SYSTEMS AND METHODS FOR DETERMINING PREVALENCE OF SARS COV 2 IN A POPULATION

A system configured to determine the load in a liquid sample of predetermined antigens is provided. The system comprises a measurement chamber configured for receipt therein of the liquid sample, a sensor circuit, and an analysis unit. The sensor circuit comprises a plurality of working electrodes, each comprising antibodies on its surface associated with one of the predetermined antigens, at least one reference electrode, and at least one counter electrode. Proximal ends of the electrodes are disposed on a reading zone of the sensor circuit, the reading zone being disposed within the measurement chamber. The analysis unit is configured to facilitate the determination of the load of each of the antigens by measuring electrical properties of the electrodes.

TECHNOLOGICAL FIELD

The presently disclosed subject matter relates to methods for determining the prevalence of SARS-CoV-2 in a population, and to systems for performing such methods.

BACKGROUND

In order to manage a pandemic or other outbreak, health authorities are aided by current information about the spread of the disease. While individually testing individuals may provide such information, obtaining data sets which are suitably representative of different locations, in particular multiple data sets for the same population over time, is generally impractical.

Wastewater based epidemiology is one method of obtaining location-specific information about the spread of the disease. Samples of wastewater are collected and tested for the presence and load of the pathogen. Based on this information, and accounting for the locations from which each of the samples were taken, authorities can obtain a broad picture of the spread of the disease without the need to perform tests on individuals.

SUMMARY

According to an aspect of the presently disclosed subject matter, there is provided a system configured to determine the load in a liquid sample of each of a plurality of predetermined antigens, at least one of the predetermined antigens being associated with a variant of SARS-CoV-2, the system comprising:

a measurement chamber configured for receipt therein of the liquid sample, the measurement chamber being disposed between an inlet disposed upstream thereof and having a coarse filter, and an outlet disposed downstream thereof and having a fine filter, wherein the coarse filter allows passage of pathogens associated with the predetermined antigens therethrough, and the fine filter does not allow passage of pathogens associated with the predetermined antigens therethrough;

a sensor circuit comprising a plurality of working electrodes, each comprising antibodies on its surface associated with one of the predetermined antigens, at least one reference electrode, and at least one counter electrode, proximal ends of the electrodes being disposed on a reading zone of the sensor circuit, the reading zone being disposed within the measurement chamber; and

an analysis unit configured to facilitate the determination of the load of each of the antigens by measuring electrical properties of the electrodes.

It will be appreciated that herein the present description and appended claims, references to quantities of antigens (e.g., “a plurality of predetermined antigens”) refer to quantities of types of antigens, and not to the number of individual molecules and/or molecular structures, unless otherwise clear from context.

The system may further comprise a mixing chamber upstream of the inlet, the mixing chamber being configured to receive therein the liquid sample for mixing with a buffer solution.

The mixing chamber may be configured for injection therein of the buffer solution.

The buffer solution may be a buffered saline solution.

The buffer solution may constitute a redox couple.

The analysis unit may be configured to measure the frequency response of the sensor circuit.

The analysis unit may comprise a potentiostat circuit.

The working electrodes may be made of gold.

Each of the reference electrodes may be uniquely associated with one of the working electrodes.

Each of the counter electrodes may be uniquely associated with one of the working electrodes.

At least some of the reference electrodes may be associated with a plurality of the working electrodes.

Working electrodes of the plurality associated with a single reference electrode may comprise the same antibodies as one another.

At least some of the counter electrodes may be associated with a plurality of the working electrodes. Working electrodes of the plurality associated with a single counter electrode may comprise the same antibodies as one another.

One or more of the predetermined antigens may comprise an S1 subunit of a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may comprise an S2 subunit of a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may be a whole-virus antigen.

The system may be further configured to determine the load in the liquid sample of one or more predetermined antigens being associated with a pathogen other than SARS-CoV-2.

At least two of the predetermined antigens may be associated with different variants of SARS-CoV-2.

According to another aspect of the presently disclosed subject matter, there is provided a system configured to determine the load in a liquid sample of each of a plurality of predetermined antigens, the system comprising:

a measurement chamber configured for receipt therein of the liquid sample;

a sensor circuit comprising a plurality of working electrodes, each comprising antibodies on its surface associated with one of the predetermined antigens, at least one reference electrode, and at least one counter electrode, proximal ends of the electrodes being disposed on a reading zone of the sensor circuit, the reading zone being disposed within the measurement chamber; and

an analysis unit configured to facilitate the determination of the load of each of the antigens by measuring electrical properties of the electrodes.

The measurement chamber may be disposed between an inlet disposed upstream thereof and having a coarse filter, and an outlet disposed downstream thereof and having a fine filter, wherein the coarse filter allows passage of pathogens associated with the predetermined antigens therethrough, and the fine filter does not allow passage of pathogens associated with the predetermined antigens therethrough.

The system may further comprise a mixing chamber upstream of the inlet, the mixing chamber being configured to receive therein the liquid sample for mixing with a buffer solution.

The mixing chamber may be configured for injection therein of the buffer solution.

The buffer solution may be a buffered saline solution.

The buffer solution may constitute a redox couple.

The analysis unit may be configured to measure the frequency response of the sensor circuit.

The analysis unit may comprise a potentiostat circuit.

The working electrodes may be made of gold.

Each of the reference electrodes may be uniquely associated with one of the working electrodes.

Each of the counter electrodes may be uniquely associated with one of the working electrodes.

At least some of the reference electrodes may be associated with a plurality of the working electrodes.

Working electrodes of the plurality associated with a single reference electrode may comprise the same antibodies as one another.

At least some of the counter electrodes may be associated with a plurality of the working electrodes. Working electrodes of the plurality associated with a single counter electrode may comprise the same antibodies as one another.

One or more of the predetermined antigens may comprise an S1 subunit of a peplomer. According to some examples, the peplomer is a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may comprise an S2 subunit of a peplomer. According to some examples, the peplomer is a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may be a whole-virus antigen.

At least two of the plurality of predetermined antigens may be associated with different viruses.

At least two of the plurality of predetermined antigens may be associated with different variants of the same virus.

At least one of the predetermined antigens may be associated with a variant of SARS-CoV-2.

According to another aspect of the presently disclosed subject matter, there is provided a system configured to determine the load in a liquid sample of at least one predetermined antigen being associated with a variant of SARS-CoV-2, the system comprising:

a measurement chamber configured for receipt therein of the liquid sample;

a sensor circuit comprising a plurality of working electrodes, each comprising antibodies on its surface associated with one of the at least one predetermined antigens, at least one reference electrode, and at least one counter electrode, proximal ends of the electrodes being disposed on a reading zone of the sensor circuit, the reading zone being disposed within the measurement chamber; and

an analysis unit configured to facilitate the determination of the load of each of the antigens by measuring electrical properties of the electrodes.

The measurement chamber may be disposed between an inlet disposed upstream thereof and having a coarse filter, and an outlet disposed downstream thereof and having a fine filter, wherein the coarse filter allows passage of pathogens associated with the predetermined antigens therethrough, and the fine filter does not allow passage of pathogens associated with the predetermined antigens therethrough.

The system may further comprise a mixing chamber upstream of the inlet, the mixing chamber being configured to receive therein the liquid sample for mixing with a buffer solution.

The mixing chamber may be configured for injection therein of the buffer solution.

The buffer solution may be a buffered saline solution.

The buffer solution may constitute a redox couple.

The analysis unit may be configured to measure the frequency response of the sensor circuit.

The analysis unit may comprise a potentiostat circuit.

The working electrodes may be made of gold.

Each of the reference electrodes may be uniquely associated with one of the working electrodes.

Each of the counter electrodes may be uniquely associated with one of the working electrodes.

At least some of the reference electrodes may be associated with a plurality of the working electrodes.

Working electrodes of the plurality associated with a single reference electrode may comprise the same antibodies as one another.

At least some of the counter electrodes may be associated with a plurality of the working electrodes. Working electrodes of the plurality associated with a single counter electrode may comprise the same antibodies as one another.

The predetermined antigen may comprise an S1 subunit of a SARS-CoV-2 peplomer.

The predetermined antigen may comprise an S2 subunit of a SARS-CoV-2 peplomer.

The predetermined antigen may be a whole-virus antigen.

The system may be configured to determine the load in the liquid sample of each of a plurality of predetermined antigens.

The system may be further configured to determine the load in the liquid sample of one or more predetermined antigens being associated with a pathogen other than SARS-CoV-2.

At least two of the predetermined antigens may be associated with different variants of SARS-CoV-2.

According to another aspect of the presently disclosed subject matter, there is provided a system configured to determine the load in a liquid sample of at least one predetermined antigen, the system comprising:

a measurement chamber configured for receipt therein of the liquid sample, the measurement chamber being disposed between an inlet disposed upstream thereof and having a coarse filter, and an outlet disposed downstream thereof and having a fine filter, wherein the coarse filter allows passage of pathogens associated with the predetermined antigens therethrough, and the fine filter does not allow passage of pathogens associated with the predetermined antigens therethrough;

a sensor circuit comprising a plurality of working electrodes, each comprising antibodies on its surface associated with one of the at least one predetermined antigens, at least one reference electrode, and at least one counter electrode, proximal ends of the electrodes being disposed on a reading zone of the sensor circuit, the reading zone being disposed within the measurement chamber; and an analysis unit configured to facilitate the determination of the load of each of the antigens by measuring electrical properties of the electrodes.

The system may further comprise a mixing chamber upstream of the inlet, the mixing chamber being configured to receive therein the liquid sample for mixing with a buffer solution.

The mixing chamber may be configured for injection therein of the buffer solution.

The buffer solution may be a buffered saline solution.

The buffer solution may constitute a redox couple.

The analysis unit may be configured to measure the frequency response of the sensor circuit.

The analysis unit may comprise a potentiostat circuit.

The working electrodes may be made of gold.

Each of the reference electrodes may be uniquely associated with one of the working electrodes.

Each of the counter electrodes may be uniquely associated with one of the working electrodes.

At least some of the reference electrodes may be associated with a plurality of the working electrodes.

Working electrodes of the plurality associated with a single reference electrode may comprise the same antibodies as one another.

At least some of the counter electrodes may be associated with a plurality of the working electrodes. Working electrodes of the plurality associated with a single counter electrode may comprise the same antibodies as one another.

One or more of the predetermined antigens may comprise an S1 subunit of a peplomer. According to some examples, the peplomer is a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may comprise an S2 subunit of a peplomer. According to some examples, the peplomer is a SARS-CoV-2 peplomer.

One or more of the predetermined antigens may be a whole-virus antigen.

The system may be configured to determine the load in the liquid sample of each of a plurality of predetermined antigens.

At least two of the plurality of predetermined antigens may be associated with different viruses.

At least two of the plurality of predetermined antigens may be associated with different variants of the same virus.

At least one of the predetermined antigens may be associated with a variant of SARS-CoV-2.

According to another aspect of the presently disclosed subject matter, there is provided a method of monitoring the prevalence of SARS-CoV-2 in a population, the method comprising providing one or more systems according any of the above aspects, and performing a measurement comprising:

obtaining a liquid sample, from a predetermined location, of wastewater generated by at least a portion of the population;

determining, using the system, the load of one or more antigens associated with SARS-CoV-2 in the liquid sample; and

relating the load of antigens to the prevalence of SARS-CoV-2 in the population.

The method may further comprise monitoring the prevalence of different strains of SARS-CoV-2.

The prevalence may be a relative prevalence.

The method may further comprise performing the measurement at a plurality of locations.

The method may further comprise performing the measurement a plurality of times at the same location.

The system may be configured to determine the load of one or more antigens autonomously.

The system may be configured to transmit information related to the determined load of antigens to a remote location.

DETAILED DESCRIPTION

As illustrated inFIG.1, there is provided a system, which is generally indicated at10, configured for determining the load of a predetermined antigen or antigens, associated with severe acute respiratory syndrome coronavirus 2 (hereafter SARS-CoV-2) in a liquid sample. According to some examples, the antigen comprises at least a portion of the SARS-CoV-2 peplomer (i.e., spike protein), for example the S1 and/or the S2 subunits thereof. According to some examples, the antigen is a whole-virus antigen.

It will be appreciated that descriptions herein of determining the load of SARS-CoV-2 are presented as non-limiting examples, and that according to some examples the system10may be configured to determine the load of other viruses, e.g., in addition to the load of SARS-CoV-2. Accordingly, the system 10 according to the presently disclosed subject matter may be configured to determine the respective loads of each of a plurality of predetermined antigens in a single liquid sample, of which some or all are not associated with SARS-CoV-2. It will be further appreciated that descriptions herein to the system 10 determining the load of an antigen is for convenience only, and that such descriptions include, inter alia, determining respective loads each of a plurality of antigens, unless otherwise clear from context, mutatis mutandis.

The system10may be further configured to determine and/or facilitate determining the prevalence and/or rate of spread of SARS-CoV-2 and/or other pathogens in a community, for example based on measurements performed on wastewater in that community.

The system10comprises a sampling unit12, configured to physically interact with the liquid sample, and an analysis unit14. According to some examples, e.g., as will be further described hereinbelow, the sampling unit12is configured to be repeatedly and selectively mated and disconnected from the analysis unit14, thereby allowing a single analysis unit to be mated with a plurality of sampling units, each in turn. According to other examples, the sampling unit12and analysis unit14are provided as a single integrated device.

The sampling unit12comprises a measurement chamber16configured for receipt therein of the liquid sample. According to some examples, the measurement chamber16is disposed between an inlet18for introduction therethrough of the liquid sample, and an outlet20for egress therethrough of liquid. In order to perform an initial refinement of the liquid sample, a coarse filter22rated for a size larger than the SARS-CoV-2 virion may be provided within the inlet18, and a fine filter24rated for a size smaller than the SARS-CoV-2 virion may be provided at the outlet20.

Estimates for the size of a SARS-CoV-2 virion range between 50-200 nm in diameter; accordingly, the coarse filter22may be a 300 nm rated filter, and the fine filter24may be a 50 nm rated filter. Thus, particles which owing to their size, are presumably too large to be a SARS-CoV-2 virion are prevented from entering the measurement chamber16. In addition, of the particles in the measurement chamber16, only those which owing to their size, are presumably too small to be a SARS-CoV-2 virion are permitted to leave the measurement chamber.

The sampling unit12may further comprise a mixing chamber26upstream of the inlet18of the measurement chamber16. The mixing chamber is configured for receipt therein of the liquid sample via a sample inlet28, and of a buffer solution through a buffer inlet30. The inlet18of the measurement chamber16constitutes an outlet of the mixing chamber26.

The buffer solution may be configured to dilute the liquid sample a predetermined degree. In addition, it may be a saline solution, for example constituting a redox couple.

The buffer solution may be isotonic to SARS-CoV-2 virions, for example to facilitate maintaining their integrity. The buffer solution may have a pH in the physiological range, e.g., having a pH between about 7.3 and about 7.5.

According to some examples, the buffer solution may be, but is not limited to, phosphate-buffered saline or a HEPES buffer solution.

According to some examples, the liquid sample is introduced on a swab (not illustrated) inserted through the sample inlet28.

According to some examples, the buffer solution is provided through the buffer inlet30using a syringe32. The pressure provided by the syringe32to inject the buffer solution into the mixing chamber26may concurrently force the buffer solution-liquid sample mixture into the measurement chamber16through its inlet18.

The sampling unit12may further comprise one or more sensor circuits34, each with a reading zone36thereof disposed within the measurement chamber16, thereby facilitating measurement thereby of the liquid sample.

Each sensor circuit34comprises one or more working electrodes38, each comprising (e.g., being biofunctionalized with) antibodies for SARS-CoV-2, as will be further discussed below. Each working electrode38may comprise antibodies for a single variant, for multiple variants (e.g., as below) of SARS-CoV-2, and/or for one or more SARS-CoV-2 variants and at least one other pathogen. According to some examples, the antibodies comprise monoclonal antibodies (mAb's) and/or polyclonal antibodies. The working electrode may be made of gold.

The sensor circuit34further comprises one or more reference electrodes40, each associated with one or more of the working electrodes38. The sensor circuit34further comprises one or more counter electrodes42, each associated with one or more of the working electrodes38. A proximal end of each of the electrodes38,40,42extends into the reading zone36.

According to other examples, for example as illustrated inFIG.2, each sensor circuit34comprises a plurality of working electrodes38, one or more reference electrodes40, and one or more counter electrodes42. All of the working electrodes38in a single sensor circuit34may comprise antibodies for the same variant of SARS-CoV-2. According to some modifications, a single circuit may comprise a plurality of working electrodes38, at least some of which comprise antibodies for a variant different than that of other working electrodes, e.g., some working electrodes may comprise antibodies for the B.1.1.7 lineage (per the PANGO nomenclature; commonly referred to as “the UK variant”), some working electrodes in the same sensor circuit34may comprise antibodies for the B.1.351 lineage (commonly referred to as “the South Africa variant”), some working electrodes in the sensor circuit may comprise antibodies of the P.1 lineage (commonly referred to as “the Brazil variant”), etc.

According to further modifications, some individual working electrodes38may comprise a mixture of antibodies for different variants of SARS-CoV-2, and different working electrodes in the same sensor circuit34may comprise different combinations thereof.

According to some examples, each reference electrode40is uniquely associated with a corresponding one of the working electrodes38, i.e., there is a one-to-one relationship between working and reference electrodes.

According to other examples, at least some of the reference electrodes40are associated with a plurality of working electrodes38. In this case, electrical properties of the working electrodes38associated with a single reference electrode may be measured (for example as described below) simultaneously using the same electrical parameters, or simultaneously. According to some modifications, all of the working electrodes associated with a single reference electrode40are similarly biofunctionalized, i.e., they comprise antibodies which correspond to the same antigens as the other working electrodes associated with the same reference electrode.

According to some examples, a single counter electrode42is associated with several or all of the working electrodes38; according to other examples, each counter electrode is uniquely associated with one of the working electrodes.

According to some examples, the sensor circuit34comprises a single working electrode38and a single reference electrode40.

Providing the antibodies on the working electrodes38may be accomplished by any suitable method, some of which are known in the art. It may be accomplished via covalent immobilization of the SARS-CoV-2 capsid-specific mAb's.

According to some examples, the sensitivity of the sensor circuit34is calibrated, for example by designing a dosing strategy, e.g., using a model system known in the art. SARS-CoV-2 capsid-specific mAb's may be used as the biorecognition element. mAb's integrated onto the working electrode 38 may then be validated for the detection of a model virus pseudo-typed with spike protein or any other Sars-CoV-2 antigen, e.g., a SARS-CoV-2 spike protein-pseudo-typed Vesicular stomatitis virus (VSV) and/or real sample. According to some examples, pVSV-AG-GFP plasmid may be transfected with plasmids encoding the VSV nucleocapsid (N), phosphoprotein (P), and large polymerase subunit (L), together with plasmid encoding the SARS-CoV-2 peplomer.

According to some examples, various lentivirus vectors pseudo-typed with SARS-CoV-2 spike protein may be used as a model. In a different example, a pseudo-typed virus model with the SARS-CoV-2 infection property can be developed, based on the HIV-1 backbone consisting of an S expression plasmid and the HIV-1 packaging system incorporating luciferase reporter genes.

The mAb's may be obtained from a commercial source, and/or may be produced, e.g., using phage bio-panning with naive human and immunized mice single-chain fragment variable (scFv) libraries, for example as is well-known in the art. High throughput sequencing may be employed to enhance mAb discovery throughout phage selection cycles.

The analysis unit14is configured to measure electrical properties of each of the sensor circuits26. According to some examples, for example as illustrated inFIG.3, the analysis unit14may comprise a potentiostat circuit44, connected to distal ends of each of the electrodes38,40,42.

As mentioned above, the sampling unit12may be configured to be repeatedly and selectively mated and disconnected from the analysis unit14, thereby allowing a single analysis unit to be mated with a plurality of sampling units, each in turn. According to some examples, the analysis unit14comprises a connector, for example per the Universal Serial Bus (USB) standard, with distal ends of the electrodes38,40,42being formed as or in electrical contact with a connector formed to mate with the connector of the analysis unit. According to some modifications, the analysis unit14in configured to pair with a portable computing device, for example a smartphone, to facilitate analysis, for example as described below.

The potentiostat circuit44comprises an input source46configured to provide an electrical current at a constant voltage, an electrometer48configured to measure the voltage difference between the working and reference electrodes38,40, a current-to-voltage (I/E) converter50configured to measure the current of the electrochemical cell realized when the reading zone36of the sensor circuit34is submerged within the liquid sample, and a control amplifier52configured to maintain the voltage between the working and reference electrodes, by adjusting its output. As potentiostats are well-known, one having ordinary skill in the art will be able to implement and operate the potentiostat circuit44.

It will be appreciated that the potentiostat circuit44described above with reference to and as illustrated inFIG.3is provided as a non-limiting example only, and one having skill in the art will recognize that other circuit designs may be employed in place thereof in order to realize the required measurement.

In use, when, e.g., pathogens carrying the target antigen come in contact with the working electrode38, they bind to them, thereby causing an impedance change of the sensor circuit34. As the impedance change varies with the amount of pathogens which have bound to the proximal end of the working electrode38, the change in impedance can be correlated with the antigen load in the liquid sample.

Accordingly, the analysis unit14may be configured to measure the frequency response of the sensor circuit34after it has been exposed to the liquid sample for a predetermined amount of time. According to some examples, the predetermined amount of time is about ten minutes.

The analysis unit14may be configured to perform faradaic electrochemical impedance spectroscopy, for example as is well-known in the art, on the sensor circuit34when exposed to the liquid sample and to the redox couple in the buffer solution. A Nyquist plot (for example as illustrated inFIG.4) may be used to visually represent real (Zreal) and imaginary (Zimag) components of the impedance of the sensor circuit34at different frequencies.

Curve A (delineated by squares inFIG.4) represents the frequency response of the sensor circuit34in which the working electrode38does not comprise any antibodies. Curve B (delineated by circles inFIG.4) represents the frequency response of the sensor circuit34in which the working electrode38has been biofunctionalized with antibodies. Curve C (delineated by triangles inFIG.4) represents the frequency response of the sensor circuit34in which the working electrode38has been biofunctionalized with antibodies and has been exposed to a liquid sample with a known load of antigens. Similar curves may be used to calibrate the analysis unit, so that the antigen load of an unknown liquid sample may be accurately measured.

An equivalent electronic circuit, for example a Randles circuit, may be used to model the data obtained from the electrochemical impedance spectroscopy, for example as is well-known in the art.

As illustrated inFIG.5, a method100may be provided to utilize the system10as described above with reference to and as illustrated inFIGS.1through4, to monitor the prevalence of SARS-CoV-2 in a population. The prevalence measured may be a relative prevalence, e.g., how much greater/lower the prevalence of SARS-CoV-2 is in one area of the population compared to another, how much greater/lower the prevalence of SARS-CoV-2 is compared to an earlier time, etc.

In step110of the method, a liquid sample is taken from wastewater which has been generated by at least a portion of the population, at a predetermined location which is associated with that portion of the population. According to some examples, the predetermined location is a branch of a wastewater collection system which serves only the relevant portion of the population. According to other examples, the predetermined location is a wastewater treatment plant. It will be appreciated that liquid samples may be taken from multiple locations in a wastewater collection system, in order to obtain detailed information about a population.

In step112of the method, the system10is used to determine the load of SARS-CoV-2 antigens in the liquid sample taken at each location.

According to some examples, obtaining the samples is performed manually. Accordingly, a user may obtain the sample, for example on a swab, and insert it into the mixing chamber26of the system10. A suitable buffer solution may be injected, for example as described above. The buffer solution mixes with the liquid sample in the mixing chamber26, and enters the measurement chamber16. After a suitable amount of time has elapsed, for example between about10minutes and about30minutes, the electrical properties of the working electrodes38of the system10are measured, for example as described above. In this way the load of one or more antigens in the liquid sample may be determined.

According to some examples, the system10is configured to perform the measurements autonomously. According to further examples, the system10is configured to transmit information related to the determined load of antigens to a remote location. This information may comprise the determined loads, and/or the data read directly from the electrodes, so that the final determination may be performed at a remote location, for example taking into account other readings which were performed, e.g., simultaneously at other locations, at the same location at other times, etc.

In step114of the method, the prevalence of SARS-CoV-2 in the population is determined, based at least partially on the loads which were determined in step112. This may be done using in any suitable way, for example using one or more mathematical techniques which are well-known in the art.

It will be recognized that examples, embodiments, modifications, options, etc., described herein are to be construed as inclusive and non-limiting, i.e., two or more examples, etc., described separately herein are not to be construed as being mutually exclusive of one another or in any other way limiting, unless such is explicitly stated and/or is otherwise clear. Those skilled in the art to which this invention pertains will readily appreciate that numerous changes, variations, and modifications can be made without departing from the scope of the presently disclosed subject matter, mutatis mutandis.