavirus, Meningitis virus, Severe Acute Respiratory Syndrome (SARS) virus, respiratory syncytial virus, virulent avian influenza viruses; most preferably, the pathogen is in the aerosolized sample is selected from: SARS-CoV-2 virus, and Influenza virus.

[0092] Preferably the sample to be aerosolized by the aerosol generator (1, 1’) of the system of the invention (100, 100’) is a liquid suspension.

[0093] The (bio)aerosol generator (1, 1’) of the aerosol system (100, 100’) is then configured to produce an aerosol, preferably a bioaerosol, from a sample in the form of a liquid suspension and to direct it into the at least one aerosol chamber (2, 2’, 2”).

[0094] Preferably, the sample to be aerosolized is a liquid suspension comprising viral particles, more preferably having a viral titer of at least IxlO5TCID o / mL, or greater, as calculated according to the Reed and Muench method.

[0095] Preferably, the aerosol generator (1, 1’) comprises a cruet filled with the sample to be aerosolized. The cruet comprises an inlet in fluid connection with the aerosol generator (1, 1’) and an outlet in fluid connection with the at least one aerosol treatment chamber (2, 2’, 2”). When the aerosol generator is operated, the sample in the cruet is aerosolized and transferred into the at least one aerosol treatment chamber (2, 2’, 2”).

[0096] Any aerosol generators known in the art can be used in the aerosol system of the invention. An exemplary one is Omron C101 Essential (or a similar device) compressor nebulizer.

[0097] Preferably, the connection between the cruet outlet and the inlet of the at least one aerosol treatment chamber is sealed, e.g. with silicon, in order to maximize aerosol flow inside the at least one aerosol treatment chamber (2, 2’, 2”). Optionally said connection comprises a tube. Preferably, the elements of the aerosol system (100, 100’) that are in fluid communication with each other are connected by tubes, such as plastic and / or rubber tubes.

[0098] Preferably, fluid connections between the elements of the aerosol system (100, 100’) are regulated by means of valves, in order to guide the aerosol flux through the aerosol system. Preferably, the aerosol system (100, 100’) further includes one or more valves configured to regulate the fluid communication between the aerosol generator (1, 1’), the at least one aerosol treatment chamber (2, 2’, 2”) and / or the at least one sample collector (3).

[0099] More preferably, the aerosol system (100, 100’) comprises at least one valve (5) upstream of the at least one aerosol treatment chamber (2, 2’, 2”), configured to regulate the fluid communication between the aerosol generator (1, 1’) and the at least one aerosol treatment chamber (2, 2’, 2”) and / or at least one valve (6) downstream of the at least one aerosol treatment chamber (2, 2’, 2”), configured to regulate the fluid communication between the aerosol treatment chamber (2) and the at least one sample collector (3). The presence of at least one valve (6) downstream of the at least one aerosol treatment chamber (2, 2’, 2”), configured to regulate the fluid communication between the aerosol treatment chamber (2) and the at least one sample collector (3) is particularly advantageous as it allows to modulate the residence time of the aerosol in the treatment chamber, without the need of operating drastic changes to the system.

[0100] Advantageously, the at least one valve (5) upstream of the at least one aerosol treatment chamber (2, 2’, 2”), regulating the fluid communication between the aerosol generator (1, 1’) and the at least one aerosol treatment chamber (2, 2’, 2”), can maximize the aerosolized sample flow into the at least one aerosol treatment chamber (2, 2’, 2”).

[0101] Preferably, during aerosolization, the at least one valve (5) upstream of the at least one aerosol treatment chamber (2, 2’, 2”) is open and the at least one valve (6) downstream of the at least one aerosol treatment chamber (2, 2’, 2”) is closed.

[0102] Preferably, the at least one aerosol treatment chamber (2, 2’, 2’ ’) comprises an air inlet that can be opened or closed by an air inlet valve (7). Advantageously, the air inlet valve (7) can be opened when the vacuum is applied. Preferably, the aerosol generator of the system of the invention can produce aerosol particles of up to 10 pm in diameter, more preferably of 4-8 pm. Said size is the size of aerosol particles exhaled by either children and adults.

[0103] In preferred embodiments, the aerosol system (100, 100’) comprises a single aerosol treatment chamber (2).

[0104] In other preferred embodiments, the aerosol system (100, 100’) comprises more than one aerosol treatment chamber, such as two treatment chambers (2’, 2”).

[0105] According to preferred embodiments, the aerosol system (100, 100’) comprises a first treatment chamber (2’) and a second treatment chamber (2”) in series, and in fluid communication with each other; more preferably, the at least one treatment means (10, 10’) is placed between the first and the second treatment chambers (2’, 2”); in this way, the first treatment chamber (2’) can contain the aerosolized sample before treatment and the second treatment chamber (2”) can contain the treated aerosolized sample, e.g. the disinfected aerosolized sample.

[0106] In preferred embodiments, the aerosol treatment chamber (2, 2’, 2”) consists of an air-proof container for containing the aerosolized sample. Preferably, the container is a plastic container, a plexiglass container, or a glass container.

[0107] The at least one aerosol treatment chamber (2, 2’, 2”) of the aerosol system (100, 100’) is suitable for interacting with the at least one aerosol treatment means (10, 10’, 30, 30’), in particular for allowing treatment of aerosol, and of pathogens therein if any, by means of the aerosol treatment means.

[0108] The interaction of the at least one aerosol treating means (10, 10’, 30, 30’) with the at least one aerosol treatment chamber (2, 2’, 2”) can be physical and / or functional. In other words, in certain embodiments the treating means (10, 10’, 30, 30’) is physically connected to the at least one treatment chamber (2), while in other embodiments the at least one aerosol treatment chamber (2, 2’, 2”) and treating means (10, 10’) are not physically connected but treating means (10, 10’) can interact functionally the aerosol treatment chamber, in such a way that the aerosol treating means (10, 10’) is capable of treating the aerosolized sample contained in the treatment chamber.

[0109] According to preferred embodiments, the aerosol treating means is a disinfecting means (10’). Preferred disinfecting means (10’) of the apparatus (200, 200’) of the inventions are: microwave emitter, UV lamp, ionizer, or filter.

[0110] According to preferred aspects, the aerosolized sample comprising pathogens is disinfected by said at least one disinfecting means (10’), then the disinfected sample is collected in the collector (3). Optionally, the aerosol cruet of the aerosol generator (1, 1’) is placed inside the at least one aerosol treatment chamber (2, 2’, 2”).

[0111] Optionally, the at least one treating means (10, 10’, 30, 30’) is placed inside the at least one aerosol treatment chamber (2, 2’, 2”).

[0112] Preferably, when the at least one aerosol treating means is a sensor (30, 30’), the aerosol treating means is placed inside the at least one aerosol treatment chamber (2, 2’, 2”).

[0113] Preferably, during operation of the sensor, the at least one valve (6) downstream of the at least one aerosol treatment chamber (2, 2’, 2”) is closed.

[0114] According to preferred embodiments, the at least one sample collector (3) comprises, or consists of, an impinger.

[0115] As known in the field, an impinger is an instrument for collecting particles suspended in air in a sample, such as particles in an aerosol, by a stream of the suspension on a surface or in a liquid. The impinger comprises an inlet that is configured to receive an aerosol flux and an outlet connected to means for suction of air; the impinger further comprises at least one tangential nozzle through which air flows and at least one surface (impaction surface) towards which air flows exiting the nozzle(s).

[0116] When vacuum is applied, air is drawn into the impinger using a vacuum pump, and the air stream flows through the one or more nozzles toward an impaction surface, where particles are separated from the air stream by their inertia. Larger particles collect on the impaction surface, while small particles that do not impact follow the air stream.

[0117] The impinger is capable of collecting the aerosolized sample (21, 21’) that exits from the at least one aerosol treatment chamber (2, 2’, 2”) and of recovering the particles in the aerosolized sample. In particular, wherein the aerosolized sample transferred into the aerosol treatment chamber(s) comprises a pathogen, the impinger collects the pathogens in the bioaerosol that impact directly onto a liquid medium at the bottom of the impinger, such as a cell culture medium. In this way the recovered pathogens can be directly employed to infect target cells and transferred to a laboratory incubator for being tested.

[0118] Advantageously, live pathogens can be recovered from the aerosol system of the invention. Optionally, the recovered pathogen is frozen and stored as such, for instance at -80°C, until use. Therefore, the aerosol system (100, 100’) of the invention preferably comprises a sample collector being an impinger (3) and a vacuum pump (4) connected to the impinger (3).

[0119] When the sample is collected, the vacuum pump is turned on for suctioning the aerosolized sample from the at least one aerosol chamber (2, 2’, 2”). Preferably, a valve (6) downstream the at least one aerosol chamber (2, 2’, 2”) and an air inlet valve (7), regulating the air flow through the aerosol chamber (2, 2’, 2”), are opened, then the sample is collected in the impinger.

[0120] Preferably when the aerosolized sample comprises a pathogen, the pathogen is collected in a medium suitable for maintaining the pathogen alive, such as a culture medium. The pathogen can then be recovered from the collector in a liquid medium.

[0121] According to preferred embodiment, about 5 minutes are necessary to collect sample: the vacuum pump (4) is then turned on for 2-10 minutes, more preferably for 5-10 minutes, once the valves (6) and (7) are opened.

[0122] The pathogen recovered from the disinfected sample in the collector can be analysed in an assay suitable for testing the viability of the pathogen, and / or the effectiveness of the disinfection means.

[0123] A preferred assay is an in vitro assay for determining viral replicative capacity, such as an assay for determining the viral titer.

[0124] Viral titer after treatment by the aerosol-treatment means provides information on the neutralization effectiveness of the aerosol treatment means. Preferably, the sample before aerosolization is also analysed in the same assay for comparison.

[0125] Preferably, the size of the system as a whole is such that the system can stay inside a regular biosafety cabinet (e.g. a cabinet having a working area of about 50 x 50 x 120 cm), in order to avoid contaminations of the environment by biohazardous pathogens in the aerosolized sample. It should be understood that all the possible combinations of the preferred aspects of the present invention are also described, and therefore similarly preferred.

[0126] Examples of preferred embodiments of the present invention are provided below for illustrative and non-limiting purposes.

[0127] As shown also in the following examples, the aerosol system and apparatus of the invention are effective, reliable and can be easily adapted to test very different aerosol treatment means.

[0128] Moreover, different means, such as disinfecting means and sensors, can even be tested on the same aerosolized sample, thus making it possible to obtain results that can be directly compared. Moreover, the aerosol system has been configured in order to achieve an optimal recovery of live pathogens in the collected sample and to carry out the whole method of the invention in a very short time, such as a time of less than 30 minutes / sample.

[0129] EXAMPLES

[0130] Example 1

[0131] 3 ml of a concentrated viral suspension (viral titer >105TCID o / mL, tissue culture infectious dose 50%) were inserted into the cruet of the aerosol generator (!’) of an aerosol system (100’) according to a preferred embodiment of invention, as exhibited in Fig. 2.

[0132] Aerosol generator (E) was turned on for 10 minutes, with the valve (5) upstream of the aerosol chamber (2) open and the valve (6) downstream of the aerosol chamber closed.

[0133] Aerosolized virus flew inside the aerosol treatment chamber (2).

[0134] The aerosol treatment chamber is a plastic chamber.

[0135] The aerosol generator (F) was turned off and both valves were closed. Aerosolized virus was then treated by an aerosol treatment means (10’) to be tested.

[0136] Aerosolized treated sample was collected from the aerosol treatment chamber (2) after treatment, inside the impinger (3), filled with 20 mL of culture medium.

[0137] Collection was carried out by turning on the vacuum pump (4) for 5 minutes at a flux of 12 L / min, opening the valve (6) downstream of the aerosol chamber (2) and the air inlet valve (7) of the aerosol chamber.

[0138] Different viruses (SARS-CoV-2 Wuhan strain, Omicron variant, Delta variant; H1N1 virus) were successfully aerosolized and recovered from the collected sample.

[0139] In particular, live virus was recovered, as determined by testing the recovered virus in an in vitro assay for evaluating the viral replicative capacity (i.e., viral titer).

[0140] The assay was carried out as follows.

[0141] Target cells (Vero E6 cells, African green monkey kidney, for SARS-CoV-2 strains, MDCK, Madin-Darby canine kidney, for H1N1 virus) were seeded at a density of 20,000 cells / well into a 96-well plate in complete medium (DMEM with 10% bovine serum) at 37° C and 5% CO2. Twenty-four hours after seeding, the cells reached -90% confluency and the fetal bovine serum concentration was decreased to 2% to avoid interference with the viral infection. The cells were then infected with 10-fold serial dilutions (8 replicates for each dilution) of the viral suspensions obtained after the impingement procedures for aerosol collection. The cells were then cultured for 3 to 7 days, and infection was determined by observing the cytopathic effect under an inverted microscope.

[0142] The viral titer of the recovered virus was also calculated according to the Reed and Muench method and expressed as TCIDso / mL.

[0143] Table 1 hereafter shows viral titer of recovered virus after aerosolization:

[0144] Table 1.

[0145] High titers of alive virus have been recovered from the aerosol system of the invention.

[0146] Example 2

[0147] The effectiveness of a disinfecting RF device has been determined in an apparatus (200’) according to a preferred embodiment of the invention, as depicted in Fig. 2.

[0148] A virus-containing aerosolized sample (11) was generated by a commercially available aerosol generator (Omron) (F) integrated in an aerosol system (100’) according to the invention. A viral suspension of the infectious SARS-CoV-2 (original Wuhan strain), with high titer virus (10E+06 TCIDso / mL), was used to generate the aerosol. The aerosolized sample particles had a size up to 10 pm.

[0149] The aerosolized sample was introduced in a treatment chamber (2) consisting of a plastic, airproof, container of 0.5 liters volume.

[0150] During the test, the treatment chamber (2) containing the infected aerosol sample was subjected to irradiation by the disinfecting device (10’), emitting an electromagnetic signal at different frequencies and amplitudes.

[0151] Compared to classical pathogen inactivation, electromagnetic wave radiation can penetrate and heat rapidly and homogeneously, and resonate with microorganisms.

[0152] After treatment, the treated aerosolized sample (21) was collected by means of active impingement directly in a complete medium with 2% serum contained in a glass collector (3). The collector used is an impinger comprising an inlet and tangential nozzles, through which air from the aerosol treatment chamber is sucked when vacuum is applied. A vacuum pump connected to the impinger was operated as previously described. Viral particles were absorbed in this medium and recovered. Recovered viral particles were then cultured in laboratory for viral titer determination.

[0153] A control (untreated) virus was processed in the same experimental conditions with the disinfecting device turned off. The replicative capacity (i.e., viral titer) of the virus treated with microwaves (compared to untreated virus) was then determined in an in vitro system as follows. Vero E6 cells were seeded at a density of 20,000 cells / well into a 96-well plate in complete medium at 37° C and 5% CO2. Twenty-four hours after seeding the cells reached -90% confluency the fetal bovine serum concentration was decreased to 2% to avoid interference with the viral infection. The cells were then infected with 10-fold serial dilutions (8 replicates for each dilution) of the viral suspensions obtained after the impingement procedures for aerosol collection and control. The cells were then cultured for seventy-two hours, and infection was determined by observing the cytopathic effect under an inverted microscope. The viral titer of the recovered virus was determined according to the Reed and Muench method and expressed as TCIDso / mL. The results of the SARS-CoV-2 inactivation by the tested disinfecting device, at different potencies of electromagnetic waves (400 V / m in experiment 1, 100 V / m in experiment 2, 40 V / m in experiment 3) and 6 V / m in experiment 4) are shown in table 2.

[0154] Table 2

[0155] The viral inactivation ratio by the tested disinfecting device, at different values of frequency step (10 MHz step in experiments 4 and 5, 20 MHz step in experiments 6 and 7) and frequency dwell time (3.2 sec dwell in experiments 6 and 7, 0.05 sec dwell in experiments 8 and 9), are reported in Tables 3 and 4, respectively.

[0156] Table 3

[0157] Table 4 The disinfecting device was then tested in the apparatus of the invention, by verifying the antiviral activity in the assay described above, at different exposure times (for 5 min in experiment 9, 3 min in experiment 10 and 1 min in experiment 11). Results are shown in table 5.

[0158] Table 5

[0159] The effectiveness of the microwave disinfecting means tested has been correctly and reliably verified by means of the apparatus of the invention. Moreover, the apparatus of the invention has permitted to optimize the operativity of the disinfecting device tested.

[0160] Example 3

[0161] The apparatus (200’) depicted in Fig. 3, according to a preferred embodiment of the invention, has been employed to test viral inactivation effectiveness of a polymer within a filter.

[0162] In said apparatus (200’) two identical aerosol treatment chambers (2’, 2”) have been employed, connected with each other through a filter holder. Bioaerosol was produced as described in previous examples and the pathogen recovered by means of the impinger (3) and vacuum pump (4).

[0163] Filter efficiency was calculated by comparing viral titer in the treated sample obtained after treatment with filter to viral titer in untreated sample obtained from an aerosol system without the filter.

[0164] The results of the assay on the recovered viruses are shown in Table 6 below.

[0165] Table 6

[0166] The effectiveness of the filter tested has been correctly and reliably verified by means of the apparatus of the invention.

[0167] Example 4

[0168] The apparatus (200’) depicted in Fig. 4, according to a preferred embodiment of the invention, has been employed to test viral inactivation effectiveness of an ionizer device.

[0169] In such configuration a larger aerosol treatment chamber (2) was employed in order to accommodate the air disinfection device inside.

[0170] Aerosol was produced as described above and the treated sample recovered as previously reported.

[0171] The ionizer neutralizing effectiveness was calculated by comparing the viral titer of the sample collected after treatment compared to the viral titer of an untreated sample, obtained with the ionizer turned off. The results of the assay on the recovered viruses are shown in Table 7 below.

[0172] Table 7.

[0173] The effectiveness of the ionizer tested has been correctly and reliably verified by means of the apparatus of the invention.

[0174] Example 5

[0175] The apparatus (200’) depicted in Fig. 5, according to a preferred embodiment of the invention, has been employed to test viral inactivation effectiveness of a UV lamp treatment.

[0176] In such configuration a larger aerosol treatment chamber was employed in order to accommodate the UV lamp device inside.

[0177] Aerosol was produced as described above and the treated sample recovered as previously reported.

[0178] Device efficiency was calculated by comparing of the sample collected after treatment compared to the viral titer of an untreated sample, obtained with the UV lamp turned off.

[0179] The results of the assay on the recovered viruses are shown in Table 8 below. Table 8.

[0180] The effectiveness of the UV lamp treatment tested has been correctly and reliably verified by means of the apparatus of the invention.

[0181] Example 6

[0182] An electrochemical sensor (30) was placed inside the treatment chamber (2) of an aerosol apparatus (300) according to a preferred embodiment of invention, as exhibited in Fig. 6.

[0183] The sensor was connected to a computer (40) through an electric cable.

[0184] Aerosol generator (1) was turned on for 10 minutes, with the valve (5) upstream of the aerosol chamber (2) open and the valve (6) downstream of the aerosol chamber closed.

[0185] Aerosolized sample flew inside the aerosol treatment chamber (2).

[0186] The sensor (30) detected the aerosolized sample, and transmitted information to a computer, connected to it by an electric cable, for elaboration by a software.

[0187] The valve (6) downstream of the aerosol chamber was then opened in order to collect the sample.

[0188] Example 7

[0189] A biosensor (30’) for virus detection was placed inside the treatment chamber of an aerosol apparatus (300’) according to a preferred embodiment of invention, as exhibited in Fig. 7.

[0190] 3 ml of a concentrated viral suspension (viral titer >105TCID o / mL, tissue culture infectious dose 50%) were inserted into the cruet of the aerosol generator (1’).

[0191] Aerosol generator (1’) was turned on for 10 minutes, with the valve (5) upstream of the aerosol chamber (2) open and the valve (6) downstream of the aerosol chamber closed.

[0192] Aerosolized virus flew inside the aerosol treatment chamber (2).

[0193] After exposing the biosensor (30’) to the virus aerosol for a suitable time, the biosensor was collected from the chamber and then connected to a computer (40’) to elaborate a readout of the sensor outcome. The valve (6) downstream of the aerosol chamber was then opened in order to collect the sample and the results obtained were compared.

[0194] Example 8

[0195] A biosensor (30’) for virus detection was placed inside the treatment chamber of an aerosol apparatus (400’) according to a preferred embodiment of invention, as exhibited in Fig. 8.

[0196] The sensor was connected to a computer (40) through an electric cable. 3 ml of a concentrated viral suspension (viral titer >105TCID o / mL, tissue culture infectious dose 50%) were inserted into the cruet of the aerosol generator (1’).

[0197] Aerosol generator (1’) was turned on for 10 minutes, with the valve (5) upstream of the aerosol chamber open and the valve (6) downstream of the aerosol chamber closed. Aerosolized sample flew inside the aerosol treatment chamber (2).

[0198] The sensor (30’) detected the aerosolized sample, and transmitted information to a computer, connected to it by an electric cable. The computer transmitted a signal to a microwave disinfecting means (10’) activating it.

[0199] After a suitable time, the disinfecting means is turned off and the sensor was operated again to verify efficacy of the disinfecting means. The valve (6) downstream of the aerosol chamber was then opened to further check that information obtained from the sensor was correct.

Claims

CLAIMS1. An aerosol system (100, 100’) for treating an aerosolized sample, comprising, in sequence:- an aerosol generator (1, 1’), configured to aerosolize a sample,- at least one aerosol treatment chamber (2, 2’, 2”), configured to contain the aerosolized sample (11,11’) generated by the aerosol generator (1, 1’),- at least one sample collector (3), configured to collect an aerosolized sample (21, 21’) that exits from the at least one aerosol treatment chamber (2, 2’, 2”), wherein the aerosol generator (1, 1’), the at least one aerosol treatment chamber (2, 2’, 2”) and the at least one sample collector (3) are in fluid communication, wherein the aerosol system further comprises at least one valve (6) downstream of the at least one aerosol treatment chamber (2, 2’, 2”), configured to regulate the fluid communication between the aerosol treatment chamber (2) and the at least one sample collector (3).

2. An apparatus for testing aerosol-treating means, comprising: a) the aerosol system (100, 100’) of claim 1, and b) at least one aerosol-treating means (10, 10’, 30, 30’), interacting with the at least one aerosol treatment chamber (2, 2’, 2”).

3. The apparatus of claim 2 being an apparatus (200’) for testing disinfecting means, wherein: a) the aerosol system (100’) comprises:- the aerosol generator being a bioaerosol generator (1’), configured to aerosolize a sample comprising a pathogen, and- the at least one aerosol treatment chamber (2, 2’, 2”) being configured to contain a bioaerosol containing a pathogen (11’), and wherein: b) the at least one aerosol-treating means comprise at least one disinfecting means (10’) for inactivating pathogens, configured to interact with the at least one aerosol treatment chamber (2, 2’, 2”).

4. The apparatus (200’) of claim 3, wherein at least one disinfecting means (10’) is selected from: a radiofrequency device, a UV lamp, an ionizer, or a filter.

5. The apparatus of claim 2 being an apparatus (300, 300’) for testing sensors, wherein: b) theat least one aerosol-treating means comprise at least one sensor (30, 30’).

6. The apparatus (300, 300’) of claim 5, wherein the at least one sensor is a biosensor (30’).

7. The apparatus of anyone of claims 3-6 being an apparatus (400’) wherein b) the aerosoltreating means comprise at least one sensor (30, 30’) and at least one disinfecting means (10’).

8. The apparatus (400’) of claim 7, wherein the at least one sensor (30, 30’) is configured to transmit a signal to the at least one disinfecting means (10’), optionally wireless, preferably wherein said signal activates the at least one disinfecting means (10’) once received by the at least one disinfecting means.

9. The aerosol system (100, 100’) of claim 1 or the apparatus (200’, 300, 300’, 400’) of anyone of claims 2-8, further comprising: at least one valve (5) upstream of the at least one aerosol treatment chamber (2, 2’, 2”), configured to regulate the fluid communication between the aerosol generator (1, 1’) and the at least one aerosol treatment chamber (2, 2’, 2”), preferably wherein the at least one aerosol treatment chamber (2, 2’, 2”) further comprises an air inlet valve (7) regulating the flux of air through an air inlet in the at least one aerosol treatment chamber (2, 2’, 2”).

10. The aerosol system (100, 100’) of claim 1 or the apparatus (200’, 300, 300’, 400’) of anyone of claims 2-9, comprising at least two treatment chambers (2’, 2”), preferably wherein the at least two treatment chambers (2’, 2”) are in fluid communication; optionally wherein the at least one treatment means (10, 10’) is placed between the at least two treatment chambers (2’, 2”).

11. The aerosol system (100, 100’) of claim 1 or the apparatus (200’, 300, 300’, 400’) of anyone of claims 2-10, wherein the at least one sample collector (3) comprises, or consists of, an impinger, preferably connected to a vacuum pump (4).

12. A method for testing disinfecting means for inactivating airborne pathogens, carried out by the apparatus (200’, 300, 300’, 400’) of claim 3-11 when depending from claim 3, the method comprising the steps of:- providing a sample comprising a pathogen, preferably in the form of a liquid suspension;- aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11’) comprising the pathogen;- transferring the aerosolized sample (11’) into the at least one aerosol treatment chamber (2, 2’, 2”),- disinfecting the aerosolized sample (11’) by means of the at least one disinfecting means (10’), obtaining a disinfected aerosolized sample (21’),- collecting the disinfected aerosolized sample (21’) by means of the at least one sample collector (3),- recovering particles in the disinfected aerosolized sample (21’), and- analysing the particles in an assay that is suitable to verify the viability of the pathogen and / or effectiveness of the disinfecting means.

13. A method for testing sensors (30, 30’), carried out by the apparatus (300’, 400’) of anyone of claims 5-11, when depending on claim 5, comprising the steps of:- providing a sample, optionally comprising a pathogen;- aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11, 11’) optionally comprising the pathogen;- transferring the aerosolized sample into the at least one aerosol treatment chamber (2, 2’, 2”), said at least one aerosol treatment chamber containing at least one sensor (30’) configured to detect airborne pathogens,- operating the sensor and obtaining information on the aerosolized sample (11, 11’).

14. A method for testing disinfecting means for inactivating airborne pathogens, carried out by the apparatus (400’) of claims 7 or 8, the method comprising the steps of:- providing a sample comprising a pathogen, preferably in the form of a liquid suspension;- aerosolizing said sample by means of the bioaerosol generator (1’) of the aerosol system (100’), obtaining an aerosolized sample (11’) comprising the pathogen;- transferring the aerosolized sample (11’) into the at least one aerosol treatment chamber (2, 2’, 2”), said at least one aerosol treatment chamber containing at least one sensor (30’) configured to detect airborne pathogens;- operating the sensor and obtaining information on the aerosolized sample (11’); - transmitting a signal to at least one disinfecting means (10’) activating the at least one disinfecting means (10’);- disinfecting the aerosolized sample (11’) by means of the at least one disinfecting means (10’),and obtaining a disinfected aerosolized sample (21’),- operating the sensor (30’) and obtaining information on the disinfected aerosolized sample (21’); optionally the method further comprising the steps of:- collecting the disinfected aerosolized sample (21’) by means of the at least one sample collector (3),- recovering particles in the disinfected aerosolized sample (21’), and- analysing the particles in an assay that is suitable to verify the viability of the pathogen and / or effectiveness of the disinfecting means.

15. The method of any one of claims 12-14, wherein the assay is an in vitro assay for evaluating the pathogen replicative capacity.

16. The apparatus (200’, 300, 300’, 400’) of anyone of claims 3-11 or the method of any one of claims 12-15, wherein the pathogen is selected from: coronavirus, rhinovirus, Influenza virus, Varicella virus, Measles virus, Mumps virus, Hantavirus, Meningitis virus, Severe Acute Respiratory Syndrome (SARS) virus, respiratory syncytial virus, virulent avian influenza virus.

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