Methods and systems for detection of biohazard signatures in complex clinical and environmental samples
Inventors
Bhartia, Rohit • Reid, Michael R. • Hug, William F. • Reid, Ray D.
Assignees
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Publication Number
US-11448598-B1
Publication Date
2022-09-20
Expiration Date
Abstract
Methods, apparatus, and systems provide improved identification of selected biohazard and/or biohazard signatures from complex in vivo or in vitro samples and include deep UV native fluorescence spectroscopic analysis for multiple locations of a sample wherein classification results for individual locations are combined and spatially correlated to provide a positive or negative conclusion of biohazard signature presence (e.g., for signatures for viruses, bacteria, and diseases including SARS-CoV-2 and its variants and COVID-19 and its variants). Improvements include one or more of reduced sample processing time (minutes to fractions of a minute), reduced sampling cost (dollars to fractions of a dollar), high conclusion reliability (rivaling real time RT-PCR). Some embodiments may incorporate a stage or scanning mirror system to provide movement of a sample relative to an excitation exposure location. Some embodiments may incorporate Raman or phosphorescence spectroscopic analysis as well as imaging systems.
Core Innovation
The invention provides methods for identifying the presence of at least one SARS-CoV2 or COVID-19 biohazard signature in a sample using deep UV excitation radiation and native fluorescence emission radiation. A plurality of locations on the sample is exposed and read, while the sample is moved relative to an exposure/emission location to enable reading of subsequent locations. The excitation wavelength is selected from below 300 nm, below 275 nm, or below 250 nm, and the emission is read in a plurality of wavelength bands.
The analysis is performed in levels starting with a location-by-location signal threshold requirement. Emission readings meeting signal threshold requirements exceed background noise by a factor selected from at least 3, at least 5, or at least 7. For each location meeting the threshold, a second-level membership analysis assigns positive or negative class membership based on whether emission radiation is more closely aligned to biohazard signature presence or to biohazard signature non-presence, including use of a KNN algorithm with a selected K value.
A further analysis uses spatial relationships between positive membership locations to conclude a sample is positive. The sample is determined to comprise a number N of neighboring locations having positive membership with no more than M intervening locations having negative membership, with selectable ranges for N and percentage limits for M. Another embodiment distinguishes useful signal groups from unusable signal groups by comparing signal strength against corresponding background signal by a predefined amount and forms a biohazard indicative conclusion based on matched useful groups and relative spacings between sample portions.
Claims Coverage
The independent claims collectively specify a multi-level spectroscopy-and-spatial-logic analysis workflow. The inventive features center on deep-UV native fluorescence sensing with multi-location scanning, thresholded signal filtering, location-wise membership classification, and spatial grouping rules used to form a sample-level conclusion.
Deep-uv excitation and multi-location native fluorescence emission acquisition
Exposing a sample to deep UV excitation radiation and reading resulting native fluorescence emission radiation in a plurality of wavelength bands from each of a plurality of locations on the sample, with the sample moved relative to an exposure/emission location for reading subsequent locations.
Background-threshold first-level location analysis
Performing a first level analysis on a location-by-location basis to determine which locations provide emission radiation that meets signal threshold requirements by exceeding background noise by a selected factor.
Knn-based positive and negative location membership assignment
For each emission radiation location meeting signal threshold requirements, performing a second level membership analysis that assigns class membership to individual locations indicative of potential relevance to a SARS-CoV2 or COVID-19 biohazard signature presence, including positive membership and negative membership based on closeness to biohazard signature presence or non-presence.
Spatial neighbor-and-intervening-location rule for sample positivity
Performing at least one additional level of analysis involving spatial relationships between positive membership locations, determining sample positivity when the sample comprises a number N of neighboring locations having positive membership with no more than M intervening locations having negative membership.
Useful-versus-unusable signal grouping by background threshold
Detecting groups of emission signals associated with different sample portions and distinguishing useful signal groups from unusable signal groups where useful groups contain at least one signal having strength greater than a corresponding background signal by a predefined amount.
Comparison to predetermined biohazard and non-biohazard indicative signal information
Providing predetermined biohazard indicative signal information and predetermined non-biohazard indicative signal information related to the selected biohazard signature, and producing a biohazard indicative status for each useful signal group based at least in part on comparison of emission signal data for that group with the predetermined indicative signal information.
Sample-level conclusion based on matched useful groups and relative spacing
Forming a biohazard indicative conclusion based at least in part on combination of biohazard indicative signal information for a plurality of useful signal groups and relative spacings between the portions of the sample associated with those useful signal groups.
Minimum threshold spatial grouping criterion from positive membership locations
Defining at least one minimum threshold spatial grouping criterion for locations with positive membership that is necessary to conclude that the sample is positive for presence of the biohazard signature.
Across the independent claims, the coverage centers on deep UV excitation and native fluorescence emission readout across multiple wavelength bands and sample locations, followed by background-based threshold filtering, location-level membership classification, and sample-level determination using spatial relationships among positive-member locations or useful signal groups.
Stated Advantages
Provides methods for identifying presence of at least one of a SARS-CoV2 or a COVID-19 biohazard signature in a sample.
Performs location-by-location analysis using signal threshold requirements relative to background noise.
Assigns positive and negative class membership for locations based on closeness to biohazard signature presence versus biohazard signature non-presence.
Determines sample positivity using spatial relationships between positive membership locations and intervening negative membership locations.
Distinguishes useful signal groups from unusable signal groups using a predefined background-strength threshold.
Produces a biohazard indicative status for each useful signal group based on comparison to predetermined biohazard indicative and non-biohazard indicative signal information.
Forms a biohazard indicative conclusion based on matched useful signal groups together with relative spacings between associated sample portions.
Documented Applications
Pilot study setup and results for SARS-CoV-2/COVID-19 using deep-UV autofluorescence spatial scanning with KNN classification, spatial filtering, and comparison to RT-PCR reporting sensitivity/specificity.
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