WO2018208783A2

WO2018208783A2 - Roving reference diagnostic system

Info

Publication number: WO2018208783A2
Application number: PCT/US2018/031609
Authority: WO
Inventors: Jr. Robert M. Lloyd
Original assignee: Rtt Molecular Dx Usa, Inc.
Priority date: 2017-05-08
Filing date: 2018-05-08
Publication date: 2018-11-15
Also published as: WO2018208783A3

Abstract

The present disclosure relates generally to diagnostic systems and methods for analyzing and presenting data for use in determining an individual's propensity to respond effectively to a treatment regimen.

Description

ROVING REFERENCE DIAGNOSTIC SYSTEM CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

62/503,069, filed May 8, 2017. The entire contents of which is incorporated by reference herewith.

FIELD OF THE INVENTION

[0002] The present disclosure relates generally to diagnostic systems and methods for analyzing and presenting data for use in determining an individual's propensity to respond effectively to a treatment regimen. BACKGROUND OF THE INVENTION

[0003] Hepatitis C virus (HCV) is associated with substantial human suffering and important socioeconomic consequences (e.g. healthcare costs). HCV is widely transmitted across the world. The World Health Organization estimates that 3.3% (200 million) of the world population is infected with HCV, and the Centers for Disease Control and Prevention reports more than 3.5 million individual HCV infections in the United States. HCV is a highly infectious, blood-borne agent that causes liver related ailments, and a significant cause of morbidity and mortality in the human population. Left untreated, it can lead to progressive liver injury, cirrhosis, hepatocellular carcinoma, and the need for liver transplantation.

[0004] HCV is an enveloped virus containing a positive sense, linear, single-stranded RNA genome of approximately 9,600 nucleotides (9.6 kb). The single open reading frame of the HCV genome encodes a HCV polyprotein precursor that is co- and post- translationally processed by cellular and viral proteases to yield 10 mature protein products (Core, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Figure 1).

[0005] Direct acting antivirals (DAAs) against HCV have revolutionized the treatment and management of HCV. DAAs typically interrupt HCV replication by targeting specific HCV proteins, such as the NS5A protein, NS5B polymerase, and NS3/4A protease. DAAs that inhibit NS3/4A (simeprevir, paritaprevir, grazoprevir), NS5A (daclatasvir, ledipasvir, ombitasvir, elbasvir), and NS5B (sofosbuvir, dasabuvir) have been approved for treatment of HCV infection and are available in fixed-dose combinations. Additional DAAs in development may expand such options.

[0006] Although the antiviral potency of most DAAs is impressive, the rapid emergence of drug resistance in HCV must be considered as a major threat. The high mutation rate of the HCV genome, combined with selective pressure from ongoing therapy, can lead to selection of HCV variants that are resistant to DAAs (e.g. administration of a DAA to an infected individual can result in the rapid emergence of a mutant HCV having reduced susceptibility to the DAA). The efficacy of DAAs can be genotype dependent, mainly due to the presence of natural polymorphisms associated with resistance to some of these compounds in distinct viral variants (i.e. resistance associated mutations (RAMs)). Several exemplary DAA's whose efficacy can be genotype dependent include, for example, ZEPATIER, a fixed-dose combination product containing elbasvir, a HCV NS5A inhibitor, and grazoprevir, an HCV NS3/4A protease inhibitor, and DAKLINZA, a HCV NS5A inhibitor.

[0007] Given the extreme genetic variability of HCV, testing for HCV genotype and the presence of polymorphisms is recommended at baseline for patients prior to initiation of treatment in order to determine the best treatment regimen for an individual patient. Likewise, it can also be important for practitioners to monitor the HCV genotype and any resistance associated mutations in a patient during the course of treatment in order to evaluate whether any changes to the treatment regimen are needed in view of viral mutations that confer drug resistance. DAAs have side effects, require effective drug treatment regimens by health professionals, are extremely expensive, and are not equally available worldwide, and thus knowing whether a patient is likely to respond to a particular DAA is critically important.

[0008] Genotyping and assessment of the viral load in HCV patients is important for designing therapeutic strategies because the efficacy of available treatments (e.g. anti-viral drugs) for a particular viral strain are genotype dependent. The present commercial systems and methods used to determine patient- specific drug resistance profiles do not provide clinicians with adequate information to treat HCV. Therefore, new systems and methods for use in identifying mutations or polymorphisms associated with drug treatment efficacy or resistance, and new methods and systems for recommending patient-specific HCV treatment regiments, and treatment for other viral or disease conditions marked by genetic mutations, are needed.

SUMMARY OF THE INVENTION

[0009] The present disclosure provides a method for conducting a genotype assay comprising; (1) comparing a nucleotide sequence from a patient sample against each sequence in a phylogenic tree consisting of nucleotide sequences for a disorder or disease of interest; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number identifying a type and subtype of the patient's sample corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0010] The present disclosure provides a method for conducting a genotype assay comprising; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for HCV; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0011] The present disclosure provides a method for conducting a genotype assay comprising; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a viral infection, a bacterial infection, oncogenes, or tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0012] The present disclosure provides a method for conducting a genotype assay comprising: (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from viral infections (such as HCV), bacterial infections, oncogenes, or tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0013] The present disclosure provides a method for conducting a genotype assay comprising: (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from viral infections (such as HCV), bacterial infections, oncogenes, or tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, and wherein the merged consensus sequence comprises the sequence for one or more regions of the genome of the disorder or disease of interest; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0014] The present disclosure provides a method for conducting a genotype assay comprising: (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from viral infections (such as HCV), bacterial infections, oncogenes, or tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient' s sequence has the closest homology to; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence that the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, and wherein the merged consensus sequence comprises the sequence for one or more sub-regions comprising the positions where drug resistance mutations and drug resistance associated mutations occur within the genome of the disorder or disease of interest; and (6) providing a drug resistance report for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0015] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0016] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for HCV; (2) determining which sequence of the phylogenic tree has a closest homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0017] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from a group comprising; viral infections; bacterial infections; oncogenes, and tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference; and (6) providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0018] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from a group comprising; viral infections; bacterial infections; oncogenes, and tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; and (6) providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

[0019] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from a group comprising; viral infections; bacterial infections; oncogenes, and tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, and wherein the merged consensus sequence comprises the sequence for one or more regions of the genome of the disorder or disease of interest; and (6) providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient. [0020] The present disclosure provides a product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in; (1) comparing a patient's sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest, wherein the disorder or disease of interest is selected from a group comprising; viral infections; bacterial infections; oncogenes, and tumorigenic cells; (2) determining which sequence of the phylogenic tree has a closest or best fit homology to the patient's sequence; (3) assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology; (4) assigning the patient a cluster identifier corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology; (5) comparing the patient's sequence to a roving reference, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, and wherein the merged consensus sequence comprises the sequence for one or more sub- regions comprising the positions where drug resistance mutations and drug resistance associated mutations occur in the genome of the disorder or disease of interes; and (6) providing a drug resistance report such as by a print-out or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient and information about drug resistance mutations in the patient's sequence determined by comparison to the roving reference merged consensus sequence of the cluster.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Referring now to the drawings, which are meant to be exemplary and not limiting, and wherein like elements are numbered alike. The detailed description is set forth with reference to the accompanying drawings illustrating examples of the disclosure, in which use of the same reference numerals indicates similar or identical items. Certain embodiments of the present disclosure may include elements, components, and/or configurations other than those illustrated in the drawings, and some of the elements, components, and/or configurations illustrated in the drawings may not be present in certain embodiments.

[0022] Figure 1 shows a schematic of the HCV genome. [0023] Figure 2 shows a schematic containing a portion of a HCV phylogenic tree subdivided into clusters. Each cluster is assigned a merged consensus sequence.

[0024] Figure 3 shows a schematic of a sample homology search window.

[0025] Figure 4 shows a schematic after the overall homology matching of a patient's HCV genotype against each sequence in a HCV phylogenic tree and assignment of identifiers (actual Accesssion numbers blurred).

[0026] Figure 5 shows an exemplary output report, wherein the right side of Figure 5 shows exemplary types of information that could be reported and the left side merely displays exemplary information as a visual example of an output report.

[0027] Figure 6 shows a typing report based on prior diagnostic systems.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and systems described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety.

[0029] "A," "an," and "the" include plural referents, unless the context clearly indicates otherwise. For example, "a nucleic acid" as used herein is understood to represent one or more nucleic acids. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0030] "About" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a deviation of ±10% and preferably ±5%.

[0031] "Comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term "comprising," those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language "consisting essentially of and/or "consisting of." [0032] "Amplicon" or "amplification product" refers to the nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a target nucleic acid or a region thereof. Amplicons can be double stranded or single stranded and can include DNA, RNA or both. Methods for generating amplicons are known to those skilled in the art.

[0033] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid or its complement, or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Amplification, in the context of fragments, refers to production of an amplified nucleic acid that contains less than the complete target nucleic acid or its complement, e.g., produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include, for example, replicase- mediated amplification, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from RNA in a sample using reverse transcription (RT)-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification. During amplification, the amplified products can be labeled using, for example, labeled primers or by incorporating labeled nucleotides.

[0034] "Primer" refers to an enzymatically extendable oligonucleotide, generally with a defined sequence that is designed to hybridize in an antiparallel manner with a complementary, primer- specific portion of a target nucleic acid. A primer can initiate the polymerization of nucleotides in a template-dependent manner to yield a nucleic acid that is complementary to the target nucleic acid when placed under suitable nucleic acid synthesis conditions (e.g. a primer annealed to a target can be extended in the presence of nucleotides and a DNA/RNA polymerase at a suitable temperature and pH). Suitable reaction conditions and reagents are known to those of ordinary skill in the art. A primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. The primer generally is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent (e.g. polymerase). Specific length and sequence will be dependent on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. Preferably, the primer is about 5-100 nucleotides. Thus, a primer can be, e.g., 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides in length. A primer does not need to have 100% complementarity with its template for primer elongation to occur; primers with less than 100% complementarity can be sufficient for hybridization and polymerase elongation to occur. A primer can be labeled if desired. The label used on a primer can be any suitable label, and can be detected by, for example, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection means. A labeled primer therefore refers to an oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow selective detection of the target sequence.

[0035] "Sample preparation" refers to any steps or methods that prepare a sample for subsequent amplification and/or detection of target nucleic acids present in the sample. Sample preparation may include any known method of concentrating components, such as nucleic acids, from a larger sample volume. Sample preparation may include physical disruption and/or chemical lysis of cellular components to release intracellular components into a substantially aqueous or organic phase and removal of debris, such as by using filtration, centrifugation or adsorption. Sample preparation may include use of a nucleic acid oligonucleotide that selectively or non-specifically captures a target nucleic acid and separates it from other sample components.

[0036] "Separating" or "purifying" means that one or more components of a sample are removed or separated from other sample components.

[0037] "Sequence" refers to the order and identity of nucleotides in a nucleic acid. A sequence is typically read in the 5' to 3' direction. The terms "identical" or percent "identity" in the context of two or more nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403- 410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266: 131-141, Altschul et al. (1997) ""Gapped BLAST and PSI- BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Res. 7:649-656, which are each incorporated by reference. Many other optimal alignment algorithms are also known in the art and are optionally utilized to determine percent sequence identity.

[0038] "Sample" or "specimen" refers to any composition in which a target nucleic acid may exist as part of a mixture of components. Samples include any tissue or material derived from a living or dead organism which may contain a target nucleic acid, including, e.g., cells, tissues, lysates made from cells or tissues, sputum, peripheral blood, plasma, serum, cervical swab samples, biopsy tissues (e.g., lymph nodes), respiratory tissue or exudates, gastrointestinal tissue, urine, feces, semen, or other fluids or materials. A sample may be treated to physically disrupt tissue and/or cell structure to release intracellular components into a solution which may contain enzymes, buffers, salts, detergents and other compounds, such as are used to prepare a sample for analysis by using standard methods.

[0039] "Subject" or "patient" refers to a human or other mammal, including, e.g., dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, and camel. The subject can be a normal subject or the subject can be in need of a treatment (e.g. an anti-viral treatment). Subject and patient do not denote a particular age or sex and thus include adults, infants, and newborns.

[0040] As used herein, "preventative" treatment is meant to indicate a postponement of development of a disease, a symptom of a disease, or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurrence of a disease or symptom. "Curative" treatment includes reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition. [0041] "Treating" or "treatment" or "alleviation" refers to both preventative and curative therapeutic treatment wherein the object is to slow down (lessen) if not cure the targeted pathologic condition or disorder or prevent initial occurrence or recurrence of the condition. A subject is successfully "treated" if, after receiving a therapeutic amount of a therapeutic agent, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the particular disease. Reduction of the signs or symptoms of a disease may also be felt by the patient. A patient is also considered treated if the patient experiences stable disease. In some embodiments, treatment with a therapeutic agent is effective to result in the patients being disease-free 3 months after treatment, preferably 6 months, more preferably one year, even more preferably 2 or more years post treatment. These parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician of appropriate skill in the art.

[0042] The term "combination" refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound and a combination partner (e.g., another drug as explained below, also referred to as "therapeutic agent" or "co-agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non- fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0043] In embodiments, the present disclosure generally provides for typing of a sample from a patient and/or recommending a course of therapy based on the genotypic typing. Genotyping is a valuable clinical tool in determining an appropriate course of therapy.

[0044] In embodiments, the present disclosure generally provides for typing of an HCV isolate in a sample from a patient and/or recommending a course of therapy based on the identified genotype. Conventional HCV typing is ill-suited for use as a DAA response predictor (i.e. efficacy prediction). Amino acids in a viral protein, such as NS5A dictate response to DAA not the type or subtype of HCCV. Typing is not particularly useful with respect to being a surrogate for DAA response. Intensification cannot be applied without a drug resistance test, thus the warning from the FDA in the DAA product insert.

[0045] In embodiments, the present disclosure provides a diagnostic system for conditions and diseases wherein genetic variability and drift and polymorphic drift have created a diverse population of genetically distinct sequences originating from the same basic organism, wherein such variability can effect drug resistance and treatment regimen efficacy, by increasing and varying the number of drug resistance mutations (DRM) or resistance associated mutations (RAM). Non-limiting examples include, viral infections, bacterial infections, genetic makeup of oncogenes, and tumorigenic cells.

[0046] In embodiments, the present disclosure provides a diagnostic system using a roving sequence reference, wherein the roving reference is generated from a merged consensus sequence from individual sequences within a genetic cluster. The roving reference can be used for any genetic material, but is beneficially used for genetic material showing a high degree of variability, for example the HCV virus, which generally consists of Types 1-6, and wherein each Type comprises several subtypes. Such variability in the genetic material results in there being no plausible standard for generating a fixed reference useable for identifying specific RAM and/or DRM.

[0047] In embodiments, the present disclosure provides a HCV diagnostic system and methods of therapeutic tgreatment based thereon, by administration of an effective amount of a DAA based on the diagnosis..

[0048] In embodiments, the HCV diagnostic system can include a HCV phylogenic tree. In embodiments, the HCV phylogenic tree is a database of different HCV genotypes, each genotype having its own accession number. In embodiments, the HCV phylogenic tree is subdivided into clusters. Each cluster represents two or more HCV genotypes (two or more accession numbers) that are the closest relatives to one another.

[0049] In embodiments, the HCV diagnostic system can include a kit of reagents for carrying out a HCV diagnostic test. In embodiments, the kit can include reagents for separating, isolating, purifying, amplifying, detecting, and/or quantifying viral nucleic acids. In embodiments, the kit can include reagents for separating, isolating, purifying, amplifying, detecting, and/or quantifying HCV sequences. The reagents of the kits are not limited to any particular technique or any particular modification thereof. The reagents may be supplied in a solid (e.g., lyophilized) or liquid form.

[0050] For example, a kit can include reagents such as: primers; buffers or reagents for making the same (e.g. wash buffers, hybridization buffers, etc.); enzymes having reverse transcriptase and/or polymerase activity; enzyme cof actors such as magnesium or manganese; salts; nicotinamide adenine dinuclease (NAD); and deoxynucleoside triphosphates (dNTPs) such as, for example, deoxyadenosine triphosphate; deoxy guanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate, biotinylated dNTPs. In embodiments, the kits can include primers used in PCR methods for the amplification and detection of specific HCV alleles, such as but not limited to NS5A alleles.

[0051] In embodiments, the HCV diagnostic system can include a computer-implemented method. The computer-implemented method can be configured to conduct a homology search between a patient's HCV sequence and each sequence in the HCV phylogenic tree to determine the sequence or sequences in the HCV phylogenic tree having the closest homology to the patient's HCV sequence. In embodiments, the computer-implemented method can determine which sequence or sequences (e.g. top 1, 2, 3, 4, 5, 10, or 15 sequences) of the HCV phylogenic tree have the closest homology to the patient's HCV sequence. In embodiments, the computer-implemented method can assign a patient the accession number corresponding to the HCV phylogenic tree sequence that the patient's HCV sequence has the closest homology to (e.g. best fit homology). In embodiments, the computer-implemented method can assign a patient a cluster identifier (e.g. a roving reference) corresponding to the cluster identifier associated with the HCV phylogenic tree sequence that the patient's HCV sequence has the closest homology to (e.g. best fit homology).

[0052] Although embodiments of the invention are not limited in this regard, discussions utilizing terms such as, for example, "genotyping," "comparing," "determining," "assigning accession number", "assigning cluster identifier", "determining and providing a suggested treatment regimen", or the like, may refer to operation(s) and/or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulate and/or transform data represented as physical (e.g., electronic) quantities within the computer's registers and/or memories into other data similarly represented as physical quantities within the computer's registers and/or memories or other information storage medium that may store instructions to perform operations and/or processes.

[0053] Some embodiments of the present invention may further provide a computer readable medium encoded with computer executable instructions, which when accessed, cause a machine to perform operations comprising determining and providing a suggested treatment regimen to a specific patient.

[0054] For example, in embodiments, the computer-implemented method used in combination with a central processing unit can determine and provide a suggested treatment regimen for the patient based on the accession number assigned to the patient and/or the cluster identifier assigned to the patient. For example, the assigned accession number may have clinical treatment data associated therewith and may be provided or taken into consideration in a suggested treatment report for use by a clinician (e.g. provided in the form of a printout, electronic display, email, or the like). As another example, the assigned accession number may not have clinical treatment data associated therewith but a different accession number within the same cluster identifier assigned to the patient may have clinical treatment data associated therewith. In such a case, the computer-implemented method may provide or take into consideration the treatment data of the other accession numbers in the identified cluster in a suggested treatment report for use by a clinician. It will be appreciated by those of ordinary skill that any combination of treatment information from an assigned accession number or any accession number within an assigned cluster identifier can be provided or taken into consideration in a suggested treatment report for use by a clinician. Thus, for example, treatment data including treatment data for the assigned accession number and/or one or more unassigned accession numbers within the same cluster identifier may be provided or taken into consideration in a suggested treatment report for use by a clinician. In embodiments, the treatment data may also include standard treatment data (e.g. default treatment regimen based on HCV genotype/subtype)

[0055] By applying specific rules to determine which accession number, corresponding to the sequence in the HCV phylogenic tree, to which the subject's specimen has the closest homology, the diagnostic system can further determine the cluster and its corresponding cluster identifier that conventional diagnostic systems do not. In particular and as will be described in greater detail below, the specific rules facilitate determinations as to whether any drug resistances are indicated and whether any treatment regimens have shown a better efficacy for the identified subject genotype. The treatment regimens can include single drug treatments as well as combination drugs. The indicated drug resistances can be based at least in part on the closest identified HCV sequence in the phylogenic tree, as well as the other sequences contained in the identified cluster and/or the roving reference for the identified cluster. The roving reference is a merged consensus sequence for the identified cluster comprising DRM, RAM, and polymorphic changes. In embodiments the roving referenced is based on the entire HCV genome. In other embodiments the roving references is based on one or more regions or sub-regions contained within the HCV genome.

[0056] Further, the specific rules facilitate the identification and removal of duplicative and redundant data that is typically found among, for example, the various accession numbers within an identified cluster and drug resistance and treatment regimen efficacy. The duplicative and/or redundant data can include, for example, multiple accession numbers having the same DRM and/or RAM, same sub-regions free of any RAM and/or DRM, drug resistance interpretations, and the same treatment guidance. Such duplicative or redundant data can oftentimes reduce the performance of searches and/or analyses to identify, for example, treatment efficacy or drug resistances when multiple resistance associated mutations are present.

[0057] As a result, at least one technical solution of certain embodiments of the disclosure is that a diagnostic system can generate groupings of data (e.g., drug resistance) per subject, and the system can output various reports or graphical user interface (GUI) presentations, for example, with tables or lists indicative of relationships between a subjects genotype (e.g. HCV genotype) and/or subtype with a drug resistance and/or treatment guidance (Figure 5). The various reports or GUI presentations can include one or more representations of drug resistance, where the representation displays resistance analytics and/or treatment trends. Such analyses are based at least in part on the combined data set (generated based at least in part on the specific rules) that enables the system to determine whether drug resistances are indicated and whether treatment guidance exists. Further, another technical solution of certain embodiments of the disclosure is that the combined data set can be used by the system to access and present granular details about the indicated and/or the treatment guidance by way of the report or GUI presentations. In any instance, certain technical solutions of embodiments of the disclosure can improve performance and processing speed.

[0058] Genotyping is typically done by translating sequenced reads into protein coding sequences or protein sequences and running homology searches against publicly available databases. However, such methods have become inefficient due to the large number of reference sequences contained within such databases, thus limiting their value as a diagnostic system. To address this problem, the present disclosure provides a method for using a limited database, wherein the limited database only contains genes relevant to the disease or condition being diagnosed. In the presently disclosed method, using a limited database only containing sequences relevant to a particular disease or disorder. In some embodiments the limited database contains the phylogenic tree relevant to a particular disease or disorder. The sequences are clustered, and a merged consensus sequence for each cluster, containing all DRM and RAM, is created (i.e. roving reference). The database can further contain, sequence accession numbers, resistance interpretation for each resistance associated mutation, and known treatment efficacy and guidance.

[0059] In exemplary embodiments, the disclosure provides methods for using a diagnostic system. In embodiments, the methods can include one or more of the following steps: (1) providing an specimen from a patient, (2) amplifying nucleic acids from the specimen, (3) genotyping of the patient's sample, (4) comparing the patient's genotype against each sequence in a phylogenic tree for the disease or disorder of interest; (5) determining which sequence or sequences (e.g. top 1, 2, 3, 4, 5, 10, 15, or more sequences) of the phylogenic tree have the closest homology to the patient's sequence; (6) assigning the patient an accession number corresponding to the phylogenic tree sequence that the patient's sequence has the closest homology to (e.g. best fit homologywhich includes identification of the type and subtype of agent causing the disease or disorder of interest); (7) assigning the patient a cluster identifier (e.g. a roving reference) corresponding to the cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology (e.g. best fit homology); and (8) determining and providing a suggested treatment regimen for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier (i.e. roving reference) assigned to the patient.

[0060] In further exemplary embodiments, the disclosure provides methods for using a HCV diagnostic system. In embodiments, the methods can include one or more of the following steps: (1) obtaining or providing an HCV specimen from a patient, (2) amplifying HCV nucleic acids from the specimen, (3) detecting a genotype of the patient's HCV, (4) comparing the patient's HCV genotype against each sequence in a HCV phylogenic tree; (5) determining which sequence or sequences (e.g. top 1, 2, 3, 4, 5, 10, 15, or more sequences) of the HCV phylogenic tree have the closest homology to the patient's HCV sequence; (6) assigning the patient an accession number corresponding to the HCV phylogenic tree sequence to which the patient's HCV sequence has the closest homology (e.g. best fit homology); (7) assigning the patient a cluster identifier (e.g. a roving reference) corresponding to the cluster identifier associated with the HCV phylogenic tree sequence to which the patient' s HCV sequence has the closest homology (e.g. best fit homology); and (8) determining and providing a suggested treatment regimen for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier (i.e. roving reference) assigned to the patient.

[0061] In further embodiments, the invention also provides for the treatment of the patient by administering a direct acting antiviral or other drug effective, i.e., without patient resistance, to treat the HCV mutations identified.

[0062] In an example overall methodology, a computer-executable application is configured to aggregate sequence data associated an accession number. The sequence data can include: (1) drug resistance associated mutations; (2) resistance interpretation information, for example, partial resistance, association between other known resistances, region specific resistances, etc.; and (3) treatment guidance, for example, improved treatment efficacy with combination treatment, known substitute drugs, known relationships between drugs, etc. In non-limiting examples for HCV, the sequence data can include; resistance associated mutations (e.g. a K24 E/R/N, H54R, E62D, or Y93H mutation in HCV NS5A is associated with resistance to NS5A inhibitors); and treatment regimen efficacies associated with specific mutations.

[0063] The application can be configured to obtain or be granted access to the sources of sequence data by having administrative access and/or login access to multiple databases. For example, instead of the application having access to sequence data from one or more particular databases associated with the specific disease or disorder, the application can be manually updated.

[0064] Based at least in part on the aggregated sequence data associated with the specific disease or disorder, the sequences may be clustered by relatedness and at least one merged consensus sequence (cluster identifier/roving reference) can be generated to include and map out drug resistance associated mutations (RAM) and drug resistance mutations (DRM). The merged consensus sequence may be based on: the entire genome of the specific disease or disorder; select regions of the genome of the specific disease or disorder; and/or sub-regions of the genome of the specific disease or disorder. The homology of a subject's sequence with the roving reference can be analyzed by application to determine or identify well-known mutations for specific drug resistances or trends between multiple mutations and drug resistance. The at least one roving reference can be generated by selecting and applying well-known filtering rules to the aggregated sequence data to, for example, determine that only specific regions or sub-regions are associated with drug resistance and/or treatment efficacy. After determination of which roving reference should be compared to a subject, the application can generate an appropriate presentation outlining resistance interpretations and treatment guidance. A variety of available genomic software can be used to compare sequence homologiescreaqte phylogenic trees, define clusters of related nucleotide sequences with a phylogenic tree, to created a merged consensus sequence of the cluster, and to compare the consensus sequence to a sample of intrest, such as DNASTAR (using default settings).

[0065] A presentation application can further be configured to cause an output of a report or GUI presentations at one or more client computing devices. For example, a report or a GUI presentation can include tables or lists indicative of relationships between a subject's genotype (e.g. HCV genotype) and/or subtype with a drug resistance and/or treatment guidance. The various reports or GUI presentations can include one or more representations of drug resistance mutations, where the representation displays resistance analytics and/or treatment trends.

[0066] In embodiments, HCV sequences are extracted from a database or library comprising characterized and accessioned gene specific segments, including partial gene sequences for drug resistance mutations (DRM) or resistance associated mutations (RAM). The extracted sequences are subjected to phylogenetic and clustering analysis. Each identified cluster is assigned a cluster identification, and for each cluster a merged consensus sequence is generated.

[0067] It is to be understood that the merged consensus sequence may be for the entire genome of interest, preferably only for a region of the genome, more preferably only for a sub-region containing DRM and RAM sites. For example, a merged consensus sequence of the entire HCV genome would be ~ 9600 nucleotides long, whereas a merged consensus sequence for a single region, such as the NS5A encoding region would be ~ NS5A -1200 nucleotides long, and a merged consensus sequence of a sub-region comprising resistance associated mutations would be ~ 350 nucleotides long. The regional or sub-regional merged consensus sequences can be used as roving references to identify DRM and RAM, which upon identification can be used to propose a treatment regimen.

Examples

[0068] Characterized and accessioned gene sequences for the HCV genome here extracted from NIH's genetic sequence database, GENBANK, and subjected to best fit and homology comparisons, clustering and phylogenic analysis using DNASTAR (from DNASTAR, Inc., Madison, WI (default settings)). Each extracted sequence has an accession number, wherein the accession number may have clinical outcome data associated therewith, such as but not limited to, specific drug resistance mutations (DRM) or drug resistance associated mutations (RAM). The degree of relatedness of sequences defining a cluster of more than one genetic sequence can be selected by default settings in commonly available bioinformatics software, such as DNASTAR, or adjusted as desired by those skilled in the art, in order to create the merged consensus sequence. Within the created library, when available, each accession number is keyed to specific mutations, which in turn is keyed to specific DRM and RAM, and available clinical outcome data. Each identified cluster in the generated phylogenic tree is assigned a cluster identifier and a merged consensus sequence (i.e. roving reference) for the cluster is generated (Figure 2), wherein the merged consensus sequence maps out and highlights drug resistance mutations (DRM) and drug resistance associated mutations (RAM).

[0069] After providing an HCV specimen from a patient, amplifying HCV nucleic acids from the specimen, and detecting genotype of the patient's HCV, a homology search of a patient's genotype is run against the created HCV library to identify the sequences with the highest homology (Figure 3) using DNASTAR.

[0070] Once identified, the accession number associated with the highest homology is assigned to the patient's genotype. In addition, the cluster identifier and the roving reference for the cluster wherein the identified accession number is found is also assigned to the patient's HCV genotype.

[0071] One benefit to using the roving reference for identifying DRM and RAM present in a patient's HCV genotype is that the patient's genotype needs only be compared to a single sequence in order to provide a complete drug resistance landscape for the patient's genotype, wherein information of existing drug resistance mutation and drug resistance associated mutations found within the identified cluster can be extracted.

[0072] Figure 4 shows a schematic after homology matching a patient's HCV genotype against each sequence in a HCV phylogenic tree and the subsequent assigning of the accession number identifying the type and subtype of HCV corresponding to the HCV phylogenic tree sequence to which the patient' s HCV sequence has the closest homology (e.g. best fit homology), and the assigning of a cluster identifier and estabslihing a roving reference merged consensus sequence of the regions of interest from the membes of the cluster using DNASTAR.

[0073] The patient's genotype is compared to the roving reference merged consensus sequence for the identified cluster to determine the presence or absence of DRM and RAM (Figure 5) using DNASTAR. Based on identified DRM and RAM a suggested treatment regimen for the patient based on the known correlations of mutations identified with drug resistance can be provided.

[0074] Figure 6 shows a typing report based on prior diagnostic systems, outlining the extent and type of information that a physician would review. Available DAA products for treatment of various HCV genotypes list genotype lb as treatable, however, this genotype displays some drug resistance as documented in the manuscript matching the accession number. Thus, treating the patient matching product requirements for DAA treatment would not likely be successful. More particularly, this patient would likely go on treatment without intensification and would, therefore, have a higher risk of failure on therapy, i.e. drug failure and no complete viral clearance and the emergence of drug resistant variants. Only by looking at the RAM codon array for this isolate, as done by the disclosed method, could this be avoided.

[0075] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method for conducting a genotype assay comprising:

comparing a nucleotide sequence from a patient sample against each nucleotide sequence in a phylogenic tree for a disorder or disease of interest;

determining which sequence of the phylogenic tree has a closest homology to the patient's sequence;

determining a cluster of closely related sequences within the phylogenic tree to which the closest homology sequence belongs:

establishing a roving reference for the patient by creating a consensus sequence from the sequences in the cluster;

comparing the patient's sequence to the roving reference; and providing a drug resistance report for the patient based on the roving reference comparison containing known mutations causing drug resistance.

2. The method of claim 1 , wherein the disorder or disease of interest is selected from a group comprising viral infections, bacterial infections, oncogenes, and tumorigenic cells.

3. The method of claim 1, wherein the merged consensus sequence comprises the sequence for one or more regions of the genome of the disorder or disease of interest.

4. The method of claim 1, wherein the disorder or disease of interest is HCV and, wherein the drug is a direct acting antiviral (DA A).

5. The method of claim 4, wherein the nucleotide sequence encodes NS5A.

6. The method of claim 1, wherein the merged consensus sequence comprises the sequence for one or more sub-regions comprising the positions where drug resistance mutations and drug resistance associated muations occur within the genome of the disorder or disease of interest.

7. A product including a non-transitory storage medium having stored thereon instructions that, when executed by a machine, result in;

comparing a patient' s sequence against each sequence in a phylogenic tree consisting of sequences for a disorder or disease of interest;

assigning the patient an accession number corresponding to the phylogenic tree sequence to which the patient's sequence has the closest homology;

assigning the patient a cluster identifier corresponding to a cluster identifier associated with the phylogenic tree sequence to which the patient's sequence has the closest homology;

comparing the patient's sequence to a roving reference; and providing a drug resistance report or graphical user interface (GUI) for the patient based at least in part on the accession number assigned to the patient and/or the cluster identifier assigned to the patient.

8. The product of claim 7, wherein the disorder or disease of interest is HCV.

9. The product of claim 7, wherein the disorder or disease of interest is selected from a group comprising; viral infections; bacterial infections; oncogenes, and tumorigenic cells.

10. The product of claim 7, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient' s sequence has the closest homology.

11. The product of claim 7, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, and wherein the merged consensus sequence comprises the sequence for one or more regions of the genome of the disorder or disease of interest. The product of claim 7, wherein the roving reference is a merged consensus sequence for the cluster associated with the phylogenic tree sequence to which the patient's sequence has the closest homology, wherein the merged consensus sequence comprises the sequence for one or more sub-regions comprising the positions where drug resistance mutations and drug resistance associated muations within the genome of the disorder or disease of interest.