Patent Description:
The present invention concerns an apparatus and a method for sampling and detecting pathogens in air. The invention is preferably applied in air sampling, mainly in closed environments such as A&E waiting rooms, hospital rooms, supermarkets, offices, lifts, undergrounds, etc. and in aerosol emissions produced directly by the respiratory tracts of a patient and in breathing and ventilation apparatus, for detecting pathogens in the air sampled. More specifically, the apparatus of the present invention allows the detection of viruses suspended in the air sampled by said apparatus, including the SARS-CoV-<NUM> virus, which will be referred to below without loss of generality.

In the current public health emergency caused by the global and uncontrolled spread of the SARS-CoV-<NUM> virus, stringent control and prevention measures have to be introduced in order to limit the probability of contracting the disease.

Among these measures, accurate and timely methods for sampling and detecting the SARS-CoV-<NUM> virus in the air are desirable. The fundamentally important role of detection of the SARS-CoV-<NUM> virus in the air sampled, as a method that can effectively help to curb the spread of the pandemic, has been shown by various scientific studies that highlight the possible survival of the SARS-CoV-<NUM> virus in the air for several hours and over long distances.

Following said scientific evidence, the World Health Organization recommends maintaining a distance between individuals of approximately <NUM> metre in order to limit the spread of SARS-CoV-<NUM> virus infections. As reported in a recent study (<NPL>), these recommendations are the result of an estimate of the distances based on models, developed in the first half of the <NUM>, according to which infection can occur via expiratory droplets having dimensions large enough to be deposited in the immediate vicinity of the infected individuals before evaporating, whereas the smaller expiratory droplets evaporate before they are deposited. However, the report shows that, following exhalation, sneezing and coughing, in addition to the droplets that follow semi-ballistic trajectories, a turbulent gas cloud is generated that transports within it a cluster of droplets in a wide range of dimensions. The damp and warm environment of the cloud, as opposed to the droplets being in isolation, contributes to prolonging the evaporation times of the droplets, extending their life time up to a factor of <NUM> and increasing the distances travelled to approximately <NUM>-<NUM>. The droplets that are deposited along the trajectory can contaminate surfaces, the others remain trapped in the cloud until it loses momentum and allows evaporation. Droplet residues or nuclei are therefore left and can remain in suspension for hours in the air. The length of time the droplets remain in the air is regulated by the ambient conditions and by any ventilation and conditioning systems operating.

This scientific evidence is supported by other studies such as, for example, the one carried out by <NPL>, which reports the persistence of the SARS-CoV-<NUM> virus in the aerosol for at least <NUM> hours.

In addition to the expiratory droplets, SARS-CoV-<NUM> enters the air also simply by breathing, as shown by an American study that detected the SARS-CoV-<NUM> virus in air samples collected at a distance of over <NUM> metres between two patients, or in operating theatres during operations on infected patients.

At the same time, the virus can survive for several hours in ventilation equipment and aeration systems. There is also evidence of the presence of the virus on various surfaces, from the floors to the walls of hospital rooms. From these surfaces, the virus could be re-circulated in the air, for example by walking on contaminated floors.

In addition to the droplets exhaled and the breath of infected persons, a further potential carrier for transmission of the virus is atmospheric particulate. Several studies show the possible link between pollution caused by atmospheric particulate and the spread of the SARS-CoV-<NUM> epidemic, while in another study the virus was found on the particulate.

Hence the need to sample and analyse both the atmospheric particulate and the aerosol, including the aerosol emitted directly by persons potentially infected by SARS-CoV-<NUM>.

Following identification of the virus, targeted timely actions can be implemented aimed, for example, at sanitisation of the environment in which the air sampling was carried out and also of the neighbouring areas, identification of the persons who regularly visit said environment, carrying out diagnostic tests on them and directly diagnosing the virus infection if the air collected comes directly from the breath of a patient or asymptomatic persons, giving the apparatus of the present invention a diagnostic function not only of ambient type but also for medical use, alongside the commonly used nasopharyngeal swabs.

In the patent and non-patent literature, devices are known that separately perform sampling of the air with capture of the airborne pathogens and the detection of said pathogens based on chemical or biomolecular analyses or analysis of chemical-physical properties.

The devices belonging to the first category and therefore most commonly used for sampling the air and capturing the virus include solid impactors (for example Andersen or slit impactors) or liquid impactors (All-glass impingers - AGIs and similar), cyclonic samplers and filters.

As regards the methods for detecting pathogens, including viruses, alongside the traditional spectroscopic and microbiological techniques, there are the most recent devices that use lab-on-chip technology. In this regard, the patent <CIT> describes an integrated system for chemical and/or biomolecular analysis that uses the lab-on-chip technology to perform, automatically and rapidly, complex analyses on small quantities of DNA. The DNA sample amplified by the polymerase chain reaction (PCR) is analysed by means of spectroscopic techniques in emission or absorption, and the response is translated into an electric pulse thanks to the optoelectronic sensors integrated in the chip.

However, the known devices cited above, like other similar ones, have some limitations. Firstly, the sampling and detection of pathogens are carried out in separate stages and with independent devices. This requires the intervention of an operator who can withdraw the sample collected in a first device and, subsequently, prepare the sample for analysis by means of a second device. Usually, the sample collected must be treated before being analysed. For example, from the air sample collected the biological material is isolated, from which the nucleic acids (RNA and DNA) are extracted, such as macromolecules which will undergo molecular testing in order to identify infectious agents. Also to implement said processing phases of the sample collected, specific devices are used. Therefore, the intervention of an operator and likewise the use of multiple specific devices which are often not compatible entails logistical problems, in the transfer of samples from the sampling device to the analysis device after the processing phase, criticalities connected with the reliability, quality and repeatability of the results with related expenditure of time and money. Secondly, the detection devices, like those used in the sample processing phase, are usually characterised by sophisticated and often bulky instrumental apparatus installed in specialist laboratories.

Consequently, analysis of the air sampled does not take place on site but in different places and at different times from the sampling, introducing complex and delicate mechanisms for storage and conservation of the sample and prolonging the timescales for ascertaining the presence of any pathogens contained in the air sampled.

<CIT> describes an integrated device for sampling and detecting airborne pathogens.

The device comprises a sampling container where lysis of the pathogen and extraction of its nucleic acid take place, a device for amplifying the nucleic acid by Polymerase Chain Reaction (PCR), a device for detecting the pathogen and an automatic control system.

One object of the present invention is to provide an integrated easily transportable apparatus able to carry out sampling of the air, processing of the sample collected and detection of pathogens contained in the sampled air, and which optimises the transport efficiency of the pathogen from collection to detection thereof.

The present invention thus concerns to an apparatus as defined in the appended claims.

The present invention also concerns a method according to the appended claims.

Said invention therefore allows sampling and analysis of the air containing aerosol and/or atmospheric particulate, using one single compact, portable and semi-automatic apparatus which combines the functions of three modules. Therefore, said apparatus allows sampling and analysis of the air directly onsite, reducing times and costs connected with transport of the sample from the place of collection to the specialist analysis laboratories. More precisely, said apparatus allows the detection of pathogens contained in the sampled air, and preferably allows detection of the SARS-CoV-<NUM> virus. Therefore, detection of the virus is timely and simplified, and the apparatus can be used also by unskilled operators, maintaining a high level of accuracy in terms of the results obtained.

For a better understanding of the present invention some preferred embodiments are described below with reference to the attached drawings, in which:.

With reference to <FIG>, the number <NUM> indicates overall an apparatus for sampling and detecting pathogens in air according to the present invention.

The apparatus <NUM> comprises in cascade a first module A for sampling the air, a second module B for the isolation and processing of biological material contained in the sampled air and a third module C for the analysis of said material.

<FIG> schematically illustrates an implementation example of the first module A forming part of the apparatus <NUM>.

The first module A is able to sample particles and/or droplets the dimensions of which are between <NUM> and <NUM>. Said dimensional range includes both the atmospheric particulate with diameters smaller than <NUM> (PM10 and PM2. <NUM>), and the droplets contained in the aerosol emitted during respiration or via coughing and sneezing, with variable dimensions between less than <NUM> and approximately <NUM>.

The first module A comprises a casing <NUM> provided with an inlet opening <NUM>, an inlet duct <NUM> communicating with the opening <NUM>, an outlet duct <NUM> and a pump <NUM> arranged along the outlet duct <NUM> for determining an airflow through the inlet duct <NUM>. Inside the casing <NUM> and in a position facing the inlet duct <NUM> a collector <NUM> is arranged designed to intercept the particles <NUM> entrained by the airflow sucked in.

The collector <NUM> can assume different embodiments according to the nature of the particles to be sampled. For example, if the main focus is the analysis of the droplets of an aerosol, as in the case of the analysis of the air exhaled by a patient or circulating in a ventilation system, the collector <NUM> comprises a support frame <NUM> and a membrane <NUM> made of humidified cellulose, for example compressed cotton wadding impregnated with a quantity of water between <NUM> and <NUM>, which occupies a central portion of the frame <NUM>.

The membrane <NUM> of the collector <NUM> can be composed of other materials other than the cellulose, provided that the aerosol adsorbed is maintained in an aqueous solution.

The frame <NUM> is expediently metallic and is thermally regulated by a Peltier cell <NUM> so as to allow condensation of the aerosol on the membrane <NUM> and avoid re-evaporation of the aerosol droplets <NUM>.

The mixture of aerosol droplets <NUM> intercepted by the membrane <NUM> and water present in said membrane <NUM> represents a biological solution <NUM> which then undergoes molecular testing.

The droplets <NUM> and any other substances not trapped in the collector <NUM> reach the outlet duct <NUM> and return to the external environment.

The ratio between the dimension of an outlet hole <NUM> of the inlet duct <NUM> and the dimension of the collector <NUM> must be calibrated so as to ensure the impact of the droplets <NUM> on the collector <NUM>. Furthermore, the geometries in terms of length and diameter of the inlet duct <NUM> and outlet duct <NUM> respectively affect the sampling efficiency and the flow rate (volume sucked in per unit of time) of the first module A.

An implementation of the first module A illustrated in <FIG> by way of example comprises:.

The above-mentioned values can be modified by fluid-dynamic simulations provided that the dimensional sampling range of the droplets <NUM> remains unchanged and between <NUM> and <NUM>.

The biological solution <NUM>, with volume in the range between <NUM>µl and <NUM>µl, is sucked and transferred towards the second module B.

For this purpose, the module B, described in detail below, comprises at the inlet an interconnection chamber <NUM> connected to the membrane <NUM> by means of an interconnection system comprising one or more microtubes <NUM>; in <FIG> and <FIG> only one microtube <NUM> is illustrated for the sake of graphic simplicity.

The microtube <NUM> comprises one end connected to the inside of the collector <NUM>, directly or via a needle inserted in the membrane <NUM>, and an opposite end connected to the interconnection chamber <NUM> in the second module B.

The interconnection system comprises a miniaturized peristaltic pump <NUM> which allows suction of the biological solution <NUM> from the membrane <NUM> in the first module A to the interconnection chamber <NUM> in the second module B (<FIG>).

According to an embodiment variation of the invention, the collector <NUM> can be connected to a humidification system, if the aqueous component already present in the membrane <NUM> is not quantitatively sufficient, for example if the sampling protocol involves a relatively long sampling phase, during which the membrane <NUM> could dry out.

The humidification system can comprise, for example, a tank containing a distilled water reserve and a peristaltic pump (not shown in <FIG>) connected to the collector <NUM> by means of a microtube <NUM>.

According to another preferred embodiment of the invention, the first module A is configured for sampling atmospheric particulate. Said configuration entails replacement of the membrane <NUM> of the collector <NUM>, used to trap the aerosol droplets <NUM>, with a tray protected by a filter sized so as to retain the particles with dimensions larger than those of interest. The tray is connected to a humidification system as described above.

Therefore, the particulate that passes through the filter is collected by the water contained in the tray, from which it can be drawn by means of a microtube provided with needle if necessary as previously described.

Analogously to collection of the aerosol droplets <NUM>, a biological solution, obtained by mixing the particulate trapped in the tray with the aqueous solution, is sucked into the second module B thanks to the interconnection system described above.

<FIG> illustrates overall the second module B, which comprises a series of miniaturized chambers in which specific phases of the isolation and processing of the biological material starting from the biological solution <NUM> are carried out, in particular the steps for:.

In particular the second module B comprises a filtration chamber <NUM> split into a sub-chamber 21a connected to the interconnection chamber <NUM> and a sub-chamber 21b separated from the sub-chamber 21a by a transverse wall comprising a filter <NUM> configured to retain particles having dimensions greater than or equal to <NUM>.

The second module B furthermore comprises a reaction chamber <NUM> in which isolation of the virus dispersed in the filtered solution <NUM> follows a process of release of the viral RNA.

The biological solution <NUM> is sucked from the interconnection chamber <NUM> to the reaction chamber <NUM> by a peristaltic pump <NUM> located downstream of the sub-chamber 21b. Therefore, the sub-chamber 21b represents the centre for the collection of a filtered biological solution <NUM>.

The solid particles retained by the filter <NUM> can be removed by means of a suction system not illustrated.

The reaction chamber <NUM> is selectively connected to a tank <NUM> containing lysis and binding reagents by means of a pump <NUM>, and to a tank <NUM> containing magnetic beads suspended in water by means of a pump <NUM>. The above-mentioned reagents and the bead suspension, in addition to other substances described below, are available in kit under the trade name Dynabeads ® SILANE Viral NA produced by ThermoFisher.

A magnetic stirring device <NUM> is arranged in the reaction chamber <NUM>, said device being designed to generate, when excited, an oscillating magnetic field having the purpose of stirring the reaction mixture contained in the reaction chamber <NUM>.

The second module B comprises an RNA extraction chamber <NUM> connected to the reaction chamber <NUM> and housing a coil <NUM> (or permanent magnets) configured to define a magnetic extraction path for transport of the beads to which the viral RNA is bound. A tank <NUM> containing a buffer washing solution and a tank <NUM> containing an elution reagent are selectively connected to the extraction chamber <NUM> by means of respective pumps <NUM>, <NUM> (said products form part of the kit mentioned above). A further pump <NUM> is designed to remove the supernatant and buffer washing solution from the extraction chamber <NUM> and convey them towards a discharge tank <NUM>.

The second module B comprises an interconnection chamber <NUM> designed to receive an RNA solution from the extraction chamber <NUM> by means of a pump <NUM>, and reagents for use in the subsequent module C, as will be better described below.

The operation of the second module B, already partly evident from the above, is described below with reference to <FIG>, which illustrates the various phases with reference to the chambers of the second module B in which they occur.

The biological solution <NUM>, coming out of the first module A, is sucked by the pump <NUM> and collected in the interconnection chamber <NUM> (block <NUM>). The sample is then conveyed into the filtration chamber <NUM> where a filtering step takes place (block <NUM>) in which the solid particles present in the air and collected by the first module A are removed. The filtered solution <NUM> is then sent to the reaction chamber <NUM> where the following phases occur in sequence: input of the magnetic beads (block <NUM>), input of the reagents for the lysis and the binding (block <NUM>), and lysis reaction of the viral capsid and binding of the beads with the viral RNA released (block <NUM>).

At the end of the reactions, the sample is sent through the magnetic path <NUM> into the RNA extraction chamber <NUM> where it is washed by the buffer solution (block <NUM>) pumped in by the pump <NUM>. Subsequently the elution reagent is introduced into the RNA extraction chamber <NUM> (block <NUM>) by means of the pump <NUM>, thus starting the elution phase (block <NUM>). Once the elution phase has been completed, requiring a few minutes, the supernatant is eliminated (block <NUM>) by means of the pump <NUM> while the RNA sample extracted <NUM> is sent towards the interconnection chamber <NUM> by means of a pump <NUM>.

Expediently, the interconnection chamber <NUM> is connected to a tank <NUM> containing a mixture of reagents for amplifying the RNA as described below by means of a pump <NUM> (block <NUM>). A reactive mixture, as a product of the interaction between the RNA solution <NUM> and the amplification reagents, is sent to the module C (block <NUM>).

The chambers where the various phases take place can be appropriately temperature-controlled.

The interconnection chamber <NUM> is connected to the third module C by means of an interconnection system comprising a pump <NUM> and a microtube <NUM> (<FIG>).

The third module C is schematically illustrated in <FIG> and comprises a lab-on-chip device <NUM> for amplifying and detecting the viral RNA in real time and a reading, control and interface electronic unit <NUM> for acquiring and processing signals from the lab-on-chip device <NUM>.

The lab-on-chip device <NUM> comprises one single monolithic substrate or several substrates coupled permanently or temporarily, inside which the following are integrated:.

In particular, the microfluidic unit <NUM> can be composed of a network of low-cost disposable microtubes or permanent microtubes that can be reused after the implementation of appropriate cleaning and/or sterilization procedures.

In the example illustrated, the microfluidic unit <NUM> comprises a microfluidic network <NUM> composed of a simple microfluidic channel connected at the inlet to the microtube <NUM>, from which it receives the sample to be treated together with the additional reagents, and at the outlet to a microtube <NUM> that can be connected to a waste tank not illustrated.

Alternatively, the microfluidic network <NUM> can be more complex and comprise various microchannels and microtanks, for example formed of wells of appropriate volume.

Detection of the amplification of the viral RNA can be carried out in fluorescence or in chemiluminescence or in bioluminescence. In the example illustrated the detection is carried out in fluorescence, and the third module (C) also comprises an excitation radiation source <NUM>.

The materials used for the lab-on-chip device <NUM> comprise, and are not limited to, one or more of the following materials: glass, polymer resins, polydimethylsiloxane (PDMS), cyclic olefin copolymers (COC), pressure-sensitive adhesives (PSA), silicon.

In the example illustrated, the microfluidic unit <NUM> is made of COC with micromilling sealed by pressure-sensitive adhesive tape.

Once the RNA solution <NUM> and the reagents have been loaded in the microfluidic channel <NUM>, the analytical phase of amplification of the viral RNA can be initiated via the implementation of known techniques such as PCR, LAMP, LAMP-BART, RT-PCR, RT-LAMP and others.

In the example illustrated, the on-chip sensors <NUM>, <NUM> and the on-chip heater <NUM> are expediently obtained on opposite faces of a sheet of glass <NUM> to form a System on Glass (SoG) coupled with the microfluidic unit <NUM>, for example as described in the scientific article<NPL>.

The electronic unit <NUM> is structured on several levels. A first level is represented by an input/output board <NUM>, below which a control board <NUM> is located. The last level consists of an interface board <NUM> which establishes connection between the lab-on-chip device <NUM> and the user interface systems (light indicators, display, computer interface). The lab-on-chip device <NUM> is connected to the input/output board <NUM> of the electronic unit <NUM> via a series of electrical contacts <NUM> which emerge from the input/output board <NUM>.

The electrical contacts <NUM> conduct the following signals or voltages:.

The input/output board <NUM> also contains low-noise transimpedance amplifiers, low-noise analog-digital converters, low-noise bias voltage generators between <NUM> and <NUM> V, low-noise bias current generators between <NUM> nA and <NUM> mA, high-efficiency current generators between <NUM> mA and <NUM> A.

The control board <NUM> contains a microcontroller or DSP or microprocessor with external interface circuitry for management of the protocol to be implemented on the lab-on-chip device <NUM> via the input/output board <NUM>, and management of the ancillary systems present in the apparatus <NUM>, for example the microfluidic pumps of the second module B.

The interface board <NUM> contains the electronics for management of the user interface (display, optical or acoustic indicator systems, data interface towards an external computer, etc.).

The boards <NUM>, <NUM> and <NUM> or a sub-assembly thereof can be implemented on one single board, or the single boards can be created by combining several modules.

The excitation radiation source <NUM>, comprised in the third module C, illuminates the lab-on-chip device <NUM> containing the sample in the microfluidic channel <NUM> and the optical sensors <NUM>. In particular, the sample to be detected comprises fluorescent substances (for example fluorophores) which emit light in fluorescence following excitation by absorption of radiations having specific wavelengths coming from the radiation source <NUM>. Preferably, the radiation source <NUM> emits UV radiations in resonance with the fluorescent substances comprised in the sample. The radiation source <NUM> can consist of LEDs.

Expediently, the optical sensors <NUM> are provided with interferential filters which absorb the radiations coming from the radiation source <NUM> and transmit the radiation emitted in fluorescence by the sample.

Further technical details can be found in the article cited and in the relative Supplementary Material.

The analysis method implemented in the third module C, in which the viral RNA is amplified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), by way of example entails the following steps:.

Basically, a sample consisting of a small volume of purified viral RNA enters the microfluidic unit <NUM> by means of the inlet microtube <NUM>, together with the relative reagents. Drawing of the sample from the interconnection chamber <NUM> to the third module C is controlled by the electronic system which drives the active components of the lab-on-chip device <NUM> and of the off-chip ancillary systems present in the apparatus <NUM>, and in particular in the second module B (microfluidic pumps, fans, etc.). The viral RNA, deposited on the lab-on-chip device <NUM>, is amplified by means of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) or by means of Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP, RT-LAMP-BART).

Once an amplification of viral RNA has been obtained sufficient for the molecular testing, the detection phase can be initiated. In the example illustrated, the SARS-COV-<NUM> virus RNA is detected in fluorescence. In fact, a fluorophore is added to the multiplied viral RNA, said fluorophore manifesting an intense photoluminescence only when it binds to the SARS-COV-<NUM> virus RNA. Alternatively, the detection can be carried out in chemiluminescence or bioluminescence.

Since the detection is based on emission spectroscopy techniques, to increase the sensitivity it is necessary to reduce the interfering radiations coming from sources external to the lab-on-chip device <NUM>. Therefore, the third module C is preferably protected by a sealed opaque casing which blocks the interfering radiations.

From an examination of the characteristics of the present invention, the advantages it offers are evident.

Claim 1:
An apparatus (<NUM>) for sampling and detecting a pathogen in air comprising:
- a first air sampling module (A) configured to collect a sample of airborne particles and comprising suction means for sucking an airflow from an external environment and a collector (<NUM>) comprising a humidified membrane (<NUM>) to intercept the airflow and retain said particles;
- a second sample processing module (B) comprising a filtration chamber (<NUM>) equipped with a filter (<NUM>) configured to retain solid particles larger than the size of the pathogen and means for extracting a nucleic acid from the sample and comprising a reaction chamber (<NUM>) equipped with stirring means and feeding means for feeding into the reaction chamber (<NUM>) magnetic beads and at least one lysis and binding reagent, and a nucleic acid extraction chamber (<NUM>) defining a magnetic extraction path (<NUM>) for the magnetic beads bound to the nucleic acid;
- a third module (C) comprising nucleic acid analysis means for the detection of pathogens including a lab-on-chip device (<NUM>) and an electronic unit (<NUM>);
- first interconnection means configured to transfer the sample from the first module (A) to the second module (B);
- second interconnection means configured to transfer the nucleic acid of the sample from the second module (B) to the third module (C); and
- at least an automatic control unit of said modules and said interconnection means.