Dried oral fluid diagnostics apparatus and method

Abstract

A dried oral fluid diagnostics apparatus includes a porous and absorbent layer of material and a sampling zone at an end of the layer. The sampling zone is configured to contact a user's tongue to receive an oral fluid. The sampling zone has a sampling periphery, top and bottom surfaces, a storage zone, and a wicking path. The sampling periphery defines at least in part a receiving portion, and the top and bottom surfaces define a thickness of the sampling zone. The storage zone is configured to receive the oral fluid initially in a moist form, and subsequently stores the oral fluid in dry form to allow processing for recovery of analytes. The wicking path is in fluid communication with the sampling periphery, the storage zone, and the top and bottom surfaces. The wicking path provides a flow path to the storage zone for the oral fluid in moist form.

Description

Attorney Docket No. 700355-088491WOPTDRIED ORAL FLUID DIAGNOSTICS APPARATUS AND METHODCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 723,877, filed on November 22, 2024, and U.S. Provisional Application No. 63 / 724,786, filed on November 25, 2024, both of which are incorporated herein by reference in their entireties.FIELD OF THE INVENTION

[0002] The present invention relates generally to dried oral fluid diagnostics, and, more specifically, to sampling oral fluid and storing it in dry form for future processing.BACKGROUND OF THE INVENTION

[0003] Historically, nasophary ngeal swabs have been the gold standard method of sample collection to diagnose respiratory viruses. Nasopharyngeal sampling requires a swab to be inserted 9-10 centimeters into the nasal cavity to collect secretions while in contact with the nasopharynx. Though effective for diagnostics, sampling via this method requires a highly trained healthcare worker, a tolerant patient, and invasive collection tools. Anterior nasal swabs inserted at least 1 centimeter into the nasal cavity to collect secretions from the nasal membrane were introduced as an alternative and substantially less invasive sampling technique. The adoption of anterior nasal swabs for respiratory virus diagnostics increased exponentially through the COVID-19 pandemic, predominately because they allow patients to self-collect a sample, which greatly reduced the risk of exposure for healthcare workers and increased monitoring capabilities for larger populations.

[0004] Though undeniably less invasive than nasopharyngeal swabs, there are still some inevitable disadvantages of using anterior nasal swabs such as (i) the lack of standardization in the sample collection method and (ii) the large volume of diluents required for sample storage and processing. Saliva is another minimally invasive matrix that has been used to detect respiratory viruses (e.g., SARS-CoV-2, Influenza A / B, RSV) and has been shown to have comparable accuracy to nasopharyngeal swabs, even for early detection. However, saliva has not achieved the same clinical adoption as anterior nasal swabs. This hesitation to change matrices may be attributed to the same lack of standardization in sample collection for saliva as anterior nasal swabs, with the added nuance of intrinsic patient-to-patient variability of theAttorney Docket No. 700355-088491WOPT matrix (e.g., viscosity, pH, native microbiome, food contamination, mouth dryness).

[0005] To circumvent these challenges, several collection technologies have been developed to collect and preserve saliva at the point of care. Methods to collect liquid saliva require the user to place a receptacle or device near their mouth and spit or drool into a sterile collection tube. For example, the SalivaBio® Saliva Collection Aid (Salimetrics) can collect roughly 1.0 to 2.0 mL of saliva using a single-use funnel and collection tube with a fill line. While supporting collection of relatively large volumes of saliva, the disadvantage of collecting drool or spit is the influence of (i) stimulated or unstimulated production of saliva, (ii) mucin aggregation, and (iii) viscosity, which can impact downstream analyses. Alternatively, saliva collection assisted by an absorbent material (e.g., swabs) requires the user to manually insert the material in their mouth and use swirling motions. For example, the Salivette" (Sarstedt) comprises an absorbent cotton swab and a collection tube, which can collect between 0.8 and 1.4 mL of saliva. Swab collection devices are often easier to use and cleaner for the user but can be limited to reduced analyte recovery from the swab material.

[0006] A need exists for an apparatus and method for sampling and storing saliva for future processing (e.g., extracting analytes) that reduces invasiveness, difficulty of administering, need for large volumes of diluents, low analyte recovery', and other difficulties associated with known technologies. The present disclosure provides a solution to these and other needs.SUMMARY OF THE INVENTION

[0007] The term embodiment and like terms, e.g., implementation, configuration, aspect, example, and option, are intended to refer broadly to all of the subject matter of this disclosure and the claims below . Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the claims below. Embodiments of the present disclosure covered herein are defined by the claims below, not this summary7. This summary is a high-level overview' of various aspects of the disclosure and introduces some of the concepts that are further described in the Detailed Description section below. This summary’ is not intended to identify key or essential features of the claimed subject matter. This summary is also not intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification of this disclosure, any or all drawings, and each claim.

[0008] According to certain aspects of the present disclosure, a dried oral fluid diagnostics apparatus includes a layer of material that is porous and absorbent and a sampling zoneAttorney Docket No. 700355-088491WOPT positioned at one end of the layer of material. The sampling zone is configured for placement in contact with a user’s tongue to receive an oral fluid in moist form. The sampling zone has a sampling periphery, a top surface and a bottom surface, a storage zone, and a wicking path. The sampling periphery defines at least in part a receiving portion of the sampling zone. The top surface and the bottom surface define a thickness of the sampling zone along a cross- sectional direction of the sampling zone. The storage zone is configured to receive the oral fluid initially in the moist form. The storage zone subsequently holds the oral fluid stored in dry form to allow processing for recovery of analytes. The wick mg path is in fluid communication with the sampling periphery, the storage zone, and the top and bottom surfaces. The wicking path provides a flow path to the storage zone for the oral fluid in moist form.

[0009] According to certain aspects of the present disclosure, the dried oral fluid diagnostics apparatus further includes a channel in fluid communication with the sampling zone. The channel receives at least some of the oral fluid in moist form. The channel includes a saturation indicator.

[0010] According to some features of the above aspects, the saturation indicator includes graduation marks indicative of saturation of the sampling zone based on how many of the graduation marks are reached by the oral fluid in the moist form.

[0011] According to some features of the above aspects, the saturation indicator includes a color generated in response to a reaction with the oral fluid in the moist form.

[0012] According to some features of the above aspects, the saturation indicator includes a change from an initial color to a subsequent color in response to the reaction with the oral fluid in the moist form.

[0013] According to certain aspects of the present disclosure, the dried oral fluid diagnostics apparatus further includes one or more sampling guides attached along an exterior periphery of the layer of material. The one or more sampling guides are indicative of an insertion end for insertion into the user’s mouth.

[0014] According to some features of the above aspects, at least one of the one or more sampling guides is foldable.

[0015] According to some features of the above aspects, the analytes are selected from a group consisting of pathogens, microorganisms, proteins, enzymes, antibodies, polysaccharides, hormones, steroids, electrolytes, metabolites, nucleic acids, therapeutics, vitamins, exosomes, and drugs of abuse or their metabolites.

[0016] According to some features of the above aspects, the pathogens are upperAttorney Docket No. 700355-088491WOPT respiratory viruses including SARS-CoV-2, Influenza A, Influenza B, Rhinovirus, or combinations thereof.

[0017] According to some features of the above aspects, the layer of material has a liquid sampling volume in a range of about 5 microliters per square centimeter (pL / cm2) to about 50 pL / cm2.

[0018] According to some features of the above aspects, the storage zone includes one or more predetermined regions of the sampling zone.

[0019] According to some features of the above aspects, the one or more predetermined regions are perforated.

[0020] According to some features of the above aspects, the one or more predetermined regions each has a conical shape for insertion into a conical tube, a shape for insertion into a microcentrifuge tube, or a shape for insertion into a micro well plate.

[0021] According to certain aspects of the present disclosure, a kit for processing of dried oral fluid includes at least one dried oral fluid diagnostics apparatus, and at least one pouch. The at least one dried oral fluid diagnostics apparatus includes a layer of material that is porous and absorbent, and a sampling zone. The sampling zone is configured for placement in contact with a user’s tongue to receive an oral fluid in moist form. The sampling zone has a sampling periphery’, a storage zone, and a wicking path. The sampling periphery' defines at least in part a receiving portion of the sampling zone. The storage zone is configured to receive the oral fluid initially in the moist form. The storage zone subsequently holds the oral fluid stored in dry form to allow processing for recovery of analytes. The wicking path is in fluid communication with the sampling periphery’ and with surfaces of the sampling zone. The wicking path provides a flow' path to the storage zone for the oral fluid in moist form. The at least one pouch is configured to seal around the at least one dried oral fluid diagnostics apparatus.

[0022] According to some features of the above aspects, the at least one dried oral fluid diagnostics apparatus includes a plurality of dried oral fluid diagnostics apparatuses. The at least one pouch includes a plurality of pouches. Each of the plurality of pouches is configured to seal around one of the plurality of dried oral fluid diagnostics apparatuses.

[0023] According to certain aspects of the present disclosure, a method of sampling dried oral fluid for molecular diagnostics includes contacting a sampling zone of a layer of porous and absorbent material to a user's tongue. The method further includes wicking an oral fluid in moist form from the user's tongue into the sampling zone of the layer of material. The methodAttorney Docket No. 700355-088491WOPT further includes dry ing the layer of material such that the sampling zone holds the oral fluid in dry form in at least one storage zone that is a subset of the sampling zone. The method further includes separating the at least one storage zone from a remainder of the sampling zone for processing. The method further includes processing the at least one storage zone for recovery of analytes from the oral fluid in dry form.

[0024] According to some features of the above aspects, the sampling zone is in fluid communication with a saturation indicator for indicating that a sufficient sample of the oral fluid in the moist form has been sampled for recovery of the analytes from the oral fluid in the dry form.

[0025] According to some features of the above aspects, the at least one storage zone is perforated.

[0026] According to some features of the above aspects, the analytes are selected from a group consisting of pathogens, microorganisms, proteins, enzymes, antibodies, polysaccharides, hormones, steroids, electrolytes, metabolites, nucleic acids, therapeutics, vitamins, exosomes, and drugs of abuse or their metabolites.

[0027] According to some features of the above aspects, the pathogens are upper respiratory viruses including SARS-CoV-2, Influenza A, Influenza B, Rhino virus, or combinations thereof.

[0028] The above summary is not intended to represent each embodiment or every aspect of the present disclosure. Rather, the foregoing summary merely provides an example of some of the novel aspects and features set forth herein. The above features and advantages, and other features and advantages of the present disclosure, vvi 11 be readily apparent from the following detailed description of representative embodiments and modes for carrying out the present invention, when taken in connection with the accompanying drawings and the appended claims. Additional aspects of the disclosure will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The disclosure, and its advantages and drawings, will be better understood from the following description of representative embodiments together with reference to the accompanying drawings. These drawings depict only representative embodiments and are therefore not to be considered as limitations on the scope of the various embodiments or claims.Attorney Docket No. 700355-088491WOPT

[0030] FIG. 1 shows a schematic plan view of an exemplary' dried oral fluid diagnostics apparatus, according to certain aspects of the present disclosure.

[0031] FIG. 2 is a cross-sectional view of the exemplary dried oral fluid diagnostics apparatus taken generally along the lines '‘2-2” of FIG. 1, according to certain aspects of the present disclosure.

[0032] FIG. 3 shows an exemplary predetermined shape for a storage zone, according to certain aspects of the present disclosure.

[0033] FIG. 4 shows another exemplary predetermined shape for a storage zone, according to certain aspects of the present disclosure.

[0034] FIG. 5 shows a further exemplary' predetermined shape for a storage zone, according to certain aspects of the present disclosure.

[0035] FIG. 6 shows a dried oral fluid diagnostics apparatus having a storage zone having a shape like a plus sign, according to certain aspects of the present disclosure.

[0036] FIG. 7 shows a test tube that has been broken apart with the pieces arranged to generally resemble a shape like a plus sign, according to certain aspects of the present disclosure.

[0037] FIG. 8 shows a plus sign shaped storage zone, similar to the storage zone shown in FIG. 4, folded within a test tube, according to certain aspects of the present disclosure.

[0038] FIG. 9 shows nine storage zones of a Fishbume Tab, and the average cycle threshold value (CT) of each of the nine zones extracted from samples of the Fishbume Tab, according to certain aspects of the present disclosure.

[0039] FIG. 10A show s a plot of average CT for several sample ty pes, according to certain aspects of the present disclosure.

[0040] FIG. 10B shows a plot of the number of punches needed for most consistent sampling from a Fishbume Tab, according to certain aspects of the present disclosure.

[0041] FIG. 10C show s a plot of punch format for best sampling from a Fishbume Tab, according to certain aspects of the present disclosure.

[0042] FIG. 11 shows the average CT for samples of liquid saliva and saliva dried on Fishbume Tabs in the top graph, and the percent of positive sample reads obtained via RT- qPCR plotted for each of the sample types (liquid and dried on Tab) in the bottom graph, according to certain aspects of the present disclosure.

[0043] FIG. 12 show s a frustration matrix of a summary of clinical sample validation in section A, and performance metrics in section B. according to certain aspects of the presentAttorney Docket No. 700355-088491WOPT disclosure.

[0044] FIG. 13 shows sample collection methods for viral diagnostics compared for invasiveness and ease of application, according to certain aspects of the present disclosure.

[0045] FIG. 14 shows factors affecting the complexity of viral analysis from a sample of saliva, according to certain aspects of the present disclosure.

[0046] FIG. 15 shows features that are desirable for improving salivary diagnostic testing, according to certain aspects of the present disclosure.

[0047] FIG. 16 shows advantages of dried sampling for viral diagnostics, according to certain aspects of the present disclosure.

[0048] FIG. 17 shows an overview of dried sampling for viral diagnostics, according to certain aspects of the present disclosure.

[0049] FIG. 18 shows the replacement of nasal swabs with dried saliva sampling for viral diagnostics, according to certain aspects of the present disclosure.

[0050] FIG. 19 shows a first aspect in the characterization of a dried saliva tab, namely the sampling volume, according to certain aspects of the present disclosure.

[0051] FIG. 20 shows a second aspect in the characterization of a dried saliva tab, namely the sample distribution, according to certain aspects of the present disclosure.

[0052] FIG. 21 shows a third aspect in the characterization of a dried saliva tab, namely analyte recovery, according to certain aspects of the present disclosure.

[0053] FIG. 22 shows a flow diagram for study participants included in a clinical validation trial, according to certain aspects of the present disclosure.

[0054] FIG. 23 shows a CT cutoff evaluation for optimal analytical performance for the Fishbume Tab method, according to certain aspects of the present disclosure.

[0055] FIG. 24A shows parameters used to estimate the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of each collection type, according to certain aspects of the present disclosure.

[0056] FIG. 24B shows CT values determined by RT-qPCR for tab-extracted samples plotted against those measured from the paired, liquid-extracted samples in section A, and a Bland- Altman analysis of the RT-qPCR results from paired samples of liquid and dried saliva in section B, according to certain aspects of the present disclosure.

[0057] FIG. 25 A shows a first sample of a dried oral fluid diagnostics apparatus using a first type of material for the layer of material, according to certain aspects of the present disclosure.Attorney Docket No. 700355-088491WOPT

[0058] FIG. 25B shows a second sample of a dried oral fluid diagnostics apparatus using a second type of material for the layer of material, according to certain aspects of the present disclosure.

[0059] FIG. 25C shows a third sample of a dried oral fluid diagnostics apparatus using a third type of material for the layer of material, according to certain aspects of the present disclosure.

[0060] FIG. 25D shows a fourth sample of a dried oral fluid diagnostics apparatus using a fourth type of material for the layer of material, according to certain aspects of the present disclosure.

[0061] FIG. 25E shows a fifth sample of a dried oral fluid diagnostics apparatus using a fifth type of material for the layer of material, according to certain aspects of the present disclosure.

[0062] FIG. 26 shows a table listing the volume of a variety’ of paper materials for a 6 mm punch and per square centimeter, according to certain aspects of the present disclosure.

[0063] FG. 27 shows an overview of a clinical validation for paired samples of liquid and dried saliva, according to certain aspects of the present disclosure.

[0064] FIG. 28 shows a linearity analysis that examines the overall deviation from measurement accuracy for data from the clinical validation shown in FIG. 27, according to certain aspects of the present disclosure.

[0065] FIG. 29 shows a schematic diagram of an exemplary kit for processing of dried oral fluid, according to certain aspects of the present disclosure.

[0066] FIG. 30 shows the steps of an exemplary method of sampling oral fluid in moist form and extracting analytes from the oral fluid in dried form, according to certain aspects of the present disclosure.DETAILED DESCRIPTION

[0067] Various embodiments are described with reference to the attached figures, where like reference numerals are used throughout the figures to designate similar or equivalent elements. The figures are not necessarily drawn to scale and are provided merely to illustrate aspects and features of the present disclosure. Numerous specific details, relationships, and methods are set forth to provide a full understanding of certain aspects and features of the present disclosure, although one having ordinary skill in the relevant art will recognize that these aspects and features can be practiced without one or more of the specific details, withAttorney Docket No. 700355-088491WOPT other relationships, or with other methods. In some instances, well-known structures or operations are not shown in detail for illustrative purposes. The various embodiments disclosed herein are not necessarily limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are necessarily required to implement certain aspects and features of the present disclosure.

[0068] For purposes of the present detailed description, unless specifically disclaimed, and where appropriate, the singular includes the plural and vice versa. The word “including” means “including without limitation.” Moreover, words of approximation, such as “about,” “almost,” “substantially,” “approximately,” and the like, can be used herein to mean “at,” “near,” “nearly at,” “within 3-5% of,” “within acceptable manufacturing tolerances of,” or any logical combination thereof. Similarly, terms “vertical” or “horizontal” are intended to additionally include “within 3-5% of’ a vertical or horizontal orientation, respectively. Additionally, words of direction, such as “top,” “bottom,” “left,” “right,” “above,” and “below” are intended to relate to the equivalent direction as depicted in a reference illustration; as understood contextually from the object(s) or element(s) being referenced, such as from a commonly used position for the object(s) or element(s); or as otherwise described herein.

[0069] Oral fluid, for example, saliva, wicked from a user's tongue into a porous and absorbent material and dried in the material provides high-quality, self-collected samples to facilitate decentralized viral infection testing. The apparatus and method disclosed herein provide a platform to collect dried saliva and detect the presence of pathogens, for example without limitation, SARS-CoV-2 RNA using RT-qPCR. Saliva samples collected using the apparatus and method disclosed herein were stored at -80 °C for 3 years. The samples were thawed and processed using RT-qPCR to correctly identify positive and negative samples with a sensitivity, specificity, and accuracy of 81%, 89%, and 87%. The apparatus and method disclosed herein provides for improved user sampling and seamless integration into current clinical workflow s.

[0070] Referring to FIG. 1, in an embodiment a dried oral fluid diagnostics apparatus 100 includes a layer of material 110 that is porous and absorbent. Materials suitable for use as the layer of material 110 include, for example without limitation, cellulosic papers, fabrics, felts, and membranes. In an embodiment, a sampling zone 120 is positioned proximate to an end of the layer of material 110. The sampling zone 120 is configured for placement in contact with a user’s tongue to receive an oral fluid, for example, saliva in moist form. The sampling zoneAttorney Docket No. 700355-088491WOPT120 includes a sampling periphery 130 defining at least in part a receiving portion of the sampling zone 120. In an embodiment, the sampling zone 120 extends to the edge of the layer of material 1 10 such that the sampling periphery 130 is coincident with the edge of the layer of material 110.

[0071] Referring to FIG. 2, the sampling zone 120 includes a top surface 140 and a bottom surface 150 defining a thickness T of the sampling zone 120 along a cross-sectional direction of the sampling zone 120. In an embodiment, the layer of material 1 10 has a liquid sampling volume in a range of about 5 microliters per square centimeter (pL / cm2) to about 50 pL / cm2

[0072] Referring to FIGs. 1 and 2, in an embodiment, the sampling zone 120 is configured to receive the oral fluid initially in a moist form, for example as a liquid that wicks into the layer of material 110. Upon absorbing a quantity of the oral fluid in the moist form, the layer of material 110 is designed to be dried for subsequent storage, shipment, and / or recover}' of analytes from the oral fluid in dried form. In an embodiment, immediately upon absorbing the quantity of the oral fluid, an RNA stabilizing component is added to the sampling zone 120 before drying.

[0073] The sampling zone 120 has one or more storage zones 160 for holding the oral fluid stored subsequently in dry form. For example, in FIG. 1, the one or more storage zones 160 are represented by nine dashed circles. As show n in FIG. 2, the one or more storage zones 160 can extend across the thickness T of the sampling zone 120.

[0074] A wicking path 170 provides a flow path for wi eking oral fluid in moist form from the user’s tongue to the one or more storage zones 160. The wicking path 170 is in fluid communication with and extends from the sampling periphery7130 and from the top and bottom surfaces 140 and 150 to the one or more storage zones 160.

[0075] In an embodiment, the one or more storage zones 160 are one or more predetermined regions of the sampling zone 120, for example, labeled as circular regions A, B, and C in FIG. 1. In an embodiment, each of the one or more storage zones 160 is marked, for example with an outline of a circle or other shape. In an embodiment each of the one or more storage zones 160 is perforated. In an embodiment the marked or perforated outlines of the one or more storage zones 160 are designed to be cut or punched out of the sampling zone 120.

[0076] In other embodiments, each of the one or more storage zones 160 can be a shape as shown in FIGs. 3-5, or some other shape as desired. For example, FIG. 6 illustrates a dried oral fluid diagnostics apparatus 100 having a storage zone 160 shaped similar to the storage zone shown in FIG. 3. Each of the one or more storage zones 160 shown in FIGs. 3-5 can have, forAttorney Docket No. 700355-088491WOPT example, a flat shape or a shape achieved upon cutting, punching, rolling, or folding that can be conical for insertion into a conical tube. To illustrate this, FIG. 7 shows a test tube that has been broken apart so that the pieces generally resemble the shapes of the storage zones 160 shown in FIGs. 3-5. FIG. 8 shows the storage zone shown in FIG. 4, folded within a test tube. For example, each of the shapes shown in FIGS. 3-5 can be folded into a shape suitable for insertion into a conical tube, a shape for insertion into a microcentrifuge tube, or a shape for insertion into a microwell plate.

[0077] Upon separation of the one or more storage zones 160 from the remaining sampling zone 120, the one or more storage zones 160 are processed for recovery7of analytes from the oral fluid stored in dried form within the one or more storage zones 160. In an embodiment, the analytes that are recoverable from the oral fluid in dried form include, without limitation, pathogens, microorganisms, proteins, enzymes, antibodies, polysaccharides, hormones, steroids, electrolytes, metabolites, nucleic acids, therapeutics, vitamins, exosomes, drugs of abuse or their metabolites, or any combination thereof. In an embodiment, the pathogens are upper respiratory7viruses including SARS-CoV-2, Influenza A, Influenza B, Rhinovirus, or combinations thereof.

[0078] FIG. 9 illustrates nine storage zones 160 of a Fishbume Tab, labeled as zones A through I for reference. To investigate if samples of oral fluid, for example, saliva, are evenly distributed from a user’s tongue throughout the layer of material 110 of the Fishbume Tab, a test was conducted. Several vials of saliva from confirmed negative patients were combined to prepare a stock of pooled naive saliva. For each of three Fishbume Tabs, 200 pL of pooled saliva spiked with 2.5xl05c / mL of heat-inactivated virus was added by pipette. After drying at ambient conditions for approximately 5 minutes, one hole punch was acquired from each of the nine sampling zones A-I. RNA was extracted from each sampling zone A-I, and SARS- CoV-2 RNA was quantified via RT-qPCR.

[0079] The average cycle threshold value (CT) of all 9 zones extracted from the layer of material 110 of the Fishbume Tabs was 34.0+1.2, which suggests that when a sample is added to the layer of material 110 of a Fishbume Tab, it does not distribute uniformly across the layer of material 110. The results demonstrated that the center zone had the lowest average CT value, 33.6, as expected for one punch sampling, and the smallest standard deviation from technical replicates (o = 0.1).

[0080] Referring to FIG. 10 A, an average CT for several sample types is plotted. Liquid samples contain 2.5xl05c / mL of heat-inactivated virus in water. Saliva samples containAttorney Docket No. 700355-088491WOPT2.5xlO5c / mL of heat-inactivated virus spiked in naive saliva. No template control samples (water or saliva containing 5.0xl03c / mL of RNaseP) did not amplify (i.e., undetermined). The average CT value for each sample type is plotted (N=3). Error bars indicate the standard error of the mean.

[0081] Referring to FIG. 10B, a plot of the number of punches needed for most consistent sampling from a Fishbume Tab is presented. All samples added to Tabs comprised 2.5x105c / mL of heat-inactivated virus in water. No template control samples did not amplify during amplification (i.e., undetermined, CT > 40). The average CT value for each sample is plotted (N=3). Error bars indicate the standard error of the mean.

[0082] Referring to FIG. 10C, a plot of punch format for best sampling from a Fishbume Tab is presented. All samples added to Tabs comprised 2.5xl05c / mL of heat-inactivated virus in water. No template control samples did not amplify during amplification (i.e., undetermined, CT > 40). The average CT value for each sample is plotted (N=3). Error bars indicate the standard error of the mean.

[0083] Referring to FIG. 11. the average CT for samples of liquid saliva and saliva dried on Fishbume Tabs are shown in the top graph. The slopes of the curves correspond to amplification efficiencies of 71.1% and 79.0%, respectively. Error bars indicate the standard error of the mean. The bottom graph shows the percent of positive sample reads obtained via RT-qPCR plotted for each of the sample types (liquid and dried on Tab). The estimated limit of detection (LOD) is about 15.6 x 103c / mL for both the liquid and dried saliva.

[0084] Referring to FIG. 12, data for clinical validation of an RT-qPCR test for dried saliva collected by Fishbume Tabs is presented. Section A shows a frustration matrix of a summary of clinical sample validation where the reference method is the liquid saliva and the tested method is the dried saliva on the Fishbume tabs. Section B shows performance metrics.

[0085] To assess the clinical utility of dried saliva collected on Fishbume Tabs, samples of saliva (liquid and dried) were collected from patients whose status for SARS-CoV-2 was known. For each clinical sample, the dried saliva tab and its corresponding vial of liquid saliva was thawed for paired analysis. Continuing, (i) two punches were obtained from the center of the Fishbume Tab and (ii) 4.5 pL of the liquid saliva (the matched volume) was obtained to extract RNA and conduct analyses via RT-qPCR. Each sample was evaluated in triplicate and the result of the assay categorized as either positive or negative by majority agreement (i.e., 2 out of 3). To maximize the sensitivity and specificity of the assay, assay performance metrics were analyzed with respect to a CT cutoff. Of note, the CT cutoff was determined post-hoc,Attorney Docket No. 700355-088491WOPT after all the samples had been tested by the reference method (i.e.. liquid saliva PCR). Since the goal was to evaluate the diagnostic performance of the dried saliva method, it was important to first establish a ground truth with a method most similar to the goal standard (i.e., comparing saliva PCR results to nasal PCR results). The results indicate that a CT cutoff of 37 can minimize misclassification of results (i.e., false negatives or false positives). A confusion matrix was constructed based on this cutoff to evaluate the accuracy of dried saliva as a diagnostic specimen (Section A of FIG. 12). The results demonstrate that dried saliva recovered from Fishbume Tabs was able to correctly identify 22 of the 26 known positive samples, and 93 of the 99 known negative samples, which yielded a sensitivity, specificity, and accuracy of 85%, 94%, and 92%, respectively (Section B of FIG. 12). Of the 99 negative samples, 6 were positive by the dried saliva method, yielding CT values of 36.7, 32.6, 36.0, 36.5, 36.9, and 36.9. As our determined cutoff was a Ct of 37, five of the six values fall very’ close to the detection threshold. We speculate that these results reflect stochastic variation near the limit of detection rather than systematic error. The error near the detection limit is likely statistical noise that is a common limitation of sensitive molecular assays. There was one sample with a much lower Ct of 32.6, which we consider to be a potential rare instance of contamination as we did not observe any procedural anomalies during processing. All dried saliva samples were stored in individual, sealed bags and the hole punch was thoroughly cleaner between uses, which minimizes but does not eliminate the likelihood of cross-contamination.

[0086] The positive predictive value (PPV) and negative predictive value (NPV) for samples of dried saliva were 79% and 96%, respectively, indicating satisfactory agreement between the matrices and effective qualitative detection of SARS-CoV-2 RNA. Based on these performance metrics, the Fishbume Tab meets the acceptable thresholds for sensitivity (> 80%), PPV (> 78%). NPV (>95%), and detection range (i.e., IO4- 106c / mL) for POC SARS- CoV-2 testing as defined by WHO guidelines. However, the specificity is slightly lower than the threshold for acceptable (target of 97%), which can be partly attributed to the size of the study: only three additional, correct classifications would be required to meet the threshold. Overall, these results suggest that self-collected, dried saliva is a suitable specimen to support POC testing and screening and presents a potential approach to standardize the collection of saliva.

[0087] Referring to FIG. 13, sample collection methods for viral diagnostics are presented and compared for invasiveness and ease of application.

[0088] Referring to FIG. 14, factors affecting the complexity of viral analysis from aAttorney Docket No. 700355-088491WOPT sample of saliva are presented.

[0089] Referring to FIG. 15, features that are desirable for improving salivary diagnostic testing are presented.

[0090] Referring to FIG. 16, advantages of dried sampling for viral diagnostics are presented.

[0091] Referring to FIG. 17, an overview of dried sampling for viral diagnostics is presented.

[0092] Referring to FIG. 18, the replacement of nasal swabs with dried saliva sampling for viral diagnostics is presented.

[0093] Referring to FIG. 19, a first aspect in the characterization of a dried saliva tab, namely the sampling volume, is presented.

[0094] Referring to FIG. 20, a second aspect in the characterization of a dried saliva tab, namely the sample distribution, is presented.

[0095] Referring to FIG. 21, a third aspect in the characterization of a dried saliva tab, namely analyte recovery, is presented.

[0096] Refernng to FIG. 22, a flow diagram for study participants included in a clinical validation trial is presented.

[0097] Referring to FIG. 23, a CT cutoff evaluation for optimal analytical performance for the Fishbume Tab method is presented. Sensitivity and specificity are plotted with respect to CT cutoff value. A cutoff of 38 was determined sufficient to minimize misclassifications of false positives and false negatives.

[0098] Referring to FIG. 24A, parameters used to estimate the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of each collection type are presented.

[0099] Referring to FIG. 24B, a quantitative analysis of Fishbume Tabs using clinical samples is presented. In the plot labeled A are plotted CT values determined by RT-qPCR for tab-extracted samples against those measured from the paired, liquid-extracted samples. The data is fit by using linear regression (Y = 1.139X). The plot labeled B shows a Bland-Altman analysis of the RT-qPCR results from paired samples of liquid and dried saliva. The difference in measured CT values is plotted against their mean. The upper and lower limits of agreement are indicated by the dotted lines.

[0100] FIGs. 25A-25E show five samples of a dried oral fluid diagnostics apparatus 100, each including a different layer of material 110. The material samples have increasingAttorney Docket No. 700355-088491WOPT absorbency going from FIG. 25 A to FIG. 25E. The materials presented are Ahlstrom 601 in FIG. 25A, Whatman 4 in FIG. 25B, TFN in FIG. 25C, Shamwow in FIG. 25D, and Kapmat in FIG. 25 E.

[0101] FIG. 26 shows a table listing the volume for a 6 mm punch and also per square centimeter for a variety' of materials.

[0102] Referring to FIG. 27, an overview of a clinical validation for paired samples of liquid and dried saliva is presented.

[0103] Referring to FIG. 28, a linearity analysis that examines the overall deviation from measurement accuracy is presented for data from the clinical validation shown in FIG. 27.

[0104] Referring again to FIG. 1, in an embodiment, a channel 180 is in fluid communication with the sampling zone 130. The channel 180 receives at least some of the oral fluid in moist form from the sampling zone 120, for example, via a wicking path 172. In an embodiment, the channel 180 includes a saturation indicator 190 that can be a graduated saturation indicator 190 or a color based saturation indicator 190. For example, in an embodiment, the saturation indicator 190 includes graduation marks 192 as shown in FIG. 1. The graduation marks 192 are calibrated to provide an indication of saturation of the sampling zone 120 based on how many of the graduation marks 192 are reached by the oral fluid in the moist form. In another embodiment, the saturation indicator 190 is calibrated to generate a color or range of colors to provide an indication of saturation of the sampling zone 120 in response to a reaction with the saturation level of the sampling zone 120 by oral fluid in the moist form. In a further embodiment, the saturation indicator 190 is calibrated to generate a change from an initial color to a subsequent color in response to the reaction with the saturation level of the sampling zone 120 by oral fluid in the moist form. For example, in an embodiment, the saturation indicator 190 calibrated to generate a color or a change in color is a pH indicator.

[0105] In an embodiment, the dried oral fluid diagnostics apparatus 100 includes one or more sampling guides 195 extending along an exterior periphery of the layer of material 110. The one or more sampling guides 195 are indicative of an insertion end for insertion of the dried oral fluid diagnostics apparatus 100 into the user’s mouth. In an embodiment, at least one of the one or more sampling guides 195 is foldable, for example, along dashed line 197.

[0106] Referring to FIG. 29, in an embodiment, a kit 600 for processing of dried oral fluid includes at least one dried oral fluid diagnostics apparatus 100 as described above. The kit 600 further includes at least one pouch 610. The pouch 610 can, for example without limitation, be a vapor barrier pouch or a sterile pouch, either of which can be resealable, for example, by aAttorney Docket No. 700355-088491WOPT closure on the pouch. The at least one dried oral fluid diagnostics apparatus 100 is configured to be sized to fit within the at least one pouch 610 as indicated by the arrow 620. Further, in an embodiment, the at least one pouch 610 is configured to seal around the at least one dried oral fluid diagnostics apparatus 100 to protect from possible contamination that can occur during shipping via mail or delivery services. The kit 600 optionally further includes a desiccant (not shown) disposed within the pouch 610, and further optionally includes an outer envelope 630 sized and configured to seal around the at least one pouch 610 as indicated by the arrow 640, and suitable for shipping via mail or delivery services.

[0107] In an embodiment, the kit 600 includes a plurality of dried oral fluid diagnostics apparatuses 100 and a plurality of pouches 610, each of the plurality of pouches 610 configured to seal around one of the plurality of dried oral fluid diagnostics apparatuses 100. In this embodiment, the kit 600 optionally further includes a desiccant (not shown) disposed within each pouch 610. The kit 600 optionally further includes the outer envelope 630 that is configured to seal around the plurality of pouches 610, allowing for a plurality of the dried oral fluid diagnostics apparatuses 100 to be shipped in a single shipment.

[0108] Referring to FIG. 30, in an embodiment, an exemplary method 700 of sampling oral fluid in moist form and extracting analytes from the oral fluid in dried form is presented. Starting at step 710, the method 700 begins with contacting the sampling zone 120 of the dried oral fluid diagnostics apparatus 100 with the tongue of a user. At step 720. the method 700 continues with wi eking an oral fluid, for example, saliva, in moist form from the user’s tongue into the sampling zone 120 of the layer of material 110. In an embodiment, the sampling zone 120 can be held in contact with the user’s tongue for as long as is required for the saturation indicator 190 to indicate that a sufficient sample of the oral fluid in the moist form has been sampled for recovery of analytes from the oral fluid in the dry form. In an embodiment, the sampling zone 120 is held in contact with the user’s tongue for about 30 seconds.

[0109] At step 730, the method 700 continues with drying the layer of material 110 such that the sampling zone 120 holds the oral fluid in dry form in at least one storage zone 160 that is a subset of the sampling zone 120. The drying can continue for as long as is needed or to a level of dryness as is needed for short or long term storage or shipping. At step 740, the method 700 continues with separating the at least one storage zone 160 from a remainder of the sampling zone 120 for processing. At step 750, the method continues with processing the at least one storage zone 160 for recovery' of analytes from the oral fluid in dry form.

[0110] Although the disclosed embodiments have been illustrated and described withAttorney Docket No. 700355-088491WOPT respect to one or more implementations, equivalent alterations and modifications will occur or be known to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

[0111] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein, without departing from the spirit or scope of the disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the above described embodiments. Rather, the scope of the disclosure should be defined in accordance with the following claims and their equivalents.

Claims

Attorney Docket No. 700355-088491WOPTCLAIMSWhat is claimed is:

1. A dried oral fluid diagnostics apparatus comprising: a layer of material that is porous and absorbent; a sampling zone positioned at one end of the layer of material, the sampling zone configured for placement in contact with a user's tongue to receive an oral fluid in moist form, the sampling zone having a sampling periphery defining at least in part a receiving portion of the sampling zone, a top surface and a bottom surface defining a thickness of the sampling zone along a cross-sectional direction of the sampling zone. a storage zone configured to receive the oral fluid initially in the moist form, the storage zone subsequently holding the oral fluid stored in dry form to allow processing for recovery7of analytes, and a wicking path in fluid communication with the sampling periphery, the storage zone, and the top and bottom surfaces, the wicking path providing a flow path to the storage zone for the oral fluid in moist form.

2. The apparatus of claim 1. further comprising a channel in fluid communication with the sampling zone, the channel receiving at least some of the oral fluid in moist form, the channel including a saturation indicator.

3. The apparatus of claim 2, wherein the saturation indicator includes graduation marks indicative of saturation of the sampling zone based on how many of the graduation marks are reached by the oral fluid in the moist form.

4. The apparatus of claim 3, wherein the saturation indicator includes a color generated in response to a reaction with the oral fluid in the moist form.

5. The apparatus of claim 4, wherein the saturation indicator includes a change from an initial color to a subsequent color in response to the reaction with the oral fluid in the moist form.Attorney Docket No. 700355-088491WOPT6. The apparatus of claim 1, further comprising one or more sampling guides attached along an exterior periphery of the layer of material, the one or more sampling guides being indicative of an insertion end for insertion into the user’s mouth.

7. The apparatus of claim 6, wherein at least one of the one or more sampling guides is foldable.

8. The apparatus of claim 1, wherein the analytes are selected from a group consisting of pathogens, microorganisms, proteins, enzy mes, antibodies, polysaccharides, hormones, steroids, electrolytes, metabolites, nucleic acids, therapeutics, vitamins, exosomes, and drugs of abuse or their metabolites.

9. The apparatus of claim 8, wherein the pathogens are upper respiratory viruses including SARS-CoV-2, Influenza A, Influenza B, Rhinovirus, or combinations thereof.

10. The apparatus of claim 1, wherein the layer of material has a liquid sampling volume in a range of about 5 microliters per square centimeter (pL / cm2) to about 50 pL / cm211. The apparatus of claim 1. wherein the storage zone includes one or more predetermined regions of the sampling zone.

12. The apparatus of claim 11, wherein the one or more predetermined regions are perforated.

13. The apparatus of claim 12, wherein the one or more predetermined regions each has a conical shape for insertion into a conical tube, a shape for insertion into a microcentrifuge tube, or a shape for insertion into a microwell plate.

14. A kit for processing of dried oral fluid, the kit comprising: at least one dried oral fluid diagnostics apparatus including: a layer of material that is porous and absorbent; a sampling zone configured for placement in contact with a user’s tongue to receive an oral fluid in moist form, the sampling zone havingAttorney Docket No. 700355-088491WOPT a sampling periphery defining at least in part a receiving portion of the sampling zone, a storage zone configured to receive the oral fluid initially in the moist form, the storage zone subsequently holding the oral fluid stored in dry form to allow processing for recovery of analytes, and a wicking path in fluid communication with the sampling periphery and with surfaces of the sampling zone, the wicking path providing a flow path to the storage zone for the oral fluid in moist form; and at least one pouch configured to seal around the at least one dried oral fluid diagnostics apparatus.

15. The kit of claim 14, wherein the at least one dried oral fluid diagnostics apparatus includes a plurality of dried oral fluid diagnostics apparatuses, wherein the at least one pouch includes a plurality of pouches, each of the plurality of pouches configured to seal around one of the plurality of dried oral fluid diagnostics apparatuses.

16. A method of sampling dried oral fluid for molecular diagnostics, the method comprising: contacting a sampling zone of a layer of porous and absorbent material to a user’s tongue; wicking an oral fluid, received in moist form from the user’s tongue, into the sampling zone of the layer of material; drying the layer of material such that the sampling zone holds the oral fluid in dry form in at least one storage zone that is a subset of the sampling zone; separating the at least one storage zone from a remainder of the sampling zone for processing; and processing the at least one storage zone for recovery of analytes from the oral fluid in dry form.Attorney Docket No. 700355-088491WOPT17. The method of claim 16, wherein the sampling zone is in fluid communication with a saturation indicator for indicating that a sufficient sample of the oral fluid in the moist form has been sampled for recovery of the analytes from the oral fluid in the dry form.

18. The method of claim 16, wherein the at least one storage zone is perforated.

19. The method of claim 16, wherein the analytes are selected from a group consisting of pathogens, microorganisms, proteins, enzymes, antibodies, polysaccharides, hormones, steroids, electrolytes, metabolites, nucleic acids, therapeutics, vitamins, exosomes, and drugs of abuse or their metabolites.

20. The method of claim 19, wherein the pathogens are upper respiratory viruses including SARS-CoV-2, Influenza A, Influenza B, Rhinovirus, or combinations thereof.

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