Patent Application
20240277322
The devices and methods provided herein address a need in the art to reliably self-collect fluid samples by an individual user for subsequent testing, such as by shipping the collected specimen to a laboratory.
Although there are diagnostics companies providing consumer or at home testing kits involving blood or saliva collection devices, those devices generally require an individual to deposit a specimen (e.g. saliva spit) into a collection tube containing some stabilization buffer solution or the like. There is a risk of the user aspirating the liquid from the tube during the collection or being exposed to hazardous volatiles from the buffer solution. For these reasons, FDA guidelines require isolating the steps of saliva collection from the transfer to the buffer solution and collection tube so that the individual is safely protected. Such a procedure, however, must preserve the integrity of the specimen for further molecular testing. As one example, U.S. Pat. No. 9,732,376 describes a sample collection device for collecting a saliva sample for subsequent transport to a laboratory for DNA analysis. That device, however, suffers from a number of disadvantages, including lack of an integrated reservoir for holding buffer, ability to reliably cut into the reservoir with an insert that fits into off-the-shelf commercially available tubes for holding samples. Those devices are also not compatible of integration with a means of processing of the sample, including for direct testing of a marker on a fluid sample.
From the forgoing, there is a need in the art for self-collection devices that can collect a sample and hold a reagent volume that is to be introduced to the sample, and/or that can process the sample in an integrated, reliable and safe manner.
The invention provides a reliable, efficient and safe platform for self-collection of biofluids (e.g., biofluids that may or may not contain a biological specimen and/or a biomarker thereof), mixing of a reagent and a fluid, and related sample collection, processing and fluid mixing methods. As described, the sample collection can include collection of saliva. The processing aspect is used broadly herein and may refer to initial sample manipulation, including cell lysis, extraction, isolation and/or stabilization. Processing may also refer to a detection assay, including to detect one or more markers in a sample, such as biomarkers associated with a disease or infection in saliva. Accordingly, processing may include use of biomarkers or antibodies in an array configuration for detecting protein or viral particles associated with a pathogen, such as in a COVID test.
The present invention addresses the need in the art for a safe self-collection device that is self-integrated with all the necessary reagents for use with a self-collected sample. Relevant components include: (1) a mouthpiece for receiving a biofluid specimen; (2) a collection tube; (3) a tube insert with features that provide cutting or piercing a sealing membrane that forms a reagent reservoir; (4) a hollow cap with a cavity to contain some liquid, solid or gas reagents and (5) a sealing membrane to close the cavity of the cap. Typically, a user will spit less than 1 ml of saliva using a mouthpiece into the collection tube equipped with the tube insert. The mouthpiece is removed and replaced by the cap which can screw onto or into the collection tube, leading to the piercing of the reservoir membrane material by the tube insert piercing end. The piercing of the reservoir membrane, along with the specially configured tube insert, allows the reagents to mix with the biofluid specimen, such as the saliva. The device is compatible with any of a desired ratio range of reagent and specimen volume, such as an about 2:1 ratio.
This device can, therefore, preserve the biological specimen and inactivate pathogens such as viral or bacterial organisms while stabilizing the nucleic acid (e.g. RNA or DNA) for further molecular analysis. These analyses can comprise omics such as gene expression, proteomics, metabolomics or standard chemistries.
The devices are also compatible with substantially real-time assays. As described, the biofluid can be collected and “processed” with the reagent volume. The membrane may then be swapped out with a biomarker detection membrane that is porous to the mixed reagent and biofluid. Inverting the collection tube can force the mixed solution through the biomarker membrane and desired biomarkers in the biofluid detected. For example, the detection may be by optical detection of a colorimetric change. In this manner, any of the devices and methods may be described as providing real-time detection of a biomarker. This can significantly improve diagnostic times by avoiding having to transport the collection device to a lab for sample analysis.
Provided herein are devices for self-collection and processing of a biofluid. In particular, the biofluid is a sample of fluid in which testing for the presence or absence of a biological condition is desired. The biological condition may be the presence of absence of a biological specimen, such as a virus, bacteria, fungus, pathogen or disease-causing organism. The biological specimen may be a polynucleotide sequence indicative of an elevated risk of cancer or a polynucleotide sequence indicative of a disease state, including cancer. With this, the devices are compatible with detecting the presence or absence of a biomarker reflecting the presence or absence of the biological specimen.
The device may comprise a mouthpiece and a collection tube fluidically connected to the mouthpiece. In this manner, the user can spit into the mouthpiece and the collection tube can then collect the saliva. A tube insert is positioned in the collection tube, wherein the tube insert comprises a piercing end. A cap is configured to connect to the collection tube and fluidically seal the collection tube from a surrounding environment. The cap comprises an integrated reservoir configured to hold a biological reagent configured for use with the biological specimen in the collection tube. The biological reagent is selected depending on the application of interest, including the type of biological specimen, the timing of the processing/handling of the sample, and the type of biomarker and biomarker detector(s). A reservoir membrane contains the biological reagent in the integrated reservoir and temporarily seals the integrated reservoir from the collection tube. The “temporarily seals” reflects that the device has a tube insert piercing end configured to pierce the reservoir membrane, and in this manner, fluidically contact the biofluid in the collection tube and the biological reagent in the cap reservoir. This configuration advantageously ensures that none of the fluids, and constituents thereof, are exposed to the external environment. This assists in ensuring safety of users and others around the device and assists in maintaining biofluid integrity without possible external contaminants.
The tube insert may further comprise a positioning feature to align and position the piercing end at a pre-determined distance from the reservoir membrane and to preserve a detection interface with a detection membrane.
The device may further comprise a detection membrane positioned at the bottom of the tube insert; a means for driving fluid flow through the detection membrane to obtain a biological measurement; and a holder for holding the detection membrane. The means may be as straightforward as being driven by gravity, flow driven by shaking of the device by a user, or an actively provided force, such as via a pressure-generated flow exerted by, for example, a syringe plunger motion or by a pump.
The reservoir membrane may be a re-sealable membrane.
The device, useful for testing, may comprise a detection membrane, wherein the reservoir membrane is removable after the biological reagent is introduced to the biofluid in the collection tube; and wherein the detection membrane is a biomarker detection membrane configured to connect to the cap after the reservoir membrane is removed to detect one or more biomarkers in the biofluid.
The re-sealable membrane may comprise an interchangeable adhesive layer; and a porous substrate comprising biomarker detectors. For example, the re-sealable membrane may itself be replaceable, so that the rest of the device could be reused. This could be particularly relevant if the same user would like to use the device for multiple serial tests, such as a repeat for a different biomarker. By simply swapping out the detection membrane, another sample can be provided for immediate testing. Accordingly, “re-sealable” is used broadly herein to refer to a membrane that can be removed and replaced without sacrificing device accuracy or reliability. This reflects that the reservoir membrane itself could correspond to the detection membrane. Of course, the membranes can also be distinct membranes, including physically separated or in a stacked configuration.
The biomarker detectors, depending on the application of interest, may be selected from the group consisting of polynucleotides, polypeptides, antibodies, nucleic acids, toxins, bacteria, virus, and biological vesicles.
The devices provided herein are compatible with multiplex detection of a plurality of biomarkers. For example, the device may comprise a plurality of unique biomarker detectors arranged in a microarray on the detection membrane for multiplex detection of biomarkers in a biofluid.
The device may further comprise a vertical flow immunoassay (VFI) and/or a lateral flow assay (LFA) integrated with the reservoir membrane or a detection membrane, including a detection membrane that is a VFI or LFA membrane comprising one or more biomarker detection elements, optionally the biomarker detection elements are antibodies.
The vertical flow immunoassay may comprise a sandwich enzyme-linked immunosorbent assay (ELISA). In a VFI assay, provided is a means for driving fluid flow through the vertical flow substrate membrane; wherein the device is configured to reflect presence of the biomarker by a change in optical color at the membrane. The change in color may be by binding of optical markers that change color of the membrane upon binding. Again, the means for driving fluid flow may be by any of a passive means (inverting to ensure flow under gravity through the membrane), active (by user-supplied force or by an electronically driven pressure controller to drive fluid flow across the membrane).
The tube insert may comprise an insert body shaped for insertion into a reservoir of the collection tube; a flange on the insert body configured to be supported by an entrance wall of the collection tube; a bottom surface having a central opening; wherein the piercing end corresponds to a spike extending from the bottom surface. This combination of enhanced structure to the tube insert is beneficial for more precise puncture of the reservoir membrane and device robustness.
The piercing end may correspond to a plurality of physically separated spikes. In this manner, piercing reliability is increased and flow exchange maximized.
Also provided herein are methods of using any of the described devices for self-collection and processing of a biological specimen, including a biological specimen that may be found in a biofluid.
For example, the method for self-collecting and processing a biofluid, including a biofluid that may have a biological specimen, may comprise the steps of providing the device and introducing the biofluid into the collection tube by the mouthpiece. The cap is connected to the collection tube, thereby piercing the reservoir membrane with the tube insert piercing end. The biofluid is mixed with the biological reagent to obtain a mixture of biofluid and biological reagent. In this manner, the biofluid is collected, and any biological specimen in the biofluid is processed.
The method may further comprise the step of detecting for the presence of a biological interaction between the detection membrane and a biomarker of a biological specimen. In this aspect, the device is an assay for determining, for example, whether the user has a biological specimen. For example, a pathogen such as a virus that can be found in the saliva, or at least a biomarker of the virus can be found in saliva.
The method may further comprise the step of determining a presence or an absence of the biological specimen from the user who provided the biofluid by introducing the biofluid to the detection membrane.
The method may further comprise the step of determining a physiological parameter from the biofluid for self-diagnostics of a health condition, including by introducing the biofluid to the detection membrane.
The tube insert may be positioned in the collection tube before the step of introducing the biofluid into the collection tube.
The method may further comprise the steps of removing the cap from the collection tube, exchanging the reservoir membrane with a biomarker detection membrane, and connecting the cap with the biomarker detection membrane to the collection tube. The collection tube may be inverted to flow the mixed biofluid and biological reagent through the biomarker detection membrane under gravity or fluid impulse by shaking. The presence or absence of the biomarker in the biofluid may be detected by detecting a physio-chemical property perturbation such as by a color change and/or an electrochemical change by an electrochemical reaction that activates or releases a volatile compound in or from the biomarker detection membrane.
The biomarker detection membrane may comprise an array of detection spots, including optionally an array for multiplex detection of a plurality of unique biomarkers.
To ensure device fidelity and functionality, the biomarker detection membrane may comprise a positive control that reflects biofluid was successfully provide to the detection membrane. If the positive control does not, for example, cause a localized color change, the assay can be identified as not correctly functioning.
The method may further comprise detecting the color change by an imaging device, including a digital imaging capture device, including a camera from a smart phone, and analyzing the detected color change from the imaging device with a data analysis algorithm software to quantify a color change parameter, including intensity, color wavelength, and distribution thereof.
Also provided herein are devices for carrying out any of the methods described herein.
Also provided herein is a device for mixing two separate fluid components. The device may comprise a collection tube for containing a fluid sample and a tube insert positioned in the collection tube, wherein the tube insert may comprise: a piercing end. A cap is configured to connect to the collection tube and fluidically seal the collection tube from a surrounding environment. The cap may comprise an integrated reservoir configured to hold a sample reagent configured for use with the fluid sample in the collection tube. A reservoir membrane seals the reservoir from the collection tube. In this manner, the tube insert piercing end is configured to pierce the reservoir membrane and fluidically contact the fluid sample in the collection tube with the sample reagent in the cap reservoir.
Any of the devices provided herein may use a fluid sample that comprises saliva with a sample reagent that comprises a buffer for chemically stabilizing and/or processing the saliva.
Any of the methods and devices may integrate a detection test with the cap. For example, specially configured membranes and/or buffers may be used to facilitate sample processing and to obtain a sample measurement, including for example, presence or absence of a biomarker, such as a protein, antibody, polynucleotide, or fragments thereof. An exemplary buffer is provided in Table 24. Any of the methods and devices may contain such a buffer in the cap integrated reservoir. Of course, the devices and methods are compatible with a range of buffers, wherein the buffers are specifically tailored for the application of interest.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
“Self-collection” refers to the ability of a user, even a non-medically trained user, to reliably and safely provide a biofluid in a manner conducive to subsequent testing. No other persons are required to obtain the biofluid that is tested, either immediately, or at a later time. In this manner, the devices provided herein can be extensively distributed, including to homes, offices and worksites, where the users at those locations can self-collect a biofluid for testing. For example, self-collection of saliva to determine the presence or absence of a pathogen that can be found in saliva. Of current relevance are viruses that cause coronavirus disease (e.g., SARS-COV-2 virus that causes COVID-19). Of course, the devices provided herein are compatible with any pathogens for which biomarker detectors are available. This typically involves polynucleotide sequences that are associated with a specific pathogen, such that complementary biomarker detectors (e.g., complementary polynucleotide sequences; capture antibodies) can be incorporated with a detection membrane.
The term “processing” is used broadly herein and refers to an action on the fluid sample useful in an assay. For example, processing can correspond to one or more of lysis, nucleic acid extraction or stabilization, as well as analysis, including in embodiments having an antibody array(s) for detecting protein or viral particles in a sample, including in a COVID test. For devices that are configured to test for COVID, the biomarker detectors may correspond to any antibodies that specifically detect a protein associated with a virus responsible for COVID, including, but not limited to, the list of antibodies against SARS-CoV-2 S protein provided in Table 25.
“Fluidically connected” refers to the connection of two components so that fluid flow may occur between them, but without adversely impacting the functionality of each component.
This example demonstrates an assay incorporated with the cap to provide a biofluid collection device with an optional assay for detecting presence or absence of a biological specimen or a biomarker in the biofluid.
In this example, a re-sealable membrane can also comprise an interchangeable adhesive layer holding a porous material, for example paper filter with pore sizes of at least 10 nm, preferably in the range of about 100 nm to a few microns, which can comprise an array of spots for testing biomarkers. The tests could comprise molecular detection of proteins, antibodies, nucleic acids (e.g. RNA or DNA) to detect viruses, bacteria but also enzymes, circulating microvesicles (e.g. exosomes) or other chemical products of a biological reactions.
For example, the membrane can be printed with antibodies for detecting a sandwich assay, ELISA like or other colorimetric test for investigating biofluids, e.g. saliva, blood, urine, ascites or other biological sample for assessing the presence of target analytes, e.g. virus, bacteria, toxins.
As illustrated in
Typically, the assay can rely on the immobilization of a capture antibody on the sealable membrane that acts as a reagent pad to which the sample of interest (with or without antigen (e.g., biomarker) to be detected) is applied. Detection of the bound antigen is subsequently achieved through the binding of an antigen specific antibody gold conjugate. This step completes a sandwich comprising a capture antibody, an antigen and finally the gold conjugate, and results in a direct and permanent visually detectable color change, including a color dot indicating the presence of the antigen. A combination of different nanoparticle conjugate probe or nanoparticle shape and materials can be used for tuning the detection in the configuration of a multiplex assay.
In an embodiment, the detection of an antibody in a sample can be done in a few steps:
After the collection of the sample in the tube using the sample collection cup, attach the cap which is prefilled with a solution of reagents 72 containing gold conjugate.
During the attachment of the cap to the tube, the sealed membrane is pierced releasing the reagents into the tube with the sample which can be mixed by shaking the tube assembly a few times.
Remove the cap and place the adhesive membrane, which has been printed with an antigen, onto the cap.
Invert the tube and cap assembly so the sample is applied to the membrane.
Observe the change in color onto the spots of the membrane on the cap indicating the outcome of the assay.
In an embodiment, the cap 70 may be configured to facilitate usability (e.g., easier to hold) and manufacturing (e.g., reduce amount of plastics). Manufacturing, in particular, is an important consideration in view of challenges in obtaining supplies, including the ongoing pandemic-related supply chain bottlenecks. As shown in
In an embodiment, the cap 70′ of
The results of the experiments are included in Table 2.
All of the caps withstood pressures up to 95 kPa for up to 24 hours. All cap/tube combinations withstood pressures up to 95 kPa for 48 hours. A failure was identified at 95 kPa at 48 hours in ˜30% of the caps. Further investigation of the cap failure identified that this was due to bond failure and not the membrane itself, as the membrane does not rupture. Instead, it appears the adhesive between the membrane and the cap failed. Considered holistically, these results indicate the membrane on the cap and the capped tube exhibit significant pressure resistance.
To further evaluate the cap and the capped/tube collection under real-world conditions, additional experiments were carried out. Of note, transportation have the following characteristics: cargo air jet=75 kPa (<8 hours), ground transportation=64.5 kPa maximum.
In Study 1 (n=50), pre-filled biological reagent filled caps were transported by FedEx cross-country (from the manufacture site in Phoenix, AZ) to Branford, CT (Friday to Monday transit time). Samples were sent such that temperatures varied throughout transit from Phoenix, AZ, to Indianapolis, IN, to East Grandby, CT, and to Branford, CT. After 72 hours in transit, no evidence of seal leakage or any alteration of the sealing was identified (n=50). Thus, no leakage was noted in caps subjected to long distance road/air transportation.
In Study 2 (n=40×2), a separate clinical study was undertaken in New York, NY. 40 kits were sent on two separate occasions by overnight shipping. Tubes were routed from Connecticut via Memphis, TN (48 hours total transport). No leaks were noted in the sealed caps after arrival in New York, NY (n=80). After saliva collection, the collected samples were returned by overnight shipping from New York, NY to Branford, CT. In this study, a capped, sealed tube with saliva was sent by FedEx (i.e., standard approach). Samples were received within 24 hours. No leakage of contents was identified (n=80). Watertight seal (cap/saliva tube with clinical samples) was identified in 100%. Thus, no leakage of contents was identified on the first phase of transport (send-out) nor on the second (returning clinical samples).
In Study 3 (n=155), usability studies were performed using 155 samples that were collected and returned to Branford, CT by overnight FedEx from two different sites in CT. In these instances, a capped, sealed tube with saliva (i.e., real-world assessment) was sent by FedEx. No leakage of contents was identified in any of the 155 tubes.
Taken together, these 3 studies identify that in formal assessment using real-world clinical conditions the cap seal is robust (n=285). They also confirm that clinical samples (capped tube with clinical material/saliva) can be transported safely without breakage or leakage (n=285). These data demonstrate the integrity of both the membrane on the cap and the capped tube under real-world conditions.
In an embodiment, the cap can be configured for robotic and high through-put usability (e.g., easier to include in robotic tube racks). As in
In an embodiment, the collection tube may be modified as shown in
The devices and methods provided herein are compatible with vertical flow immunoassay (VFI) membranes integrated into a saliva collection cap. Such a configuration is compatible with rapid and safe Covid-19 testing. For example, the integrated assay may detect presence or absence of antibodies, including antibodies that may be useful in a SARS-Cov-2 or COVID-19 test. The antibodies are representative examples of some useful potential biomarkers. See, e.g.:
The cap may incorporate pad materials used in the cap. The pad materials may be characterized as having pore sizes, such as pore sizes from 10 nm-200 microns. Preliminary data suggests 80 nm may optimum.
The pad materials may comprise cellulose micro-fibrils such as a Celova® material.
The systems provided herein are uniquely configured for the potential dual use of the biofluid processing with the buffers and the ability to run a multiplex assay within the same device. The VFI platform is compatible with gene testing, including pandemic-relevant genes, associated with influenza, Covid-19, Ebola and the like.
Ahead of efficient tests for such genes, however, is to obtain an approved saliva collection device, such as an FDA EUA device that is mass producible.
Additional aspects may include internal leading edge on the collection tube that can be modified and controlled for a best seal and fit. Volume indicators can be positioned to visually indicate sample volume. The collection tube and associated components may be configured in a straight-pull configuration. The mouthpiece may have a rounded edge for user comfort. The cap may have dimensions, such as diameter and height, corresponding to standard caps that are associated with conventional collection tubes. The tube insert and piercing element are configured to avoid damage during use.
According to an embodiment, the methods and devices of the present disclosure can be evaluated for the detection of pathogens. In particular, an exemplary device of the present disclosure can be used to collect a sample for a COVID-19 PCR test for the detection of N1, N3 and RNAseP in individuals at risk for COVID-19. The COVID-19 PCR assay was authorized on Aug. 3, 2020 by EUA201111 for upper respiratory tract samples including nasopharyngeal, oropharyngeal (throat) swab, anterior nasal swab, and mid-turbinate nasal swab samples and nasopharyngeal washes/aspirates or nasal aspirates, and bronchoalveolar lavage.
Below includes data relating to the clinical utility of the COVID-19 PCR test for the detection of SARS-COV-2 in saliva samples collected using the exemplary device of the present disclosure.
In an embodiment, the exemplary device of the present disclosure allows for a quick, easy, and effective collection strategy. As described herein, the exemplary device is trouble-free (age range 8-87 years, all education attainments: at school to PhD) and unaffected by shipping temperatures (−80° C.-+40° C.). As will be demonstrated below, results from a COVID-19 PCR test based on a sample collected by an exemplary device of the present disclosure generate an accurate determination of SARS-COV-2, similar to results using nasopharyngeal collection.
To evaluate analytical sensitivity, a limit of detection study (LoD) was performed by spiking biological reagent, or saliva stabilization buffer, with different concentrations of TWIST Bioscience SARS-COV-2 RNA control (catalogue number MT007544.1, 1,000,000 copies/uL). A total of 3 replicates were tested using the COVID-19 PCR Test per dilution. The viral concentrations ranged from approximately 1-1000 copies/uL (data included in Table 3).
A standard curve was generated to convert the Cr signal to a concentration in copies/mL using a plasmid control from IDT (200,000,000 copies/uL) that was serially diluted from 1 copy/uL to 1000 copies/uL. The LoD from these dilution series was confirmed by spiking 40 individually extracted replicates of negative (control subjects) saliva (all known COVID-19 negative) with the appropriate viral copy number (TWIST Bioscience SARS-CoV-2 RNA control). Twenty-four samples were spiked-in with 15 copies/uL (˜1.5×LoD). Ten samples were spiked in with 40-100 copies/uL (˜4-10×LoD) and six samples were spiked in with 500-1000 copies/uL (˜50-100×LoD). Ten negative controls were also evaluated (no spike-in). Using the COVID-19 PCR test, twenty-four of 24 (24/24) 1.5×LoD replicates were positive, 10/10 (˜4-10×LoD) were positive and 6/6 (˜10×LoD) were positive. All negative samples were negative (10/10) for the N1 and N3 primers. Summary data for all 40 contrived samples tested are shown below (Table 4):
Clinical Utility Study: A clinical utility study was performed. The COVID-19 PCR test introduced above was evaluated in a set of 60 COVID-19 tested and known clinical samples. This included 30 verified SARS-COV-2-positive cases and 30 negative SARS-COV-2 cases. Nasopharyngeal swabs were collected using the BD Universal Viral transport Kit (BD Catalogue #220529) and stored in UTM for evaluation of viral mRNA using the CDC EUA test (CDC 2019-nCOV Real-Time RT-PCR Diagnostic Panel, unmodified). Matched saliva was collected at the same time point using the exemplary device of the present disclosure for evaluation using the COVID-19 PCR test.
Sample types (nasopharyngeal) were orthogonally compared to the Roche 6800 test (Cobas® SARS-COV-2 test output: positive or negative). Summary data for all 60 clinical samples from each of the two collection types are shown below (Table 5), wherein the CDC detection rate is determined by the CDC SARS-COV-2 Test (N1/N2: CDC 2019-nCOV Real-Time RT-PCR Diagnostic Panel).
Agreement displayed between the results from implementation of the exemplary device of the present disclosure and the CDC assay in matched nasopharyngeal samples is included in Table 6, with a two-sided 95% score confidence interval.
The positive percent agreement was 100% (95% confidence interval: 88.4-100.0%). The negative percent agreement was 100% (95% CI: 88.4-100.0%). The accuracy was 100% (95% CI: 94.0-100.0%). The mean Cr values for each of the target genes is included in Table 7, where * indicates only the CDC assay, ** indicates only an assay based on the methods of the present disclosure, and the ‘SARS-COV-2 RT-PCR Assay’ is based on the methods of the present disclosure.
The assay performed based on methods of the present disclosure exhibited similar metrics for SARS-COV-2 viral detection in saliva as did the CDC assay. Twenty (67%) of 30 nasopharyngeal samples exhibited Cr for N1>35. This identifies these are “low viral titer” samples. The mean Cr values for this sub-cohort of clinical samples is included in Table 8, where * indicates only the CDC assay, ** indicates only an assay based on the methods of the present disclosure, and the ‘SARS-COV-2 RT-PCR Assay’ is based on the methods of the present disclosure.
The assay performed based on the methods of the present disclosure using saliva identified all “low viral titer” nasopharyngeal samples.
Orthogonal Testing Study: To perform an orthogonal testing study, the 60 nasopharyngeal samples were also evaluated using the Roche 6800 test (Cobas® SARS-COV-2 test output: positive or negative) as an orthogonal validation test for the assay performed based on the methods of the present disclosure (Table 9), where * indicates the sample status was determined by the 2019-nCOV-CDC EUA, ** indicates the detection rate was determined on the basis of the Cobas® SARS-COV-2 test (Roche 6800), #indicates an n of 4 COVID-19 positive samples (by CDC) were identified as positive using an assay based on the methods of the present disclosure but were determined as negative using the Roche test, and ‘saliva’ indicates the detection rate was determined on the basis of the methods of the present disclosure.
The agreement between the assay in saliva and the Roche test in nasopharyngeal samples is included in Table 10, where 1 is a two-sided 95% score confidence interval.
A Discordant analysis was performed on the 4 false negative results using the CDC EUA assay as authorized (unmodified). All four false negative were also positive by the CDC EUA assay. Results using the COVID-19 PCR test based on the methods of the present disclosure identifies that the test is accurate and effective for the detection of SARS-COV-2 in saliva collected using an exemplary device of the present disclosure.
Temperature Testing (ISTA 7D) 2007-based shipping) Studies: Forty-eight saliva samples were evaluated in two separate temperature studies. Samples were collected into an exemplary device of the present disclosure, the exemplary device including a biological reagent, or stabilization buffer.
In a first study (Study 1), per ISTA 7D 2007 “Summer Profile”, a total of 48 samples including 23 samples at 1-2×LoD, 5 samples at 2-5×LoD, 10 samples at 5-10×LoD and 10 controls were evaluated over a 56-hour period under variable temperatures (+22° C. to +40° C.). The temperatures and times for summer profile are included in Table 11.
The acceptance criteria were: (a) Low Positive Samples: ≥95% agreement with expected results, (b) High Positive Samples: 100% agreement with expected results, and (c) Negative Samples: 100% agreement with expected results. The results of pre-temperature testing is included in Table 12, wherein UD indicates an undetermined value.
All spike-in samples were detected (38/38, 100%) and all controls (10/10 no spike-in) were negative for SARS-COV-2 detection. The results of the summer temperature test is included in Table 13, where UD indicates an undetermined value.
Thirty-seven of 38 (97%) spike-in samples were detected in the summer profile group. Twenty-two of 23 (96%) of 1-2×LoD were detected, and all samples 2-10×LoD were detected. All controls (no spike-in) were negative for SARS-COV-2 detection. The results identify that the criteria for summer transportation are met by the biological reagent, or saliva stabilization buffer, of the present disclosure.
In a second study (Study 2), for the winter profile, various temperature ranges were evaluated (Table 14).
The results of the winter temperature test is included in Table 15, where UD indicates an undetermined result.
All spike-in samples were detected (38/38, 100%) and all controls (10/10 no spike-in) were negative for SARS-COV-2 detection. The results identify that the criteria for winter transportation met by the biological reagent, or saliva stabilization buffer, of the present disclosure.
In summer, it can be appreciated that saliva is an effective source for SARS-COV-2 detection. The LoD according to methods of the present disclosure is 10 viral copies. The clinical study (n=60, matched saliva and nasopharyngeal samples) demonstrates 100% concordance between saliva and nasopharyngeal samples even at “low” viral titers (Cr>35), where the positive % agreement is 100% (95%: 88-100%) and the negative percent agreement: 100% (95%: 88-100%). The temperature studies (n=48, LoD 1.5-10×) identify no impact of heat or cold weather.
Usability Human Factors Assessment for the Saliva Collection: Two studies were performed to evaluate a saliva collection protocol according to methods of the present disclosure. The first study (n=122) was undertaken in an industrial setting. The second study (n=33) was undertaken in a school setting. Each participant was provided with a kit (including instructions), based on the methods and devices of the present disclosure, and a questionnaire (
The demographics of the participants is included in Table 16 and Table 17.
The results of the questionnaire are included in Table 18 and Table 19, where * denotes a participant that indicated they had difficulty “making” saliva and drank water. For Table 19, 3 participants required parental assistance in order to complete the sample collection.
The results identified that participants of all ages (ranging 5-75), both genders (male/female), and a range of educational achievements (at school to post-graduate degree) had no significant difficulties using the device of the present disclosure (i.e., saliva collection tube), including following instructions and physically collecting the sample.
Moreover, the following observations were made. First, children under 10 years of age require adult supervision. Online data collection (either QR code or typing into a website) was favored by the majority (134/155=86.4%). Online data collection was associated with younger individuals (median age: 40 [range: 5-75]. Paper requisitions were associated with older individuals (median age: 58 [range: 30-72]. The shorter kit insert was used in all cases. There were no concerns regarding collection and send-out. Only 1 (0.6%) failure was identified which was related to water ingestion immediately prior to collection. For this one, the RNAseP was greater than 36, which is consistent with a diluted saliva collection. At follow-up, this individual admitted drinking water. She was provided with a second kit and collected the sample appropriately.
Child Safety Study: A child safety study was performed to determine whether a sample kit based on the methods and devices of the present disclosure is safe for children.
As background, a COVID-19 PCR Saliva Collection Kit, based on the methods and devices of the present disclosure, has been FDA-EUA authorized (201111, dated: Oct. 20, 2020) for at home collection of saliva from children under a prescription-based setting. Sample collection from children aged 5-13 years is per direct adult collection. Sample collection for 14-17 years is per direct adult supervision.
To demonstrate child-safety feasibility, a prospective, observational study, evaluating the child-safety aspects of the collection cap (which contains a guanidine-based stabilization buffer) and the post-collection tube (capped saliva-collected sample), was performed. The goals of the study were to evaluate whether regular play had an impact on the aluminum seal of the cap, the cap structure, and sealing at the cap/tube connection, evaluate whether children could break the aluminum seal of the cap and/or break the cap, and evaluate whether children could open the tube once a sample was collected.
Individual subject demographics are included in Table 20, where F is female, M is male, A is Asian, B is black, C is Caucasian, H is Hispanic/Latino, and NHL is non-Hispanic/Latino.
A total of 12 children were observed with ages ranging between 5-17 years (median: 10 years), including 7 girls and 5 boys. Race, ethnicity and school grade spanned all demographics.
For “Regular play” observations, children were provided a standard sealed cap and observed for up to 60 minutes. Most children spent a median of 20 minutes playing with the object. Play ranged from throwing and kicking it to spinning the cap. Girls tended to incorporate the object into their imaginary play (e.g., use it as a prop—e.g., a cup-in their play).
Most children threw and kicked the cap, with the majority also spinning it.
For the “breakage” observations, children were asked to remove the foil with their fingers/fingernails or to break the cap by standing on it. The same caps were used as those used in the play portion of the observation. Most children used their nails to try and unpeel the seal or used a variety of options to break the cap including pressing down with their fingernail (thumb, index or middle finger).
The individual results are included in Table 21, where NE is no effect, NI is no impact, NOD is not observed/not done by the child, and * denotes a child that was observed a second time. They were asked to do the same things (play, break) with the same outcome—no impact on the cap or the cap/tube connection.
The median observation time was 20 minutes (range: 11-57 minutes).
Individual play/breakage observations included that the foil has some give so it can be pushed inwards without breaking, the foil is recessed too deeply and even little fingers/fingernails could not access it to unpeel the foil, all caps (after regular play and breakage testing) could be screwed onto the tube and were functional (i.e., could dispense the liquid into the tube).
In summary, regular play had no impact on the aluminum seal or cap structure itself. Caps functioned normally and could be screwed onto tubes. Children were unable to unpeel the aluminum seal or break the foil with their fingernail. They could not break the cap. Further, a tightened cap (post-collection) requires significant force to open it. Children could not open the cap after it was tightened.
For post-collection cap removal observations, the same group was provided with capped saliva collection tubes. They were asked to remove the caps using any means. Children were observed using a variety of grips including thumb/first digit, thumb/multiple digits, power grip, two hands, single twist (holding the tube with one hand, using the other hand to untwist), and double twisting (one hand on tube, the other on the cap and untwisting—working in opposite directions). Children were encouraged to use multiple approaches.
The individual results are included in Table 22, where NE is no effect, NI is no impact, NOD is not observed/not done by the child, and * denotes children that were observed a second time. They were asked to unscrew the cap on a second occasion. The result was the same outcome-no impact on the cap/tube connection.
The median time observed to unscrew the cap was 5.5 minutes (range: 4-12 minutes). Ultimately, all capped tubes were returned to the laboratory for the technician to remove. In all instances, the caps cannot be opened using thumb and first/second fingers. A power grip was required with a double twist, to open the caps.
A summary of observations is included in Table 23, where * denotes cases of cap breakage, ** denotes cases of cap breakage, * indicates whether the cap could be screwed onto a tube after play/breakage attempts, and * indicates whether the cap could be opened by a child after it was screwed onto a tube.
Based on these observations, it can be concluded that the cap and the capped collection tube is difficult to open and that the 10 children aged 5-13 years were not able to break the seal, break the cap during play or intentionally and they cannot open the sample collection system once closed.
All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and sub-combinations possible of the group are intended to be individually included in the disclosure.
Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated.
Whenever a range is given in the specification, for example, a size range, number range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
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This application claims the benefit of priority of U.S. Provisional Patent App. No. 63/086,929, filed Oct. 2, 2020, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/53233 | 10/1/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63086929 | Oct 2020 | US |
