WO2022051416A1 - Compositions and methods for detection of pharyngitis - Google Patents

Compositions and methods for detection of pharyngitis Download PDF

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WO2022051416A1
WO2022051416A1 PCT/US2021/048739 US2021048739W WO2022051416A1 WO 2022051416 A1 WO2022051416 A1 WO 2022051416A1 US 2021048739 W US2021048739 W US 2021048739W WO 2022051416 A1 WO2022051416 A1 WO 2022051416A1
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pharyngitis
analytes
gas
host
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PCT/US2021/048739
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French (fr)
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Gregory STORCH
Jinsheng Yu
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Washington University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/14—Disorders of ear, nose or throat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • This present disclosure provides methods for rapid testing performed on host samples that allow for detection of the pathogenic etiology of pharyngitis. This approach overcomes many of the limitations of current methods allowing the determination of the etiology of symptoms which aids in making treatment decisions.
  • Pharyngitis is one of the most common reasons for acute medical care visits. According to the National Ambulatory Care Survey, there were more than 10 million visits to physicians for “symptoms related to throat” in 2016, the most recent year for which data are available. Group A Streptococcus (GAS) is the most common bacterial cause of acute pharyngitis, accounting for 5-15% of sore throat visits in adults and 20-30% in children. Recognition of GAS pharyngitis is important because appropriate antibiotic therapy can prevent progression to acute rheumatic fever, shorten the period of the acute illness, prevent local complications, and limit transmission.
  • GAS Group A Streptococcus
  • FIG. 1 shows symptomatic cases and asymptomatic controls enrolled in a study of acute pharyngitis. Positive results of testing for group A Streptococcus, adenovirus, other respiratory viruses, and Epstein-Barr virus are shown. Subgroups of cases and controls are indicated by color codes and are also designated within the colored boxes. Abbreviations: group A Streptococcus: GAS; adenovirus: AdV; rhinovirus: RV; Epstein-Barr virus: EBV; human coronavirus 229E: HCoV229E. Color codes are used consistently in other figures for designated subgroups.
  • FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D depict total white blood cell count, percent neutrophils, C-reactive protein, and procalcitonin in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls. Median values are designated by long horizontal lines and 25th and 75th percentiles are designated by short horizontal lines. Statistically significant (P ⁇ 0.05) and borderline (P ⁇ 0.10) differences between groups are shown with horizontal lines between the groups.
  • FIG. 2A shows WBC count in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
  • FIG. 1 shows WBC count in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
  • FIG. 2B shows the number of mature neutrophils in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
  • FIG. 2C shows the amount of C- reactive protein in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
  • FIG. 2D shows the amount of procalcitonin in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
  • FIG. 3A and FIG. 3B show the unsupervised hierarchical clustering analysis of differentially-expressed genes (DEG) which reveals differences between symptomatic cases and asymptomatic controls.
  • FIG. 3A is a Venn diagram showing 1464 genes which represent the union of DEG resulting from comparisons of symptomatic subgroups each compared to virus-negative controls (nCtrl). Subjects with “other pharyngitis” were not included in this analysis.
  • FIG. 3B shows z score-normalized gene expression values, gene clusters and sample clusters were determined using the unsupervised hierarchical clustering method with Euclidean distance and Ward’s clustering algorithms.
  • Results are displayed in a heat map with intensity showing the level of up (red) - or down (blue) - regulated gene expression. Colored bars underneath the dendrogram above the heatmap correspond to clinical subgroups used for analysis. Individual sample identifications are displayed above the heat map.
  • Two main sample clusters were defined, designated Cluster 1 and Cluster 2. Cluster 1 included all symptomatic cases plus 2 asymptomatic controls. Cluster 2 included all other asymptomatic controls.
  • FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show DEG resulting from comparisons of subject subgroups versus virus-negative controls (nCtrl) and from comparisons of subjects with symptomatic GAS (sGAS) versus other subgroups.
  • FIG. 4A shows a Venn diagram displaying comparison of clinical subgroups versus nCtrl.
  • FIG. 4B shows a unique gene expression profiles for all clinical subgroups based on the 1864 DEGs that represent the union of comparisons of clinical subgroups versus nCtrl.
  • FIG. 4C shows a Venn diagram displaying comparison of individual clinical subgroups versus sGAS.
  • 4D shows a unique gene expression profiles for all clinical subgroups based on the 535 DEGs common to comparisons of sGAS versus the other clinical subgroups. Numbers in parenthesis indicate the number of DEGs resulting from each comparison. Clinical subgroups are shown at the left side of the heat maps and individual identifications are listed between the two heat maps. Gene clusters displayed above the heat maps were determined by the unsupervised hierarchical clustering method.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E depict receiver operating characteristic (ROC) curves showing performance of classifier genes used to distinguish between subjects with symptomatic GAS (sGAS) and other clinical subgroups.
  • Classifier genes were identified by a classification and prediction analysis method: PAM (Prediction Analysis of Microarrays) applied to 535 differential genes derived by comparing gene expression profiles of subjects with symptomatic GAS to those of other clinical subgroups as shown in FIG. 4.
  • Individual panels show ROC curves for comparisons of sGAS versus specific subgroups: FIG. 5A shows comparison with virus-negative controls (nCtrl), FIG. 5B shows comparison with virus-positive asymptomatic controls (vCtrl), FIG.
  • FIG. 5C shows comparison with asymptomatic controls positive for GAS (asGAS)
  • FIG. 5D shows comparison with symptomatic adenovirus infection (sAdV)
  • FIG. 5E shows comparison with all asymptomatic control subjects (Asymp), showing all other clinical subgroups except sOP (All others).
  • L1 O-CV signifies the “10-fold leave-one-out cross validation” procedure that was included in the classification analysis.
  • FIG. 6 shows heat maps showing expression of classifier genes used to distinguish sGAS from other clinical subgroups. Also shown are fold-change values from corresponding comparisons for each of the 6 classification models established with the PAM method. Colored bars at the top of the figure designate the clinical subgroups. Subject identifications are displayed underneath the colored bars. The heat map was created with TMM-normalized Iog2 counts per million values.
  • FIG. 7 shows significantly enriched biological pathways defined using the analysis tool (Enrichr) on genes that are upregulated in subjects with symptomatic GAS infection. The top 20 non-redundant terms are shown with percent of genes mapped and enrichment P-values.
  • FIG. 8 shows the comparison of transcriptional profiles generated using transcriptomes from all 37 subjects (Set37) compared to profiles generated excluding 3 subjects with GAS and probable asymptomatic viral infections (Set34). For illustrative purposes, comparisons of sGAS vs nCtrl and asGAS vs nCtrl are displayed.
  • FIG. 9A and FIG. 9B depict the effect of outliers on the clustering of gene expression profiles.
  • FIG. 9A shows the differential expression and clustering analyses with the 3 outliers included in the analysis and in the heat map. Two clusters were defined, with cluster 1 comprising all 16 symptomatic cases and 2 asymptomatic controls, and cluster 2 comprising the remaining 19 asymptomatic controls. Two of the 3 outliers (P021 , P024) clustered with the symptomatic cases and 1 (P003) clustered with the asymptomatic controls.
  • FIG. 9B shows the same analysis as in (A) but excluding the 3 outliers from the analysis and from the heatmap. The clustering heatmap again yielded 2 clusters with all 16 symptomatic cases in cluster 1 and all 18 asymptomatic controls in cluster 2.
  • FIG. 10 depicts ROC curves for additional comparisons between clinical subgroups.
  • FIG. 11 depicts the comparison of fold-changes in classifier genes used to distinguish between sGAS vs. nCtrl classifier genes generated in the present study with fold-changes in the same genes from 4 human gene expression studies of bacterial infections retrieved from GEO. Each of those 4 studies analyzed gene expression using microarrays, unlike the present study with used RNA-seq. Black bars show a significant fold-change at P ⁇ 0.05 and open bars indicate a non-significant foldchange with P >0.05.
  • *1 was from GSE72829 [discovery set] (21 ), including 52 samples with bacterial infection: 10 Streptococcus pneumoniae, 10 Streptococcus pyogenes, 17 Neisseria meningitis, 4 group B Streptococcus (GBS), 11 others, and 52 healthy controls). Linkage of microarray profiles with individual species was not available.
  • *2 from GSE42026 (25), including 18 samples with gram-positive bacterial infection: 12 S. pneumoniae, 4 S. pyogenes, 2 Staphylococcus aureus, and 33 healthy controls). Linkage of microarray profiles with individual species was also not available.
  • GSE64456 [R2 set] (26), including 55 samples with bacterial infection: 36 Escherichia coli, 3 Enterococcus, 6 GBS, 3 S. aureus, 7 others, and 14 healthy controls), and *4 from GSE40396 (20), including 8 samples with bacterial infection: 2 E. coli, 2 methicillin-susceptible S. aureus (MSSA), 2 methicilliin-resistant S. aureus (MRSA), 2 others, and 22 healthy controls).
  • MSSA methicillin-susceptible S. aureus
  • MRSA methicilliin-resistant S. aureus
  • Pharyngitis commonly known as sore throat, is an inflammation of the pharynx, resulting in a sore throat. Thus, pharyngitis is a symptom, rather than a condition.
  • the etiology is usually infectious, with most cases being of viral origin. Bacterial causes of pharyngitis are also self-limiting but are concerning because of suppurative and nonsuppurative complications.
  • the most significant bacterial agent causing pharyngitis in both adults and children is group A Streptococcus (GAS) infection, and the most common viruses are rhinovirus and adenovirus. Streptococcal infections are characterized by local invasion and release of extracellular toxins and proteases.
  • M protein fragments of certain serotypes of GAS are similar to myocardial sarcolemma antigens and are linked to rheumatic fever and subsequent heart valve damage.
  • Acute glomerulonephritis may result from antibody-antigen complex deposition in glomeruli.
  • a weakness of any diagnostic approach for GAS that relies on direct detection of the organism is the common occurrence of asymptomatic colonization (also referred to as the carrier state), especially in children who are also at greatest risk for symptomatic GAS.
  • asymptomatic colonization also referred to as the carrier state
  • a recent meta-analysis reported that GAS carriage was present in 12% of well children. The frequency of carriage is lower in children less than 5 years of age and in adults.
  • Antibiotic therapy is not indicated for carriers, and failure to distinguish between symptomatic infection and carriers can be misleading and result in unnecessary antibiotic related to treating carriers with symptomatic viral infection.
  • the magnitude of this problem is underscored by a recent estimate that 1 .5 million cases of GAS carriage are present in individuals with pharyngitis in the US, potentially translating into a large volume of unnecessary antibiotic usage.
  • the present disclosure generally relates to detection methods that overcome many of the limitations of current methods for the determination of the etiology of pharyngitis. Applicants have discovered that certain methods to quantify and analyze host analytes can be used to classify asymptomatic carrier, symptomatic infection and healthy subjects. The present disclosure optimizes treatment of a subject having or suspected of having pharyngitis. As such, the present disclosure provides methods for guiding appropriate use of antimicrobials and evaluate the clinical efficacy of certain therapeutic interventions, thereby aiding against the rising tide of antimicrobial resistance.
  • One aspect of the present disclosure encompasses determining the amount of an analyte expression signature which is useful to indicate a symptomatic GAS infection or viral infection cause of pharyngitis in a subject.
  • Suitable analytes were identified according to Example 1 and describe herein. The present disclosure is not limited to the specific analytes recited in the examples as a skilled artisan could identify other suitable analytes using the techniques disclosed herein. Examining host analyte signature induced by a symptomatic GAS infection, an asymptomatic GAS carrier, a viral infection an asymptomatic viral carrier or healthy subject shows distinct analyte profiles which can then be used to determine the etiology of the infection and guide treatment decisions.
  • analyte refers to a substance in a biological sample that may be measured as an indication of the health/disease etiology of a subject.
  • analyte is equivalent to “feature” or “biomarker.”
  • an analyte may be an RNA molecule, may be a protein (e.g. a chemokine, toxin, an antibody, or other protein), an amino acid, a fatty acid (e.g. a short chain fatty acid), a bile acid, a carbohydrate or carbohydrate moiety (e.g.
  • analyte may be extracellular, or the biological sample may be treated to release an intracellular analyte using means known in the art. Alternatively, an analyte may be present on the surface of a cell in a biological sample. Such samples, in some embodiments, may be treated to release the analyte from the cell membrane or cell surface using means known in the art.
  • An analyte signature refers to a characteristic expression profile of a single or a group of analytes that is indicative of an altered or unaltered biological process, medical condition, or a subject’s responsiveness/non-responsiveness to a specific pathogen.
  • the analyte signatures disclosed herein encompass characteristic profiles of at least one analyte selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, TLR5, FFAR3, SPATC1 , KCNH7, ADM, IL18RAP, LY6G6C, ARG1 , FCGR1 B, and TAS2R40 which are identified as differentially expressed in a biological sample obtained from a subject relative to a reference value. See, e.g., Example 1 below.
  • determining analyte levels can be supplemented with diagnostic assays such as assays to determine presence, absence, and/or quantity of a pathogen, clinical assays (e.g., those described in the below examples), advanced radiographic assays, diagnostic assays (e.g., PCR and/or ELISA) and aspiration.
  • diagnostic assays such as assays to determine presence, absence, and/or quantity of a pathogen, clinical assays (e.g., those described in the below examples), advanced radiographic assays, diagnostic assays (e.g., PCR and/or ELISA) and aspiration.
  • the analyte signatures as disclosed herein may represent the expression profile of at least one analyte, for example, at least 2 analytes, at least 3 analytes, at least 4 analytes, at least 5 analytes, at least 6 analytes, at least 7 analytes, at least 8 analytes, at least 9 analytes, at least 10 analytes, at least 15 analytes, at least 20 analytes, at least 25 analytes, at least 30 analytes, at least 35 analytes, at least 40 analytes, at least 45 analytes, at least 50 analytes or more.
  • the analyte signature may comprise multiple analytes with increased levels relative to a reference value. In other examples, the analyte signature may comprise multiple analytes with decreased levels relative to a reference value. In yet other examples, the analyte signature may comprise both increased and decreased analyte levels relative to a reference value.
  • the analyte signature may comprise multiple analytes involved in multiple biological pathways, for example, 2 biological pathways, 3 biological pathways, 4 biological pathways, 5 biological pathways, 6 biological pathways, 7 biological pathways, 8 biological pathways, 9 biological pathways, 10 biological pathways, 11 biological pathways, 12 biological pathways, 13 biological pathways, 14 biological pathways, or 15 biological pathways.
  • a modulated host analyte signature may comprise any analyte with altered expression during the innate immune reaction of the host (e.g., the immune response of the pharyngeal tissue or blood).
  • the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis relative to a subject that does not have a pharyngitis. In another embodiment, the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis caused by a GAS relative to a subject that does not have a pharyngitis or a subject that is an asymptomatic GAS carrier.
  • the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis caused by a viral infection relative to a subject having pharyngitis caused by a GAS infection or a subject that is an asymptomatic viral carrier.
  • signature refers to a set of biological analytes and the measurable quantities of said analytes whose particular combination signifies the presence or absence of the specified biological state or etiology thereof. These signatures are discovered in a plurality of subjects with known status (e.g., with a confirmed bacterial infection, or viral infection), and are discriminative (individually or jointly) of one or more categories or outcomes of interest.
  • measurable analytes can be (but are not limited to) nucleic acid expression levels, protein or peptide levels, metabolite levels, white blood cell levels or neutrophil levels.
  • the analyte signature as disclosed herein may be combined with testing to detect the etiology of the infection (e.g., culturing pathogens from host samples, detecting viral or bacterial proteins or nucleotides).
  • the "signature” is a particular combination of analytes whose expression levels discriminate a condition such as a bacterial GAS pharyngitis, a viral pharyngitis, an asymptomatic viral carrier or a GAS asymptomatic carrier. See, for example, Table 5.
  • the nucleotide and amino acid sequence information for the various host analytes suitable for use in the methods of the present disclosure can be found using a reference data base known in the art, for example, UniProt.
  • a modulated host analyte suitable for use in the present methods includes one or more of, CD177 (see, e.g., NCBI Reference Sequence: XM_017027021 .2 and XP_016882510), FAM20A (see, e.g., NCBI Reference Sequence: NM_001243746.2 and NP_001230675.1 ), CASP5 (see, e.g., NCBI Reference Sequence: NM_001136109.3 and NP_001129581.1 ), ZDHHC19 (see, e.g., NCBI Reference Sequence: XM_006713494.2 and XP_006713557.1 ), FCGR1A (see, e.g., NCBI Reference Sequence: NM_000566.4 and NP_000557.1 ), ITGA7 (see, e.g., NCBI Reference Sequence: NM_001144996.2 and NP_001138468.1 ), S0CS3 (see, e.g.
  • the methods may comprise determining the expression level of at least 1 analyte, at least 2 analytes, at least 3 analytes, at least 4 analytes, at least 5 analytes, at least 6 analytes, at least 6 analytes, at least 7 analytes, at least 8 analytes, at least 9 analytes, at least 10 analytes, at least 1 1 analytes, at least 12 analytes, at least 13 analytes, at least 14 analytes, at least 15 analytes, at least 16 analytes, at least 17 analytes, at least 18 analytes, at least 19 analytes, at least 20 analytes, at least 21 analytes, at least 22 analytes, at least 23 analytes, at least 24 analytes, at least 25 analytes, at least 26 analytes, at least 27 analytes, at least 28 analytes, at least
  • "Rule in” or “rule out” tests support appropriate medical decision making and efficient utilization of resources when well-understood by health care providers.
  • Various embodiments comprise a method to rule in pharyngitis mediated by a GAS infection, or pharyngitis meditated by a viral infection using one or more host modulated analytes but other embodiments may comprise determining the level of two, three or more modulated host analytes as noted above.
  • the disclosure provides a method comprising determining the level of two or more modulated host analytes, the level of one or more of the modulated host analytes may be measured as an indicator of sample quality.
  • tests to measure the level of two or more analytes may be combined with tests for the presence of a virus or bacterium.
  • the disclosure provides a method to detect modulated levels of host analytes in a biological sample obtained from a subject.
  • biological sample means a biological material isolated from a subject. Any biological sample containing any biological material suitable for detecting the desired analytes may be used and may comprise cellular and/or non-cellular material obtained from the subject is suitable. Non-limiting examples include blood, plasma, serum, urine, and tissue. Frequently the sample will be a "clinical sample” which is a sample derived from a patient.
  • Typical clinical samples include, but are not limited to, bodily fluid samples such as synovial fluid, sputum, blood, urine, blood plasma, blood serum, sweat, mucous, saliva, lymph, bronchial aspirates, peritoneal fluid, cerebrospinal fluid, and pleural fluid, and tissues samples such as blood-cells (e.g., white cells), tissue or fine needle biopsy samples and abscesses or cells therefrom.
  • bodily fluid samples such as synovial fluid, sputum, blood, urine, blood plasma, blood serum, sweat, mucous, saliva, lymph, bronchial aspirates, peritoneal fluid, cerebrospinal fluid, and pleural fluid
  • tissues samples such as blood-cells (e.g., white cells), tissue or fine needle biopsy samples and abscesses or cells therefrom.
  • blood sample refers to a biological sample derived from blood, preferably peripheral (or circulating) blood.
  • the blood sample can be whole blood, plasma or serum, although whole blood is typically preferred
  • the sample may include a respiratory sample.
  • respiratory sample means any sample from a subject containing RNA or proteins a plurality of which is generated by cells in the upper or lower respiratory tract.
  • Non-limiting examples include nasal swabs, nasopharyngeal swabs, nasopharyngeal aspirate, oral swab, oropharyngeal swab, pharyngeal (throat) swab, throat wash or gargle, sputum, tracheal aspirate, bronchoalveolar lavage or saliva or transport medium exposed to any of these sample types.
  • the sample is a nasopharyngeal sample.
  • a "nasopharyngeal sample” as used herein means any sample from a subject containing RNA and/or proteins, a plurality of which is generated by cells in the upper respiratory tract. Non-limiting examples include nasal swabs, nasopharyngeal swabs, nasopharyngeal aspirate, oral swab, oropharyngeal swab, pharyngeal swabs, throat swabs, saliva or medium exposed to any of these sample types.
  • the biological sample is obtained using a midturbinate swab.
  • the method of collecting a biological sample can and will vary depending upon the nature of the biological sample and the type of analysis to be performed. Any of a variety of methods generally known in the art may be utilized to collect a biological sample. Generally speaking, the method preferably maintains the integrity of the sample such that the mRNA or protein can be accurately detected and the amount measured according to the disclosure.
  • a single sample is obtained from a subject to detect a host gene signature in the sample.
  • a host gene signature may be detected in samples obtained over time from a subject.
  • more than one sample may be collected from a subject over time.
  • 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or more samples may be collected from a subject over time.
  • 2, 3, 4, 5, or 6 samples are collected from a subject over time.
  • 6, 7, 8, 9, or 10 samples are collected from a subject over time.
  • 10, 11 , 12, 13, or 14 samples are collected from a subject over time.
  • 14, 15, 16 or more samples are collected from a subject over time.
  • samples may be collected every 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more hours. In some embodiments, samples are collected every 0.5, 1 , 2, 3, or 4 hours. In other embodiments, samples are collected every 4, 5, 6, or 7 hours. In yet other embodiments, samples are collected every 7, 8, 9, or 10 hours. In other embodiments, samples are collected every 10, 11 , 12 or more hours. Additionally, samples may be collected every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more days. In some embodiments, a sample is collected about every 6 days. In some embodiments, samples are collected every 1 , 2, 3, 4, or 5 days. In other embodiments, samples are collected every 5, 6, 7, 8, or 9 days. In yet other embodiments, samples are collected every 9, 10, 11 , 12 or more days.
  • determining the level of expression is meant an assessment of the absolute or relative quantity of an analyte in a sample at the nucleic acid or protein level, using technology available to the skilled artisan to detect a sufficient portion of any marker.
  • the term “amount of nucleic acid expression” or “level of nucleic acid expression” as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of mRNA transcript expressed or a specific variant or other portion of the RNA.
  • the term “nucleic acid” includes DNA and RNA and can be either double stranded or single stranded, although RNA is typically preferred.
  • suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses.
  • determining the amount of a RNA comprises, in non-limiting examples, measuring the level of mRNA expression, tRNA expression, rRNA expression, snRNA expression miRNA expression, IncRNA expression, tmRNA expression, and/or snoRNA expression.
  • the amount of nucleic acid expression may be determined by using an array, such as a microarray.
  • an array such as a microarray.
  • Methods of using a nucleic acid microarray are well and widely known in the art.
  • a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array.
  • the term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid.
  • the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
  • probe refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence.
  • the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence complementary thereof.
  • the length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence.
  • the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.
  • the methods include any know techniques for amplifying nucleic acids, including but not limited to PCR, loop-mediated isothermal amplification, and nucleic acid sequence-based amplification (NASBA).
  • the amount of nucleic acid expression may be determined using PCR.
  • a nucleic acid may be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any combination thereof. Specifically, the amount of nucleic expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art. In such an embodiment, the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene.
  • primer refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used.
  • a primer typically contains 15-25 or more nucleotides, although it can contain less or more. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.
  • the amount of nucleic acid expression may be measured by measuring an entire RNA transcript for a nucleic acid sequence or measuring a portion of the RNA transcript for a nucleic acid sequence.
  • the array may comprise a probe for a portion of the RNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full RNA of the nucleic acid sequence of interest.
  • the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence.
  • primers there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest.
  • Methods of designing primers are known in the art.
  • Methods of extracting RNA from a biological sample are known in the art.
  • the level of expression may or may not be normalized to the level of a control nucleic acid.
  • a control nucleic acid should not specifically hybridize with an RNA nucleotide sequence of the invention. This allows comparisons between assays that are performed on different occasions.
  • a method of the disclosure comprises detecting the expression levels of one or more host analytes at the protein level.
  • a host protein may be detected using methods normally used in the art for detecting a specific protein in a sample.
  • methods of detecting a protein adduct may include chromatography, mass spectrometry, an antibody-based detection method, or a combination thereof, and may be as discussed in Ausubel et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • host protein levels are detected using mass spectrometry.
  • Mass spectrometry may be tandem mass spectrometry, quadrupole mass spectrometry, MALDI-TOF mass spectrometry, inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), and spark source mass spectrometry (SSMS).
  • ICP-MS inductively coupled plasma-mass spectrometry
  • AMS accelerator mass spectrometry
  • TMS thermal ionization-mass spectrometry
  • SSMS spark source mass spectrometry
  • host protein levels are detected using a mass spectrometry method capable of detecting a specific protein.
  • mass spectrometry methods capable of detecting a specific protein include MALDI-TOF mass spectrometry and high-resolution tandem mass spectrometry.
  • host protein levels may be detected in a sample using methods based on epitope binding agents.
  • suitable epitope binding agents include agents selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, modified nucleic acids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.
  • an epitope binding agent is an antibody
  • host protein levels are detected using antibody-based methods.
  • antibodies that may be used include polyclonal antibodies, ascites, Fab fragments, Fab' fragments, monoclonal antibodies, single chain antibodies, humanized antibodies, and other fragments that contain the epitope binding site of the antibody.
  • Antibody based methods that may be used to detect a protein are known in the art.
  • Non-limiting examples of methods based on antibodies may include Western blotting, enzyme-linked immunosorbent assays (ELISA), or other solid phase immunoassays, a sandwich immunoassay, radioimmunoassay, nephelometry, electrophoresis, immunofluorescence, immunoblot, flow cytometry, immunohistochemistry, an array or other methods (see Ausubel, F. M. et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, including supplements through 2001 ).
  • an antibody-based method of detecting and measuring an amount of a protein comprises contacting some or all of the sample comprising a protein with an antibody specific for the protein under conditions effective to allow for formation of a complex between the antibody and the protein.
  • the entire sample is not needed, allowing one skilled in the art to repeatedly detect and measure the amount of one or more proteins in the sample over time.
  • the method may occur in solution, or the antibody or protein may be immobilized on a solid surface.
  • suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers. Attachment to the substrate may occur in a wide variety of ways, as will be appreciated by those in the art.
  • the substrate and the antibody may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups.
  • the antibody may be attached directly using the functional groups or indirectly using linkers.
  • the antibody may also be attached to the substrate non-covalently.
  • a biotinylated antibody may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • an antibody may be synthesized on the surface using techniques such as photopolymerization and photolithography.
  • Contacting the sample with an antibody under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibody to bind to any antigen present. After this time, the complex may be washed and then the complex is detected and the amount measured by any method well known in the art. Methods of detecting and measuring an amount of an antibody-polypeptide complex are generally based on the detection of a label or marker.
  • label refers to any substance attached to an antibody, or other substrate material, in which the substance is detectable by a detection method.
  • Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, glucose oxidase and luciferase). Methods of detecting and measuring an amount of an antibody-polypeptide complex based on the detection of a label or marker are well known in the art.
  • an antibody-based method is an immunoassay.
  • Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immmunoassays and non-competitive immunoassays.
  • a competitive immunoassay an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away, and the bound analyte is measured.
  • the antibody is labeled, not the analyte.
  • Non-competitive immunoassays may use one antibody (e.g. the capture antibody is labeled) or more than one antibody (e.g. at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.
  • an antibody-based method is an immunoblot or Western blot.
  • an antibody-based method is flow cytometry.
  • an antibody-based method is immunohistochemistry (IHC).
  • IHC uses an antibody to detect and quantify antigens in intact tissue samples.
  • the tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art.
  • an antibody-based method is an array.
  • An array comprises at least one address, wherein at least one address of the array has disposed thereon an antibody specific for a host analyte as disclosed herein.
  • Arrays may comprise from about 1 to about several hundred thousand addresses.
  • substrates suitable for the construction of arrays are known in the art, and one skilled in the art will appreciate that other substrates may become available as the art progresses. Suitable substrates are also described above.
  • the array comprises at least one host protein antibody attached to the substrate is located at one or more spatially defined addresses of the array.
  • an array may comprise at least one, at least two, at least three, at least four, or at least five host protein specific antibodies, each antibody recognizing the same or different protein, and each antibody may be may be at one, two, three, four, five, six, seven, eight, nine, ten or more spatially defined addresses.
  • a protein may be first isolated or enriched before detection.
  • proteins may be enriched or isolated using liquid chromatography, by precipitation, electrophoresis, or affinity purification.
  • proteins are enriched or purified using liquid chromatography.
  • proteins are enriched or purified using electrophoresis.
  • proteins are enriched or purified by affinity purification before detection.
  • proteins are enriched or purified by affinity purification using antibodies with specificity to the protein.
  • Methods of enriching a sample for a protein or purifying a protein using affinity purification are known in the art.
  • affinity purification comprises incubating a sample with a solid support, such as beads, a culture plate, or a membrane, that facilitates later steps.
  • a solid support may be coated with antibodies specific to a protein, causing the protein to attach to the solid support.
  • a sample may be incubated with a first antibody with specificity to a protein, and the protein-antibody complex may be isolated by incubating with a solid support coated with a second antibody with specificity against a second site on said first antibody, causing a protein-antibody complex to attach to the solid support.
  • the proteins may then be purified or enriched by washing other material in the sample that is not bound to the solid support, or, if the solid support is superparamagnetic beads, proteins attached to the beads (expressing the antigen) may be separated from the sample by attraction to a strong magnetic field.
  • the protein may then be detected in the enriched or purified sample using any of the methods described above.
  • the disclosure also provides that multiple proteins in the same biological sample may be measured simultaneously. Additionally, the disclosure provides that the proteins and corresponding non-specific proteins may be detected in the same biological sample. As such, the disclosure provides a useful method for screening changes in synthesis and clearance of host proteins on a large scale (i.e. , proteomics/metabolomics) and provides a sensitive means to detect and measure host gene signatures in response to infection.
  • proteins for use within the methods include procalcitonin and c-reactive protein.
  • Classification refers to a method of assigning a subject suffering from or at risk for pharyngitis symptoms to one or more categories or outcomes (e.g., a subject has a symptomatic infected with a pathogen or is a asymptomatic carrier of a pathogen, another categorization may be that a patient is infected with a virus or infected with a bacterium).
  • a subject may be classified as having a symptomatic infection with a specific bacteria or virus.
  • a subject may be classified to more than one category.
  • the outcome, or category is determined by the expression signature of the subject being tested, which may be compared to a “reference value” which may be a reference signature, confidence level, or limit. In other scenarios, the probability of belonging to a particular category may be given.
  • the disclosure is directed to a method of detecting etiology of pharyngitis in a subject.
  • the method generally comprises obtaining a biological sample (e.g., peripheral blood) and determining the level at least one host analyte and comparing the level of the host analyte with its respective reference level.
  • a "reference level" of an analyte means a level of the analyte that is indicative of the absence of a particular disease state, phenotype, or etiology.
  • when the level of an analyte in a subject is above the reference level of the analyte it is indicative of the presence of a disease state, phenotype, or etiology.
  • the level of an analyte in a subject when the level of an analyte in a subject is above the reference level of the analyte it is indicative of the lack of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is below the reference level of the analyte it is indicative of the presence of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is below the reference level of the analyte it is indicative of the lack of a particular disease state, phenotype, or etiology.
  • the reference value may be the level of at least one host analyte in a normal control subject or normal control population.
  • normal control it is meant a subject or population without pharyngitis and is not a carrier of GAS or virus known to cause pharyngitis.
  • the reference value may be the level of at least one host analyte in an asymptomatic GAS carrier subject or asymptomatic GAS carrier subject population.
  • the reference value may be the level of at least one host analyte in an asymptomatic viral carrier subject or asymptomatic viral carrier subject population. In still another aspect, the reference value may be the level of at least one host analyte in a GAS positive subject with pharyngitis or population of a GAS positive subjects with pharyngitis. In still yet another aspect, the reference value may be the level of at least one host analyte in a viral positive subject with pharyngitis or population of a viral positive subjects with pharyngitis.
  • the term "indicative" when used with analyte expression levels means that the analyte expression levels are up-regulated or down- regulated, altered, or changed compared to the expression levels in alternative biological states (e.g., bacterial pharyngitis or viral pharyngitis) or control.
  • alternative biological states e.g., bacterial pharyngitis or viral pharyngitis
  • expression of the modulated host analyte is determined by measuring mRNA. In other embodiments, expression is determined by measuring protein. In embodiments comprising the measurement of protein, a biological sample may be used in an immunoassay. In embodiments comprising the measurement of mRNA, a biological sample may be optionally centrifuged to form a pellet of cells and cell debris which is then added to lysis buffer. Total nucleic acid is isolated from the pellet and DNA is digested using, by way of non-limiting example, DNase I. The RNA is then reverse transcribed into cDNA. The cDNA is then analyzed to determine the level of at least one modulated host analyte.
  • the level of the at least one modulated host analyte is determined by reverse transcription quantitative polymerase chain reaction (rt-qPCR) although the skilled artisan will appreciate that there are other ways that the level of the at least one modulated host analyte may be determined by the analysis of mRNA and these methods are encompassed by the invention in its various embodiments.
  • rt-qPCR reverse transcription quantitative polymerase chain reaction
  • the present disclosure further provides methods for determining whether a patient has a symptomatic bacterial infection, a symptomatic viral infection, an asymptomatic viral infection, an asymptomatic bacterial infection or normal healthy control.
  • the method for making this determination relies upon the host analyte signature as taught herein.
  • the methods may include: a) measuring the expression levels of predefined sets of modulated host analytes; optionally normalizing analyte expression levels for the technology used to make said measurement; and b) comparing the levels of one or more host analytes with a reference value. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of the presence of a symptomatic bacterial infection.
  • the level of the one or more host analytes when the level of the one or more host analytes is below the reference level it is indicative of the presence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of an asymptomatic bacterial infection.
  • the level of the one or more host analytes when the level of the one or more host analytes is about the reference level it is indicative of the presence of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of the presence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of the presence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of a symptomatic viral infection.
  • the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the expression level of one or more host analytes selected from CD177, FAM20A, FCGR1A, and, CD274; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, FCGR1A, and, CD274 exhibit increased expression relative to a healthy control reference value.
  • the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a healthy control reference value.
  • the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a viral carrier reference value.
  • the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to
  • the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to an asymptomatic GAS carrier reference value.
  • the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a healthy control reference value.
  • the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a symptomatic viral infection reference value.
  • the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host genes selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 exhibit increased expression relative to reference value.
  • the methods may further comprise administering an anti-bacterial drug when the subject is determined to have pharyngitis caused by bacterial etiology or administering an anti-viral drug when the subject is determined to have pharyngitis caused by viral etiology.
  • a host analyte as disclosed herein may be classified as differentially expressed or aberrant when it has an increased or decreased amount relative to a reference value.
  • Any suitable reference value known in the art may be used.
  • a suitable reference value may be the amount of a host analyte in a biological sample obtained from a subject, or group of subjects, of the same species that has no clinically detectable symptom of a pharyngitis.
  • a suitable reference value may be the amount of a host analyte in a biological sample obtained from a subject, or group of subjects, of the same species that has no detectable pharyngitis infection pathology.
  • a suitable reference value may be the background signal of the assay as determined by methods known in the art.
  • a suitable reference value may be a measurement of the amount of a host analyte in a reference sample obtained from the same subject.
  • the reference sample comprises the same type of biological sample as the test sample and may be obtained from a subject when the subject had no clinically detectable symptom of pharyngitis.
  • a skilled artisan will appreciate that it is not always possible or desirable to obtain a reference sample from a subject when the subject is otherwise healthy.
  • a reference sample may be a sample obtained from a subject before therapy or at an earlier point in the disease.
  • a subject may have a risk of infection but may not have other symptoms of an infection or the subject may have one or more other symptom of a pharyngitis.
  • a suitable reference sample may be a biological sample from an individual or group of individuals that has been shown not to have pharyngitis.
  • the reference value may be a sample of the same type of biological sample obtained from one or more individuals that has not been administered therapy but has a respiratory infection.
  • the amount of the host analyte in the biological sample compared to the reference value is increased at least 1-fold.
  • the amount of the host analyte in the sample compared to the reference value is increased at least 1-fold, at least 1 .25-fold, at least 1 .5-fold, at least 1 .75-fold, at least 2-fold, at least 5- fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold.
  • the amount of the host analyte in the sample compared to the reference value is increased at least
  • the amount of the host analyte in the biological sample compared to the reference value is decreased at least 1-fold.
  • the amount of the host analyte in the sample compared to the reference value is decreased at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30- fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold.
  • the increase or decrease in the amount of a host analyte is measured using p-value.
  • p-value a RNA is identified as being differentially expressed between a biological sample and a reference value when the p-value is less than 0.1 , preferably less than 0.05, more preferably less than 0.01 , even more preferably less than 0.005, the most preferably less than 0.001 .
  • the disclosure provides a method of treating a subject exhibiting symptoms of a pharyngitis, the method comprising: a) measuring the expression levels of one or more modulated host analytes; optionally normalizing analyte expression level for the technology used to make said measurement; b) comparing the levels of one or more host analytes with a reference value; and, wherein if the normalized level of the one or more host analyte is above or below the reference level the subject is classified for the presence or absence of a GAS pharyngitis or viral pharyngitis.
  • the method may further comprise, testing the subject for the presence of at least one virus or bacteria.
  • symptoms of pharyngitis include sore throat; dry, scratchy throat; pain when swallowing; and pain when speaking
  • the invention provides a method of treating a subject exhibiting symptoms of pharyngitis, the method comprising: a) measuring the expression levels of one or more modulated host analytes; optionally normalizing analyte expression level for the technology used to make said measurement; b) comparing the levels of one or more host analytes with a reference value; and, wherein if the normalized level of the one or more host analyte is above or below the reference level the subject is classified for the presence or absence of a GAS pharyngitis or viral pharyngitis, the subject receives an appropriate treatment regimen for pharyngitis.
  • the method may further comprise, testing the subject for the presence of at least one respiratory virus or bacteria; or determining the number of white blood cells and/or neutrophils in a biological sample obtained from the subject.
  • a therapeutically effective amount of a pharmaceutical composition may be administered to a subject.
  • Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system.
  • Peripheral administration includes but is not limited to oral, inhalation, intravenous, intraperitoneal, intra-articular, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • Local administration includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation. The route of administration may be dictated by the disease or condition to be treated.
  • compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate.
  • pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate.
  • a therapeutically effective amount of a composition of the invention is administered to a subject.
  • a “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response.
  • Actual dosage levels of active ingredients in a therapeutic composition of the invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, age, the symptoms, and the physical condition and prior medical history of the subject being treated.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the severity, duration and/or progression of a disease or disorder or one or more symptoms thereof resulting from the administration of one or more therapies. Such terms refer to a reduction in the replication of a virus or bacteria, or a reduction in the spread of a virus or bacteria to other organs or tissues in a subject or to other subjects.
  • the term “effective amount” refers to an amount of a therapeutic agent that is sufficient to exert a physiological effect in the subject.
  • a therapeutic agent for treating a subject having bacteremia is an antibiotic which include, but are not limited to, penicillins, cephalosporins, fluroquinolones, tetracyclines, macrolides, and aminoglycosides.
  • a therapeutic agent for treating a subject having a viral respiratory infection includes, but is not limited to, oseltamivir, RNAi antivirals, inhaled ribavirin, monoclonal antibody respigam, zanamivir, and neuraminidase blocking agents.
  • the invention contemplates the use of the methods of the invention to determine treatments with antivirals or antibiotics that are not yet available.
  • a therapeutic agent(s) capable of producing reduction of symptoms associated with a disease state.
  • therapeutic agents include, but are not limited to, NSAIDS, acetaminophen, anti-histamines, beta-agonists, anti-tussives or other medicaments that reduce the symptoms associated with the disease process.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
  • the frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms.
  • the timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.
  • Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.
  • a subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoological animal.
  • a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears.
  • the subject is a human.
  • a subject in need thereof may be a subject suffering from, suspected of suffering from or at risk of pharyngitis.
  • the present invention provides articles of manufacture and kits containing materials useful for treating the conditions described herein.
  • the article of manufacture may include a container of a composition as described herein with a label.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition comprising reagents to detect a respiratory illness in a patient and instructions for the use thereof, wherein the instructions comprise: analyzing one or more host analytes to determine a level of at least one modulated host analyte; comparing the level of the at least one modulated host analyte with a predetermined reference level; and, wherein if the level of the at least one modulated host analyte is above or below the respective reference level, the patient is determined to have GAS pharyngitis, viral pharyngitis, or a GAS carrier.
  • 'About as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1 %, and still more preferably ⁇ 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.
  • Non-limiting examples include antibiotics.
  • “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means determining the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise determining the values or categorization of a subject's clinical parameters.
  • the terms "patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
  • Platinum or “technology” as used herein refers to an apparatus (e.g., instrument and associated parts, computer, computer-readable media comprising one or more databases as taught herein, reagents, etc.) that may be used to measure a signature, e.g., gene expression levels, in accordance with the present disclosure.
  • platforms include, but are not limited to, an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a nucleic acid sequencing platform, a hybridization and multi-signal coded (e.g., fluorescence) detector platform, etc., a nucleic acid mass spectrometry platform, a magnetic resonance platform, and combinations thereof.
  • the platform is configured to measure gene expression levels semi-quantitatively, that is, rather than measuring in discrete or absolute expression, the expression levels are measured as an estimate and/or relative to each other or a specified marker or markers (e.g., expression of another, "standard” or "reference,” gene).
  • semi-quantitative measuring includes "real- time PCR” by performing PCR cycles until a signal indicating the specified mRNA is detected, and using the number of PCR cycles needed until detection to provide the estimated or relative expression levels of the genes within the signature.
  • a real-time PCR platform includes, for example, a TaqMan® Low Density Array (TLDA), in which samples undergo multiplexed reverse transcription, followed by real-time PCR on an array card with a collection of wells in which real-time PCR is performed.
  • a real-time PCR platform also includes, for example, a Biocartis IdyllaTM sample-to-result technology, in which cells are lysed, DNA/RNA extracted and real-time PCR is performed and results detected.
  • a real-time PCR platform also includes, for example, CyTOF analysis: CyTOF (Fludigm) is a recently introduced mass- cytometer capable of detecting up to 40 markers conjugated to heavy metals simultaneously on single cells.
  • a magnetic resonance platform includes, for example, T2 Biosystems® T2 Magnetic Resonance (T2MR®) technology, in which molecular targets may be identified in biological samples without the need for purification.
  • T2MR® T2 Magnetic Resonance
  • arrays are interchangeable and refer to an arrangement of a collection of nucleotide sequences presented on a substrate. Any type of array can be utilized in the methods provided herein.
  • arrays can be on a solid substrate (a solid phase array), such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane.
  • Arrays can also be presented on beads, i.e. , a bead array. These beads are typically microscopic and may be made of, e.g., polystyrene.
  • the array can also be presented on nanoparticles, which may be made of, e.g., particularly gold, but also silver, palladium, or platinum.
  • the nucleotide sequences can be DNA, RNA, or any permutations thereof (e.g., nucleotide analogues, such as locked nucleic acids (LNAs), and the like). In some embodiments, the nucleotide sequences span exon/intron boundaries to detect gene expression of spliced or mature RNA species rather than genomic DNA.
  • the nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences.
  • the arrays may additionally comprise other compounds, such as antibodies, peptides, proteins, tissues, cells, chemicals, carbohydrates, and the like that specifically bind proteins or metabolites.
  • An array platform includes, for example, the TaqMan® Low Density Array (TLDA) mentioned above, and an Affymetrix® microarray platform.
  • TLDA TaqMan® Low Density Array
  • a hybridization and multi-signal coded detector platform includes, for example, NanoString nCounter® technology, in which hybridization of a color-coded barcode attached to a target-specific probe (e.g., corresponding to a gene expression transcript of interest) is detected; and Luminex® xMAP® technology, in which microsphere beads are color coded and coated with a target-specific (e.g., gene expression transcript) probe for detection; and Illumina® BeadArray, in which microbeads are assembled onto fiber optic bundles or planar silica slides and coated with a target-specific (e.g., gene expression transcript) probe for detection.
  • NanoString nCounter® technology in which hybridization of a color-coded barcode attached to a target-specific probe (e.g., corresponding to a gene expression transcript of interest) is detected
  • Luminex® xMAP® technology in which microsphere beads are color coded and coated with a target-specific (e.g., gene expression transcript) probe for detection
  • a nucleic acid mass spectrometry platform includes, for example, the Ibis Biosciences Plex-ID® Detector, in which DNA mass spectrometry is used to detect amplified DNA using mass profiles.
  • a thermal cycler platform includes, for example, the FilmArray® multiplex PCR system, which extract and purifies nucleic acids from an unprocessed sample and performs nested multiplex PCR; the RainDrop Digital PCR System, which is a droplet-based PCR platform using microfluidic chips; and the GenMark eSensor or ePlex systems.
  • genetic material refers to a material used to store genetic information in the nuclei or mitochondria of an organism's cells.
  • examples of genetic material include, but are not limited to, double-stranded and single-stranded DNA, cDNA, RNA, and mRNA.
  • nucleic acid oligomers refers to two or more nucleic acid oligomers, which can be DNA or RNA.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. General Techniques
  • Example 1 Host analyte analysis to improve the detection of group A streptococcal pharyngitis
  • RNA sequencing uses RNA sequencing to generate transcriptomes from 37 children, including 10 with acute GAS pharyngitis, 5 asymptomatic GAS carriers, 3 with adenoviral pharyngitis, 3 with pharyngitis of unknown etiology, and 16 asymptomatic children negative for GAS. Transcriptional profiles from each group were distinct.
  • 1357 genes were upregulated in the children with symptomatic GAS compared to those with asymptomatic carriage.
  • a panel of 13 genes distinguished between children with acute GAS and all others with 91 % accuracy.
  • Each subject also had the following research samples obtained: two throat swabs, a throat wash, a sample of oral secretions, a blood sample drawn into a Tempus blood RNA tube (Thermo Fisher, Waltham, MA) for RNA preservation, a complete blood count, and serum for C-reactive protein, procalcitonin, and IgM antibodies for Epstein-Barr virus.
  • the throat swabs were collected using ESwabs (Copan Diagnostics, Murietta, CA).
  • One throat swab was placed in Universal Transport Media (Copan), stored at -80°C and tested for respiratory viruses using the GenMark eSensor respiratory virus panel (Genmark, Carlsbad, CA).
  • the eSensor testing was performed using a research protocol that detected the following viruses: influenza A and B, respiratory syncytial virus A and B, parainfluenza virus types 1-4, rhinovirus, human metapneumovirus, adenovirus groups B/E and C, coronaviruses OC43, 229E, NL63 and HKU1 .
  • the throat wash was collected by asking subjects to gargle with a volume of 6 ml of normal saline, which was spit into a sterile container.
  • the oral secretion sample was collected by asking the subject to spit 2.0 ml of oral secretions into an Oragene- RNA tube.
  • RNA-seq Libraries and Sequencing were generated using 200 ng of globin-reduced total RNA from each sample. Library construction was performed using the TruSeq Stranded mRNA library kit (Illumina, San Diego, CA). cDNA quantity was determined with the Qubit Fluorometer (Life Technologies, Grand Island, NY) and quality assessed using the Agilent Bioanalyzer 2100 (Santa Clara, CA). Libraries were sequenced (single end reads) on the Illumina HiSeq 2500 (Illumina, San Diego, CA) to generate 20 million reads/sample.
  • RNA-seq reads were aligned to the Ensembl release 76 primary assembly with STAR program (version 2.5.1a) (A. Dobin et al., Bioinformatics 29, 15-21 (2013)). Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount (version 1.4.6-p5) (Y. Liao et al., Bioinformatics 30, 923-930 (2014)). Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC (version 2.6.2) (L. Wang et al., Bioinformatics 28, 2184-2185 (2012)).
  • PAM cross-validates the classifiers with a 2-level nested leave-one-out cross validation procedure (L10-CV), with options for the number of folds of data division and number of model cut-offs to run. Classifiers were defined using the default of 30 cutoffs and 10-fold L10-CV.
  • L10-CV 2-level nested leave-one-out cross validation procedure
  • Biological Pathway Enrichment A web-based pathway enrichment tool “Enrichr” (27) was utilized to analyze Gene Ontology Biological Processes (http://amp.pharm.mssm.edu/Enrichr/). Enrichr determines the significance of genes in the gene-list compared to the background of random gene-lists. The resulting list of mapped terms was ranked by the Benjamini-Hochberg (BH) FDR-adjusted p-value.
  • BH Benjamini-Hochberg
  • Results of microbial testing for GAS, adenovirus, other viruses, and other bacteria are shown in FIG. 1 and Table 2. Subjects were considered positive for GAS if GAS was detected by either culture or NAT. Using this definition, 11 (61 %) of the 18 cases and 5 (24%) of the 21 controls were positive for GAS. Throat culture and NAT results for each subject are shown in Tables 3 and 4.
  • AdV was detected in 4 cases and in 1 control.
  • the 4 cases included 2 that were positive only for AdV, 1 that was positive for AdV and RV, and 1 that was positive for AdV and Streptococcus dysgalactiae.
  • AdV symptomatic AdV
  • sOP symptomatic “other pharyngitis”
  • One subject with symptomatic GAS and one subject with symptomatic adenovirus infection were each also positive for RV. These subjects were classified as sGAS and sAdV respectively because RV was most likely a bystander since it is not recognized as a cause of severe pharyngitis.
  • One subject with symptomatic GAS and one control subject who was a GAS carrier were each positive for EBV IgM. The symptomatic subject was classified as sGAS because the EBV was not clinically significant based on absence of lymphocytosis. The control subject was classified as asGAS since the asymptomatic child did not have a clinically significant EBV infection.
  • WBC counts, percent neutrophils, CRP, and procalcitonin for the sGAS, sAdV, asGAS, vCtrl and nCtrl subgroups are shown in FIG. 2.
  • vCtrl and nCtrl were combined as “controls”.
  • WBC counts, percent neutrophils, CRP, and PCT were higher in subjects with sGAS compared to the other groups. Differences between sGAS and other subgroups that were statistically significant were WBC, percent neutrophils, and CRP compared to subjects with asGAS; and WBC, percent neutrophils, CRP, and PCT compared to the controls. Only WBC was significantly higher in sGAS compared to sAdV.
  • Cluster 1 consisted of all 16 symptomatic subjects plus 2 controls (1 asGAS and 1 nCtrl).
  • Cluster 2 consisted of the remaining 19 asymptomatic subjects. Seven of 10 sGAS cases were clustered tightly within Cluster 1 . Clusters of genes that were generally upregulated in sGAS, upregulated in sAdV, and downregulated in sGAS were apparent (FIG. 3B). The effect of excluding 3 subjects who were positive for a respiratory virus or EBV in addition to GAS was also investigated (boxes B, C, J). As shown in Fig. 8, excluding these subjects had only a minor impact on the detection of DEG.
  • genes of interest were identified by comparing each of the subgroups (other than sOP) to nCtrl, selecting DEG with fold change ⁇ 2.0 with P ⁇ 0.05.
  • the number of DEG identified in each comparison is shown in FIG. 4A.
  • the largest group of DEG came from the comparison of sGAS versus nCtrl (1334 DEG), followed by the comparison of sAdV versus nCtrl (508 DEG). Comparisons of asGAS and vCtrl versus nCtrl yielded much smaller numbers of DEG. In all, the union of the 4 sets of DEG defined a total of 1864 unique DEG.
  • FIG. 4B Gene expression profiles for the 6 clinical subgroups generated using supervised hierarchical clustering applied to the 1864 DEG are shown in FIG. 4B. Clear differences were evident between sGAS and sAdV, each of which also differed from the asymptomatic controls. Expression profiles of the 3 subgroups of asymptomatic controls were similar to one another, indicating that the presence of GAS or a virus in these asymptomatic control subjects did not have a strong impact on their gene expression profile.
  • Subject P019 had findings suggestive of a viral infection, including a normal white blood cell count with 46% neutrophils, a modestly elevated CRP of 43.6 mg/dL, and a borderline procalcitonin value of 0.15 ng/mL.
  • This subject s gene expression profile strongly resembled those of subjects with sAdV (FIG. 2, suggesting that the subject may have had an unrecognized AdV infection.
  • the gene expression profile of the third subject with sOP did not closely resemble that of any of the other clinical subgroups.
  • each panel has a panel of 13 genes that distinguished sGAS from all other subgroups. For this clinically important comparison, the 13- gene panel had an AUC of 0.93.
  • FIG. 6 shows fold-changes in gene expression and heat maps for the classifier genes making up the panels used to distinguish sGAS from other subgroups.
  • CD177 had the highest fold-change of any of the classifier genes.
  • FCGR1A next most elevated gene
  • FCGR1A next most elevated gene
  • ARG1 next most upregulated gene
  • Enrichr (M. V. Kuleshov et al., Nucleic Acids Res 44, W90-97 (2016)) was used to analyze biological processes enriched among the upregulated DEG from the comparison of sGAS versus nCtrl.
  • FIG. 7 shows the 20 most enriched GO Biological Process terms. The most significantly enriched terms related to various aspects of neutrophil activation.
  • FIG. 7 shows neutrophil degranulation, two other neutrophil process terms were enriched to the similar level but are not shown because FIG. 7 shows only non-redundant terms. Most of the other enriched terms were related to cytokine signaling especially IL-1 beta, and components of the inflammatory response. No terms were significantly enriched among down-regulated genes.
  • RNA sequencing was used as a broad, unbiased approach to prove the hypothesis that differences in host response were present among children with symptomatic GAS, asymptomatic GAS, viral infection as exemplified by adenovirus, and pathogen-negative controls.
  • a shrunken centroid methodology was then used (R. Tibshirani et al., Proc Natl Acad Sci II S A 99, 6567- 6572 (2002)), a machine learning technique, to identify panels consisting of limited numbers of transcripts that could distinguish among the clinical subgroups under analysis.
  • a panel of 13 transcripts was identified that distinguished the subjects with symptomatic GAS from all other subjects.
  • CD177 To find small numbers of transcripts that could provide useful information in the detection and identification of pathological etiology, the gene encoding CD177 emerged as a uniquely discriminatory marker. Also known as human neutrophil antigen 2 (HNA-s), CD177 is a cell surface glycoprotein that is involved in neutrophil activation and neutrophil transmigration. Antibodies to HNA-2 are involved in transfusion-associated lung injury (TRALI). The gene is overexpressed in polycythemia vera, and mutations of the gene are associated with myeloproliferative diseases. CD177 has also been shown to be upregulated in severe bacterial infections, pneumococcal meningitis, tuberculosis, septic shock, and Kawasaki disease.
  • CD177 is uniquely responsive to GAS infection versus other bacterial species requires further investigation. However, because bacteria other than GAS are not common causes of pharyngitis, species specificity may not be required for it to be a useful marker in the setting of acute pharyngitis.
  • the present example has shown that transcriptional analysis of peripheral blood leukocytes can provide valuable information that will improve the diagnosis of GAS pharyngitis and allow more appropriate targeting of antibiotic therapy than is possible using currently available tests.
  • a key step will be to use analytes identified in this study in simple and rapid test formats that can be used at or near the point of care.
  • it will be important to evaluate the possibility of measuring the transcriptional response in pharyngeal samples, which could allow microbial detection and transcriptional analysis to be performed on the same sample.
  • the nasal transcriptome was more informative than the transcriptome in peripheral blood leukocytes for diagnosing acute viral respiratory infection (J. Yu et al., J Infect Dis 219, 1151-1161 (2019)).
  • the present study provides rapid progress along the translational pathway to provide detection methods to promote appropriately targeted antibiotic therapy for pharyngitis, an important component in the societal battle to curb unnecessary antibiotic use and prevent the further development of antibiotic resistance (Antibiotic Resistance Threats in the United States, 2019. (2019)).
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system.
  • “about” can mean within an acceptable standard deviation, per the practice in the art.
  • “about” can mean a range of up to ⁇ 20 %, preferably up to ⁇ 10 %, more preferably up to ⁇ 5 %, and more preferably still up to ⁇ 1 % of a given value.
  • the term can mean within an order of magnitude, preferably within 2-fold, of a value.

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Abstract

The present disclosure provides molecular methods to improve the rapid diagnosis of pharyngitis. In general, the present disclosure provides method of detecting host analyte signatures which aid in determining the etiology of a pharyngitis and thereby improve the treatment and limit the unnecessary use of antibiotics which is an unnecessary expensive, can produce side effects, and contributes to antibiotic resistance.

Description

COMPOSITIONS AND METHODS FOR DETECTION OF PHARYNGITIS
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 63/073,302, filed September 1 , 2020, the disclosure of which is herein incorporated by reference in its entirety.
GOVERNMENTAL RIGHTS
[0002] This invention was made with government support under HHSN272201200005C awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE TECHNOLOGY
[0003] This present disclosure provides methods for rapid testing performed on host samples that allow for detection of the pathogenic etiology of pharyngitis. This approach overcomes many of the limitations of current methods allowing the determination of the etiology of symptoms which aids in making treatment decisions.
BACKGROUND
[0004] Pharyngitis is one of the most common reasons for acute medical care visits. According to the National Ambulatory Care Survey, there were more than 10 million visits to physicians for “symptoms related to throat” in 2016, the most recent year for which data are available. Group A Streptococcus (GAS) is the most common bacterial cause of acute pharyngitis, accounting for 5-15% of sore throat visits in adults and 20-30% in children. Recognition of GAS pharyngitis is important because appropriate antibiotic therapy can prevent progression to acute rheumatic fever, shorten the period of the acute illness, prevent local complications, and limit transmission. Most cases of pharyngitis not caused by GAS are caused by viruses such as adenovirus and Epstein-Barr virus, and do not respond to antibiotic therapy. Because clinical findings do not clearly distinguish between GAS and viral pharyngitis, clinical management relies heavily on diagnostic testing. Until recently, the diagnostic tools employed were rapid antigen detection tests (RADT) and culture. RADTs are highly specific for detection of GAS but are less sensitive than culture. Thus, the common clinical algorithm in children has been a 2-step approach that starts with a RADT, followed by a culture if the RADT is negative. A disadvantage of the 2-step approach is that culture requires 48 hours of incubation before it can be finalized, leading to delayed treatment in some patients and requiring additional contacts with health care providers to learn the test results.
[0005] Therefore, a need in the art exists for a rapid practical and reliable test to guide physicians and patients in the decision-making process during suspected pharyngitis.
BRIEF DESCRIPTION OF THE FIGURES
[0006] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0007] FIG. 1 shows symptomatic cases and asymptomatic controls enrolled in a study of acute pharyngitis. Positive results of testing for group A Streptococcus, adenovirus, other respiratory viruses, and Epstein-Barr virus are shown. Subgroups of cases and controls are indicated by color codes and are also designated within the colored boxes. Abbreviations: group A Streptococcus: GAS; adenovirus: AdV; rhinovirus: RV; Epstein-Barr virus: EBV; human coronavirus 229E: HCoV229E. Color codes are used consistently in other figures for designated subgroups.
[0008] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D depict total white blood cell count, percent neutrophils, C-reactive protein, and procalcitonin in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls. Median values are designated by long horizontal lines and 25th and 75th percentiles are designated by short horizontal lines. Statistically significant (P<0.05) and borderline (P<0.10) differences between groups are shown with horizontal lines between the groups. FIG. 2A shows WBC count in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls. FIG. 2B shows the number of mature neutrophils in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls. FIG. 2C shows the amount of C- reactive protein in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls. FIG. 2D shows the amount of procalcitonin in subjects with symptomatic GAS and AdV infections, asymptomatic GAS, and asymptomatic controls.
[0009] FIG. 3A and FIG. 3B show the unsupervised hierarchical clustering analysis of differentially-expressed genes (DEG) which reveals differences between symptomatic cases and asymptomatic controls. FIG. 3A is a Venn diagram showing 1464 genes which represent the union of DEG resulting from comparisons of symptomatic subgroups each compared to virus-negative controls (nCtrl). Subjects with “other pharyngitis” were not included in this analysis. FIG. 3B shows z score-normalized gene expression values, gene clusters and sample clusters were determined using the unsupervised hierarchical clustering method with Euclidean distance and Ward’s clustering algorithms. Results are displayed in a heat map with intensity showing the level of up (red) - or down (blue) - regulated gene expression. Colored bars underneath the dendrogram above the heatmap correspond to clinical subgroups used for analysis. Individual sample identifications are displayed above the heat map. Two main sample clusters were defined, designated Cluster 1 and Cluster 2. Cluster 1 included all symptomatic cases plus 2 asymptomatic controls. Cluster 2 included all other asymptomatic controls.
[0010] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D show DEG resulting from comparisons of subject subgroups versus virus-negative controls (nCtrl) and from comparisons of subjects with symptomatic GAS (sGAS) versus other subgroups. FIG. 4A shows a Venn diagram displaying comparison of clinical subgroups versus nCtrl. FIG. 4B shows a unique gene expression profiles for all clinical subgroups based on the 1864 DEGs that represent the union of comparisons of clinical subgroups versus nCtrl. FIG. 4C shows a Venn diagram displaying comparison of individual clinical subgroups versus sGAS. FIG. 4D shows a unique gene expression profiles for all clinical subgroups based on the 535 DEGs common to comparisons of sGAS versus the other clinical subgroups. Numbers in parenthesis indicate the number of DEGs resulting from each comparison. Clinical subgroups are shown at the left side of the heat maps and individual identifications are listed between the two heat maps. Gene clusters displayed above the heat maps were determined by the unsupervised hierarchical clustering method.
[0011] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E depict receiver operating characteristic (ROC) curves showing performance of classifier genes used to distinguish between subjects with symptomatic GAS (sGAS) and other clinical subgroups. Classifier genes were identified by a classification and prediction analysis method: PAM (Prediction Analysis of Microarrays) applied to 535 differential genes derived by comparing gene expression profiles of subjects with symptomatic GAS to those of other clinical subgroups as shown in FIG. 4. Individual panels show ROC curves for comparisons of sGAS versus specific subgroups: FIG. 5A shows comparison with virus-negative controls (nCtrl), FIG. 5B shows comparison with virus-positive asymptomatic controls (vCtrl), FIG. 5C shows comparison with asymptomatic controls positive for GAS (asGAS), FIG. 5D shows comparison with symptomatic adenovirus infection (sAdV), FIG. 5E shows comparison with all asymptomatic control subjects (Asymp), showing all other clinical subgroups except sOP (All others). L1 O-CV signifies the “10-fold leave-one-out cross validation” procedure that was included in the classification analysis.
[0012] FIG. 6 shows heat maps showing expression of classifier genes used to distinguish sGAS from other clinical subgroups. Also shown are fold-change values from corresponding comparisons for each of the 6 classification models established with the PAM method. Colored bars at the top of the figure designate the clinical subgroups. Subject identifications are displayed underneath the colored bars. The heat map was created with TMM-normalized Iog2 counts per million values.
[0013] FIG. 7 shows significantly enriched biological pathways defined using the analysis tool (Enrichr) on genes that are upregulated in subjects with symptomatic GAS infection. The top 20 non-redundant terms are shown with percent of genes mapped and enrichment P-values. [0014] FIG. 8 shows the comparison of transcriptional profiles generated using transcriptomes from all 37 subjects (Set37) compared to profiles generated excluding 3 subjects with GAS and probable asymptomatic viral infections (Set34). For illustrative purposes, comparisons of sGAS vs nCtrl and asGAS vs nCtrl are displayed. Most DEG overlapped in the two comparisons (sGAS vs nCtrl: 1157/(1157+177)=86.7%, asGAS vs nCtrl: 30/(30+26)=53.6%). More importantly, the difference in the magnitude of fold-change values from the two data sets was minimal: 5 out of 1494 (0.3%) differential genes in the sGAS vs. nCtrl comparison had a 1 .5-fold or greater difference. In the asGAS vs. nCtrl comparison, 7 out of 70 (10%) differential genes had a 1 .5-fold or greater difference. Of note, the comparison between asGAS subgroup and nCtrl yielded only a minimal number of differentially expressed genes (DEG).
[0015] FIG. 9A and FIG. 9B depict the effect of outliers on the clustering of gene expression profiles. We identified 3 samples from asymptomatic controls (P003 in vCtrl, P021 in asGAS, and P024 in nCtrl) whose gene expression profiles were outliers. We applied unsupervised hierarchical clustering to a set of DEG that comprised the union of DEG from 4 individual comparisons (sGAS, sAdV, asGAS and vCtrl each vs nCtrl). Comparing RNA-seq data with and without these 3 outliers, we observed a minimal effect on the clustering of cases and controls. FIG. 9A shows the differential expression and clustering analyses with the 3 outliers included in the analysis and in the heat map. Two clusters were defined, with cluster 1 comprising all 16 symptomatic cases and 2 asymptomatic controls, and cluster 2 comprising the remaining 19 asymptomatic controls. Two of the 3 outliers (P021 , P024) clustered with the symptomatic cases and 1 (P003) clustered with the asymptomatic controls. FIG. 9B shows the same analysis as in (A) but excluding the 3 outliers from the analysis and from the heatmap. The clustering heatmap again yielded 2 clusters with all 16 symptomatic cases in cluster 1 and all 18 asymptomatic controls in cluster 2.
[0016] FIG. 10 depicts ROC curves for additional comparisons between clinical subgroups. [0017] FIG. 11 depicts the comparison of fold-changes in classifier genes used to distinguish between sGAS vs. nCtrl classifier genes generated in the present study with fold-changes in the same genes from 4 human gene expression studies of bacterial infections retrieved from GEO. Each of those 4 studies analyzed gene expression using microarrays, unlike the present study with used RNA-seq. Black bars show a significant fold-change at P <0.05 and open bars indicate a non-significant foldchange with P >0.05. Notes: *1 was from GSE72829 [discovery set] (21 ), including 52 samples with bacterial infection: 10 Streptococcus pneumoniae, 10 Streptococcus pyogenes, 17 Neisseria meningitis, 4 group B Streptococcus (GBS), 11 others, and 52 healthy controls). Linkage of microarray profiles with individual species was not available. *2 from GSE42026 (25), including 18 samples with gram-positive bacterial infection: 12 S. pneumoniae, 4 S. pyogenes, 2 Staphylococcus aureus, and 33 healthy controls). Linkage of microarray profiles with individual species was also not available. *3 from GSE64456 [R2 set] (26), including 55 samples with bacterial infection: 36 Escherichia coli, 3 Enterococcus, 6 GBS, 3 S. aureus, 7 others, and 14 healthy controls), and *4 from GSE40396 (20), including 8 samples with bacterial infection: 2 E. coli, 2 methicillin-susceptible S. aureus (MSSA), 2 methicilliin-resistant S. aureus (MRSA), 2 others, and 22 healthy controls).
DETAILED DESCRIPTION
[0018] Pharyngitis, commonly known as sore throat, is an inflammation of the pharynx, resulting in a sore throat. Thus, pharyngitis is a symptom, rather than a condition. The etiology is usually infectious, with most cases being of viral origin. Bacterial causes of pharyngitis are also self-limiting but are concerning because of suppurative and nonsuppurative complications. The most significant bacterial agent causing pharyngitis in both adults and children is group A Streptococcus (GAS) infection, and the most common viruses are rhinovirus and adenovirus. Streptococcal infections are characterized by local invasion and release of extracellular toxins and proteases. In addition, M protein fragments of certain serotypes of GAS are similar to myocardial sarcolemma antigens and are linked to rheumatic fever and subsequent heart valve damage. Acute glomerulonephritis may result from antibody-antigen complex deposition in glomeruli.
[0019] Recently rapid molecular tests for detection of GAS have been cleared by the Food and Drug Administration for use in clinical practice. These tests use the polymerase chain reaction or other nucleic acid amplification methods, have sensitivity comparable to or exceeding that of culture, and can be performed in 30 minutes or less. Some of the test methods have waived status under the Clinical Laboratory Improvement Amendments (CLIA). The simplicity, rapidity, and sensitivity of these tests suggest that they may be used without back-up culture, thus greatly simplifying the clinical approach to the patient with pharyngitis by converting the 2-step algorithm to 1-step. Clinical studies to validate this approach have not yet been published as of this writing, and professional societies have not yet released definitive recommendations for incorporating molecular rapid strep tests into practice.
[0020] A weakness of any diagnostic approach for GAS that relies on direct detection of the organism is the common occurrence of asymptomatic colonization (also referred to as the carrier state), especially in children who are also at greatest risk for symptomatic GAS. A recent meta-analysis reported that GAS carriage was present in 12% of well children. The frequency of carriage is lower in children less than 5 years of age and in adults. Antibiotic therapy is not indicated for carriers, and failure to distinguish between symptomatic infection and carriers can be misleading and result in unnecessary antibiotic related to treating carriers with symptomatic viral infection. The magnitude of this problem is underscored by a recent estimate that 1 .5 million cases of GAS carriage are present in individuals with pharyngitis in the US, potentially translating into a large volume of unnecessary antibiotic usage.
[0021] Distinguishing between bacterial and viral infection and between symptomatic and asymptomatic infection are required for accurate diagnosis of GAS pharyngitis. The present disclosure generally relates to detection methods that overcome many of the limitations of current methods for the determination of the etiology of pharyngitis. Applicants have discovered that certain methods to quantify and analyze host analytes can be used to classify asymptomatic carrier, symptomatic infection and healthy subjects. The present disclosure optimizes treatment of a subject having or suspected of having pharyngitis. As such, the present disclosure provides methods for guiding appropriate use of antimicrobials and evaluate the clinical efficacy of certain therapeutic interventions, thereby aiding against the rising tide of antimicrobial resistance.
[0022] Additional aspects of the invention are described below.
(I) METHODS
[0023] One aspect of the present disclosure encompasses determining the amount of an analyte expression signature which is useful to indicate a symptomatic GAS infection or viral infection cause of pharyngitis in a subject. Suitable analytes were identified according to Example 1 and describe herein. The present disclosure is not limited to the specific analytes recited in the examples as a skilled artisan could identify other suitable analytes using the techniques disclosed herein. Examining host analyte signature induced by a symptomatic GAS infection, an asymptomatic GAS carrier, a viral infection an asymptomatic viral carrier or healthy subject shows distinct analyte profiles which can then be used to determine the etiology of the infection and guide treatment decisions.
[0024] An analyte, as used herein, refers to a substance in a biological sample that may be measured as an indication of the health/disease etiology of a subject. As used herein, "analyte" is equivalent to “feature” or "biomarker." For instance, an analyte may be an RNA molecule, may be a protein (e.g. a chemokine, toxin, an antibody, or other protein), an amino acid, a fatty acid (e.g. a short chain fatty acid), a bile acid, a carbohydrate or carbohydrate moiety (e.g. a sugar, a starch, or a proteoglycan), a lipid or lipid moiety, a nucleotide or nucleotide sequence, or other biomolecule. An analyte may be extracellular, or the biological sample may be treated to release an intracellular analyte using means known in the art. Alternatively, an analyte may be present on the surface of a cell in a biological sample. Such samples, in some embodiments, may be treated to release the analyte from the cell membrane or cell surface using means known in the art.
(a) Host analyte Signatures [0025] An analyte signature refers to a characteristic expression profile of a single or a group of analytes that is indicative of an altered or unaltered biological process, medical condition, or a subject’s responsiveness/non-responsiveness to a specific pathogen. The analyte signatures disclosed herein encompass characteristic profiles of at least one analyte selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, TLR5, FFAR3, SPATC1 , KCNH7, ADM, IL18RAP, LY6G6C, ARG1 , FCGR1 B, and TAS2R40 which are identified as differentially expressed in a biological sample obtained from a subject relative to a reference value. See, e.g., Example 1 below. In various embodiments, determining analyte levels can be supplemented with diagnostic assays such as assays to determine presence, absence, and/or quantity of a pathogen, clinical assays (e.g., those described in the below examples), advanced radiographic assays, diagnostic assays (e.g., PCR and/or ELISA) and aspiration.
[0026] The analyte signatures as disclosed herein may represent the expression profile of at least one analyte, for example, at least 2 analytes, at least 3 analytes, at least 4 analytes, at least 5 analytes, at least 6 analytes, at least 7 analytes, at least 8 analytes, at least 9 analytes, at least 10 analytes, at least 15 analytes, at least 20 analytes, at least 25 analytes, at least 30 analytes, at least 35 analytes, at least 40 analytes, at least 45 analytes, at least 50 analytes or more. In some examples, the analyte signature may comprise multiple analytes with increased levels relative to a reference value. In other examples, the analyte signature may comprise multiple analytes with decreased levels relative to a reference value. In yet other examples, the analyte signature may comprise both increased and decreased analyte levels relative to a reference value.
[0027] In some embodiments, the analyte signature may comprise multiple analytes involved in multiple biological pathways, for example, 2 biological pathways, 3 biological pathways, 4 biological pathways, 5 biological pathways, 6 biological pathways, 7 biological pathways, 8 biological pathways, 9 biological pathways, 10 biological pathways, 11 biological pathways, 12 biological pathways, 13 biological pathways, 14 biological pathways, or 15 biological pathways. [0028] In various embodiments, a modulated host analyte signature may comprise any analyte with altered expression during the innate immune reaction of the host (e.g., the immune response of the pharyngeal tissue or blood). In various embodiments, the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis relative to a subject that does not have a pharyngitis. In another embodiment, the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis caused by a GAS relative to a subject that does not have a pharyngitis or a subject that is an asymptomatic GAS carrier. In still another embodiment, the modulated host analyte may be any analyte the expression of which changes in a subject having pharyngitis caused by a viral infection relative to a subject having pharyngitis caused by a GAS infection or a subject that is an asymptomatic viral carrier. The term "signature" as used herein refers to a set of biological analytes and the measurable quantities of said analytes whose particular combination signifies the presence or absence of the specified biological state or etiology thereof. These signatures are discovered in a plurality of subjects with known status (e.g., with a confirmed bacterial infection, or viral infection), and are discriminative (individually or jointly) of one or more categories or outcomes of interest. These measurable analytes can be (but are not limited to) nucleic acid expression levels, protein or peptide levels, metabolite levels, white blood cell levels or neutrophil levels. In some embodiments, the analyte signature as disclosed herein may be combined with testing to detect the etiology of the infection (e.g., culturing pathogens from host samples, detecting viral or bacterial proteins or nucleotides).
[0029] In some embodiments as disclosed herein, the "signature" is a particular combination of analytes whose expression levels discriminate a condition such as a bacterial GAS pharyngitis, a viral pharyngitis, an asymptomatic viral carrier or a GAS asymptomatic carrier. See, for example, Table 5. The nucleotide and amino acid sequence information for the various host analytes suitable for use in the methods of the present disclosure can be found using a reference data base known in the art, for example, UniProt. In some embodiments, a modulated host analyte suitable for use in the present methods includes one or more of, CD177 (see, e.g., NCBI Reference Sequence: XM_017027021 .2 and XP_016882510), FAM20A (see, e.g., NCBI Reference Sequence: NM_001243746.2 and NP_001230675.1 ), CASP5 (see, e.g., NCBI Reference Sequence: NM_001136109.3 and NP_001129581.1 ), ZDHHC19 (see, e.g., NCBI Reference Sequence: XM_006713494.2 and XP_006713557.1 ), FCGR1A (see, e.g., NCBI Reference Sequence: NM_000566.4 and NP_000557.1 ), ITGA7 (see, e.g., NCBI Reference Sequence: NM_001144996.2 and NP_001138468.1 ), S0CS3 (see, e.g., NCBI Reference Sequence: NM_O01378932.1 and NP_001365861.1 ), MGAM2 (see, e.g., NCBI Reference Sequence: NM_001293626.2 and NP_001280555.1 ), CACNA1 E (see, e.g., NCBI Reference Sequence: NM_000721 .4 and NP_000712.2), MCEMP1 (see, e.g., NCBI Reference Sequence: NM_174918.3 and NP_777578.3), ANXA3 (see, e.g., NCBI Reference Sequence: NM_005139.3 and NP_005130.1 ), CD274 (see, e.g., NCBI Reference Sequence: NM_001267706.2 and NP_001254635.1 ), TLR5 (see, e.g., NCBI Reference Sequence: NM_003268.6 and NP_003259.2), FFAR3 (see, e.g., NCBI Reference Sequence: NM_005304.5 and NP_005295.1 ), SPATC1 (see, e.g., NCBI Reference Sequence: NM_001134374.2 and NP_001127846.1 ), KCNH7 (see, e.g., NCBI Reference Sequence: NM_033272.4 and NP_150375.2), ADM (see, e.g., NCBI Reference Sequence: NM_001124.3 and NP_001115.1 ), IL18RAP (see, e.g., NCBI Reference Sequence: NM_001393488.1 and NP_001380417.1 ), LY6G6C (see, e.g., NCBI Reference Sequence: NM_025261.3 and NP_079537.1 ), ARG1 (see, e.g., NCBI Reference Sequence: NM_000045.4 and NP_000036.2), FCGR1 B (see, e.g., NCBI Reference Sequence: NR_045213.2 and NR_164758.1 ), and TAS2R40 (see, e.g., NCBI Reference Sequence: NM_176882.2 and NP_795363.1 ).
[0030] In some embodiments, the methods may comprise determining the expression level of at least 1 analyte, at least 2 analytes, at least 3 analytes, at least 4 analytes, at least 5 analytes, at least 6 analytes, at least 6 analytes, at least 7 analytes, at least 8 analytes, at least 9 analytes, at least 10 analytes, at least 1 1 analytes, at least 12 analytes, at least 13 analytes, at least 14 analytes, at least 15 analytes, at least 16 analytes, at least 17 analytes, at least 18 analytes, at least 19 analytes, at least 20 analytes, at least 21 analytes, at least 22 analytes, at least 23 analytes, at least 24 analytes, at least 25 analytes, at least 26 analytes, at least 27 analytes, at least 28 analytes, at least 29 analytes, at least 30 analytes, at least 31 analytes, at least 32 analytes, at least 33 analytes, at least 34 analytes, at least 35 analytes, at least 36 analytes, at least 37 analytes, at least 38 analytes, at least 39 analytes, at least 40 analytes, at least 41 analytes, at least 42 analytes, or at least 43 analytes, at least 44 analytes, at least 45 analytes, at least 46 analytes, at least 47 analytes, at least 48 analytes, at least 49 analytes, at least 50 analytes, at least 51 analytes, at least 52 analytes, at least 53 analytes, at least 54 analytes, at least 55 analytes, at least 56 analytes, at least 57 analytes, at least 58 analytes, at least 59 analytes, at least 60 analytes, at least 61 analytes, at least 62 analytes, at least 63 analytes, at least 64 analytes, at least 65 analytes, at least 66 analytes, at least 67 analytes, at least 68 analytes, at least 69 analytes, at least 70 analytes, at least 71 analytes, at least 72 analytes, at least 73 analytes, at least 74 analytes, at least 75 analytes, at least 76 analytes, at least 77 analytes, at least 78 analytes, at least 79 analytes, at least 80 analytes, at least 81 analytes, at least 82 analytes, at least 83 analytes, at least 84 analytes, at least 85 analytes, at least 86 analytes, at least 87 analytes, at least 88 analytes, at least 89 analytes, at least 90 analytes, at least 91 analytes, at least 92 analytes, at least 93 analytes, at least 94 analytes, at least 95 analytes, at least 96 analytes, at least 97 analytes, at least 98 analytes, at least 99 analytes, at least 100 analytes, at least 101 analytes, at least 102 analytes, at least 103 analytes, at least 104 analytes, at least 105 analytes, at least 106 analytes, at least 107 analytes, at least 108 analytes, at least 109 analytes, at least 110 analytes at least 111 analytes, at least 112 analytes, at least 113 analytes, at least 114 analytes, at least 115 analytes, at least 116 analytes, at least 117 analytes, at least 118 analytes, at least 119 analytes, at least 120 analytes, at least 121 analytes, at least 122 analytes, at least 123 analytes, at least 124 analytes, at least 125 analytes, at least 126 analytes, at least 127 analytes, at least 128 analytes, at least 129 analytes, at least 130 analytes, at least 131 analytes, at least 132 analytes, at least 133 analytes, at least 134 analytes, at least 135 analytes, at least 136 analytes, at least 137 analytes, at least 138 analytes, at least 139 analytes, at least 140 analytes, at least 141 analytes, at least 142 analytes, at least 143 analytes, at least 144 analytes, at least 145 analytes, at least 146 analytes, at least 147 analytes, at least 148 analytes, at least 149 analytes , at least 150 analytes, at least 151 analytes, at least 152 analytes, at least 153 analytes, at least 154 analytes, at least 155 analytes, at least 156 analytes, at least 157 analytes, at least 158 analytes, at least 159 analytes, at least 160 analytes, at least 161 analytes, at least 162 analytes, at least 163 analytes, at least 164 analytes, at least 165 analytes, at least 166 analytes, at least 167 analytes, at least 168 analytes, at least 169 analytes, at least 170 analytes, at least 171 analytes, at least 172 analytes, at least 173 analytes, at least 174 analytes, at least 175 analytes, at least 176 analytes, at least 177 analytes, at least 178 analytes, at least 179 analytes, at least 180 analytes, at least 181 analytes, at least 182 analytes, at least 183 analytes, at least 184 analytes, at least 185 analytes, at least 186 analytes, at least 187 analytes, at least 188 analytes, at least 189 analytes, at least 190 analytes, at least 191 analytes, at least 192 analytes, at least 193 analytes, at least 194 analytes, at least 195 analytes, at least 196 analytes, at least 197 analytes, at least 198 analytes, at least 199 analytes, at least 200, at least 250, at least 300 or at least 350 analytes.
[0031] "Rule in" or "rule out" tests (with threshold set for high positive predictive value or high negative predictive value respectively) support appropriate medical decision making and efficient utilization of resources when well-understood by health care providers. Various embodiments comprise a method to rule in pharyngitis mediated by a GAS infection, or pharyngitis meditated by a viral infection using one or more host modulated analytes but other embodiments may comprise determining the level of two, three or more modulated host analytes as noted above. In certain embodiments, the disclosure provides a method comprising determining the level of two or more modulated host analytes, the level of one or more of the modulated host analytes may be measured as an indicator of sample quality. In some embodiments, tests to measure the level of two or more analytes may be combined with tests for the presence of a virus or bacterium.
(b) Biological Sample
[0032] In an aspect, the disclosure provides a method to detect modulated levels of host analytes in a biological sample obtained from a subject. As used herein, the term “biological sample” means a biological material isolated from a subject. Any biological sample containing any biological material suitable for detecting the desired analytes may be used and may comprise cellular and/or non-cellular material obtained from the subject is suitable. Non-limiting examples include blood, plasma, serum, urine, and tissue. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Typical clinical samples include, but are not limited to, bodily fluid samples such as synovial fluid, sputum, blood, urine, blood plasma, blood serum, sweat, mucous, saliva, lymph, bronchial aspirates, peritoneal fluid, cerebrospinal fluid, and pleural fluid, and tissues samples such as blood-cells (e.g., white cells), tissue or fine needle biopsy samples and abscesses or cells therefrom. As used herein, the term “blood sample” refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample can be whole blood, plasma or serum, although whole blood is typically preferred.
[0033] In a specific embodiment, the sample may include a respiratory sample. The term "respiratory sample" as used herein means any sample from a subject containing RNA or proteins a plurality of which is generated by cells in the upper or lower respiratory tract. Non-limiting examples include nasal swabs, nasopharyngeal swabs, nasopharyngeal aspirate, oral swab, oropharyngeal swab, pharyngeal (throat) swab, throat wash or gargle, sputum, tracheal aspirate, bronchoalveolar lavage or saliva or transport medium exposed to any of these sample types. In one aspect, the sample is a nasopharyngeal sample. A "nasopharyngeal sample" as used herein means any sample from a subject containing RNA and/or proteins, a plurality of which is generated by cells in the upper respiratory tract. Non-limiting examples include nasal swabs, nasopharyngeal swabs, nasopharyngeal aspirate, oral swab, oropharyngeal swab, pharyngeal swabs, throat swabs, saliva or medium exposed to any of these sample types. In an exemplary embodiment, the biological sample is obtained using a midturbinate swab.
[0034] As will be appreciated by a skilled artisan, the method of collecting a biological sample can and will vary depending upon the nature of the biological sample and the type of analysis to be performed. Any of a variety of methods generally known in the art may be utilized to collect a biological sample. Generally speaking, the method preferably maintains the integrity of the sample such that the mRNA or protein can be accurately detected and the amount measured according to the disclosure.
[0035] In some embodiments, a single sample is obtained from a subject to detect a host gene signature in the sample. Alternatively, a host gene signature may be detected in samples obtained over time from a subject. As such, more than one sample may be collected from a subject over time. For instance, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or more samples may be collected from a subject over time. In some embodiments, 2, 3, 4, 5, or 6 samples are collected from a subject over time. In other embodiments, 6, 7, 8, 9, or 10 samples are collected from a subject over time. In yet other embodiments, 10, 11 , 12, 13, or 14 samples are collected from a subject over time. In other embodiments, 14, 15, 16 or more samples are collected from a subject over time.
[0036] When more than one sample is collected from a subject over time, samples may be collected every 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more hours. In some embodiments, samples are collected every 0.5, 1 , 2, 3, or 4 hours. In other embodiments, samples are collected every 4, 5, 6, or 7 hours. In yet other embodiments, samples are collected every 7, 8, 9, or 10 hours. In other embodiments, samples are collected every 10, 11 , 12 or more hours. Additionally, samples may be collected every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more days. In some embodiments, a sample is collected about every 6 days. In some embodiments, samples are collected every 1 , 2, 3, 4, or 5 days. In other embodiments, samples are collected every 5, 6, 7, 8, or 9 days. In yet other embodiments, samples are collected every 9, 10, 11 , 12 or more days.
(c) Detecting Host analytes
[0037] Methods for assessing an amount of host analyte expression in samples are well known in the art, and all suitable methods for assessing an amount of nucleic acid or protein expression known to one of skill in the art are contemplated within the scope of the invention. By the phrase "determining the level of expression" is meant an assessment of the absolute or relative quantity of an analyte in a sample at the nucleic acid or protein level, using technology available to the skilled artisan to detect a sufficient portion of any marker.
[0038] The term “amount of nucleic acid expression” or “level of nucleic acid expression” as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of mRNA transcript expressed or a specific variant or other portion of the RNA. The term “nucleic acid” includes DNA and RNA and can be either double stranded or single stranded, although RNA is typically preferred. Non-limiting examples of suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses. In a specific embodiment, determining the amount of a RNA comprises, in non-limiting examples, measuring the level of mRNA expression, tRNA expression, rRNA expression, snRNA expression miRNA expression, IncRNA expression, tmRNA expression, and/or snoRNA expression.
[0039] In one embodiment, the amount of nucleic acid expression may be determined by using an array, such as a microarray. Methods of using a nucleic acid microarray are well and widely known in the art. For example, a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array. The term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. The term “probe” as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence complementary thereof. The length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length. [0040] In general, the methods include any know techniques for amplifying nucleic acids, including but not limited to PCR, loop-mediated isothermal amplification, and nucleic acid sequence-based amplification (NASBA). In one aspect, the amount of nucleic acid expression may be determined using PCR. A nucleic acid may be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any combination thereof. Specifically, the amount of nucleic expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art. In such an embodiment, the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene. The term “primer” as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less or more. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art.
[0041] The amount of nucleic acid expression may be measured by measuring an entire RNA transcript for a nucleic acid sequence or measuring a portion of the RNA transcript for a nucleic acid sequence. For instance, if a nucleic acid array is utilized to measure the amount of RNA expression, the array may comprise a probe for a portion of the RNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full RNA of the nucleic acid sequence of interest. Similarly, in a PCR reaction, the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence. One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest. Methods of designing primers are known in the art. Methods of extracting RNA from a biological sample are known in the art.
[0042] The level of expression may or may not be normalized to the level of a control nucleic acid. Such a control nucleic acid should not specifically hybridize with an RNA nucleotide sequence of the invention. This allows comparisons between assays that are performed on different occasions.
[0043] In other embodiments, a method of the disclosure comprises detecting the expression levels of one or more host analytes at the protein level. In essence, a host protein may be detected using methods normally used in the art for detecting a specific protein in a sample. As such, non-limiting examples of methods of detecting a protein adduct may include chromatography, mass spectrometry, an antibody-based detection method, or a combination thereof, and may be as discussed in Ausubel et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.
[0044] In some embodiments, host protein levels are detected using mass spectrometry. Mass spectrometry may be tandem mass spectrometry, quadrupole mass spectrometry, MALDI-TOF mass spectrometry, inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), and spark source mass spectrometry (SSMS). In specific embodiments, host protein levels are detected using a mass spectrometry method capable of detecting a specific protein. Non-limiting examples of mass spectrometry methods capable of detecting a specific protein include MALDI-TOF mass spectrometry and high-resolution tandem mass spectrometry.
[0045] In other embodiments, host protein levels may be detected in a sample using methods based on epitope binding agents. Non-limiting examples of suitable epitope binding agents, depending upon the target molecule, include agents selected from the group consisting of an aptamer, an antibody, an antibody fragment, a double-stranded DNA sequence, modified nucleic acids, nucleic acid mimics, a ligand, a ligand fragment, a receptor, a receptor fragment, a polypeptide, a peptide, a coenzyme, a coregulator, an allosteric molecule, and an ion.
[0046] In some specific alternatives of the embodiments, an epitope binding agent is an antibody, and host protein levels are detected using antibody-based methods. Non-limiting examples of antibodies that may be used include polyclonal antibodies, ascites, Fab fragments, Fab' fragments, monoclonal antibodies, single chain antibodies, humanized antibodies, and other fragments that contain the epitope binding site of the antibody. Antibody based methods that may be used to detect a protein are known in the art. Non-limiting examples of methods based on antibodies may include Western blotting, enzyme-linked immunosorbent assays (ELISA), or other solid phase immunoassays, a sandwich immunoassay, radioimmunoassay, nephelometry, electrophoresis, immunofluorescence, immunoblot, flow cytometry, immunohistochemistry, an array or other methods (see Ausubel, F. M. et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, including supplements through 2001 ).
[0047] In general, an antibody-based method of detecting and measuring an amount of a protein comprises contacting some or all of the sample comprising a protein with an antibody specific for the protein under conditions effective to allow for formation of a complex between the antibody and the protein. Typically, the entire sample is not needed, allowing one skilled in the art to repeatedly detect and measure the amount of one or more proteins in the sample over time. The method may occur in solution, or the antibody or protein may be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers. Attachment to the substrate may occur in a wide variety of ways, as will be appreciated by those in the art. For example, the substrate and the antibody may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the antibody may be attached directly using the functional groups or indirectly using linkers. The antibody may also be attached to the substrate non-covalently. For example, a biotinylated antibody may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, an antibody may be synthesized on the surface using techniques such as photopolymerization and photolithography.
[0048] Contacting the sample with an antibody under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibody to bind to any antigen present. After this time, the complex may be washed and then the complex is detected and the amount measured by any method well known in the art. Methods of detecting and measuring an amount of an antibody-polypeptide complex are generally based on the detection of a label or marker. The term “label”, as used herein, refers to any substance attached to an antibody, or other substrate material, in which the substance is detectable by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, glucose oxidase and luciferase). Methods of detecting and measuring an amount of an antibody-polypeptide complex based on the detection of a label or marker are well known in the art.
[0049] In some embodiments, an antibody-based method is an immunoassay. Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immmunoassays and non-competitive immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away, and the bound analyte is measured. In a noncompetitive immunoassay, the antibody is labeled, not the analyte. Non-competitive immunoassays may use one antibody (e.g. the capture antibody is labeled) or more than one antibody (e.g. at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.
[0050] In other embodiments, an antibody-based method is an immunoblot or Western blot. In yet other embodiments, an antibody-based method is flow cytometry. In different embodiments, an antibody-based method is immunohistochemistry (IHC). IHC uses an antibody to detect and quantify antigens in intact tissue samples. The tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art.
[0051] In alternative embodiments, an antibody-based method is an array. An array comprises at least one address, wherein at least one address of the array has disposed thereon an antibody specific for a host analyte as disclosed herein. Arrays may comprise from about 1 to about several hundred thousand addresses. Several substrates suitable for the construction of arrays are known in the art, and one skilled in the art will appreciate that other substrates may become available as the art progresses. Suitable substrates are also described above. In some embodiments, the array comprises at least one host protein antibody attached to the substrate is located at one or more spatially defined addresses of the array. For example, an array may comprise at least one, at least two, at least three, at least four, or at least five host protein specific antibodies, each antibody recognizing the same or different protein, and each antibody may be may be at one, two, three, four, five, six, seven, eight, nine, ten or more spatially defined addresses.
[0052] For each of the foregoing embodiments, a protein may be first isolated or enriched before detection. For instance, proteins may be enriched or isolated using liquid chromatography, by precipitation, electrophoresis, or affinity purification. In some embodiments, proteins are enriched or purified using liquid chromatography. In other embodiments, proteins are enriched or purified using electrophoresis.
[0053] In specific embodiments, proteins are enriched or purified by affinity purification before detection. In particularly specific embodiments, proteins are enriched or purified by affinity purification using antibodies with specificity to the protein. Methods of enriching a sample for a protein or purifying a protein using affinity purification are known in the art. In short, affinity purification comprises incubating a sample with a solid support, such as beads, a culture plate, or a membrane, that facilitates later steps. A solid support may be coated with antibodies specific to a protein, causing the protein to attach to the solid support. Alternatively, a sample may be incubated with a first antibody with specificity to a protein, and the protein-antibody complex may be isolated by incubating with a solid support coated with a second antibody with specificity against a second site on said first antibody, causing a protein-antibody complex to attach to the solid support. The proteins may then be purified or enriched by washing other material in the sample that is not bound to the solid support, or, if the solid support is superparamagnetic beads, proteins attached to the beads (expressing the antigen) may be separated from the sample by attraction to a strong magnetic field. Upon enrichment or purification of a protein, the protein may then be detected in the enriched or purified sample using any of the methods described above.
[0054] The disclosure also provides that multiple proteins in the same biological sample may be measured simultaneously. Additionally, the disclosure provides that the proteins and corresponding non-specific proteins may be detected in the same biological sample. As such, the disclosure provides a useful method for screening changes in synthesis and clearance of host proteins on a large scale (i.e. , proteomics/metabolomics) and provides a sensitive means to detect and measure host gene signatures in response to infection. Non-limiting examples of proteins for use within the methods include procalcitonin and c-reactive protein.
(d) Classification of a Subject
[0055] "Classification" refers to a method of assigning a subject suffering from or at risk for pharyngitis symptoms to one or more categories or outcomes (e.g., a subject has a symptomatic infected with a pathogen or is a asymptomatic carrier of a pathogen, another categorization may be that a patient is infected with a virus or infected with a bacterium). In some, embodiments, a subject may be classified as having a symptomatic infection with a specific bacteria or virus. In some cases, a subject may be classified to more than one category. The outcome, or category, is determined by the expression signature of the subject being tested, which may be compared to a “reference value” which may be a reference signature, confidence level, or limit. In other scenarios, the probability of belonging to a particular category may be given.
[0056] In various embodiments the disclosure is directed to a method of detecting etiology of pharyngitis in a subject. The method generally comprises obtaining a biological sample (e.g., peripheral blood) and determining the level at least one host analyte and comparing the level of the host analyte with its respective reference level. A "reference level" of an analyte means a level of the analyte that is indicative of the absence of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is above the reference level of the analyte it is indicative of the presence of a disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is above the reference level of the analyte it is indicative of the lack of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is below the reference level of the analyte it is indicative of the presence of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is below the reference level of the analyte it is indicative of the lack of a particular disease state, phenotype, or etiology. In some embodiments, when the level of an analyte in a subject is within the reference level of the analyte it is indicative of a lack of a particular disease state, phenotype, or etiology. In one aspect, the reference value may be the level of at least one host analyte in a normal control subject or normal control population. By normal control it is meant a subject or population without pharyngitis and is not a carrier of GAS or virus known to cause pharyngitis. In another aspect, the reference value may be the level of at least one host analyte in an asymptomatic GAS carrier subject or asymptomatic GAS carrier subject population. In another aspect, the reference value may be the level of at least one host analyte in an asymptomatic viral carrier subject or asymptomatic viral carrier subject population. In still another aspect, the reference value may be the level of at least one host analyte in a GAS positive subject with pharyngitis or population of a GAS positive subjects with pharyngitis. In still yet another aspect, the reference value may be the level of at least one host analyte in a viral positive subject with pharyngitis or population of a viral positive subjects with pharyngitis.
[0057] As used herein, the term "indicative" when used with analyte expression levels, means that the analyte expression levels are up-regulated or down- regulated, altered, or changed compared to the expression levels in alternative biological states (e.g., bacterial pharyngitis or viral pharyngitis) or control.
[0058] In some embodiments expression of the modulated host analyte is determined by measuring mRNA. In other embodiments, expression is determined by measuring protein. In embodiments comprising the measurement of protein, a biological sample may be used in an immunoassay. In embodiments comprising the measurement of mRNA, a biological sample may be optionally centrifuged to form a pellet of cells and cell debris which is then added to lysis buffer. Total nucleic acid is isolated from the pellet and DNA is digested using, by way of non-limiting example, DNase I. The RNA is then reverse transcribed into cDNA. The cDNA is then analyzed to determine the level of at least one modulated host analyte. In some embodiments the level of the at least one modulated host analyte is determined by reverse transcription quantitative polymerase chain reaction (rt-qPCR) although the skilled artisan will appreciate that there are other ways that the level of the at least one modulated host analyte may be determined by the analysis of mRNA and these methods are encompassed by the invention in its various embodiments.
[0059] The present disclosure further provides methods for determining whether a patient has a symptomatic bacterial infection, a symptomatic viral infection, an asymptomatic viral infection, an asymptomatic bacterial infection or normal healthy control. The method for making this determination relies upon the host analyte signature as taught herein. The methods may include: a) measuring the expression levels of predefined sets of modulated host analytes; optionally normalizing analyte expression levels for the technology used to make said measurement; and b) comparing the levels of one or more host analytes with a reference value. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of the presence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of the presence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of a symptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of an asymptomatic bacterial infection. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of the presence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of the presence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of a symptomatic viral infection. In some embodiments, when the level of the one or more host analytes is above the reference level it is indicative of the presence of an asymptomatic viral infection. In some embodiments, when the level of the one or more host analytes is below the reference level it is indicative of the presence of an asymptomatic viral infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the presence of an asymptomatic viral infection. In some embodiments, when the level of the one or more host analytes is about the reference level it is indicative of the absence of an asymptomatic viral infection. [0060] In one aspect, the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the expression level of one or more host analytes selected from CD177, FAM20A, FCGR1A, and, CD274; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, FCGR1A, and, CD274 exhibit increased expression relative to a healthy control reference value.
[0061] In another aspect, the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a healthy control reference value. In another embodiment, the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a viral carrier reference value.
[0062] In yet another aspect, the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to a healthy control reference value. In another embodiment, the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to an asymptomatic GAS carrier reference value.
[0063] In still another aspect, the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host analytes selected from CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a healthy control reference value. In another embodiment, the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a symptomatic viral infection reference value.
[0064] In another aspect, the disclosure provides a method of determining etiology of pharyngitis in a subject, the method comprising a) obtaining at least one biological sample from the subject, wherein the biological sample is optionally a blood sample; b) detecting the level or one or more host genes selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5; and c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 exhibit increased expression relative to reference value.
[0065] In each of the above embodiments, the methods may further comprise administering an anti-bacterial drug when the subject is determined to have pharyngitis caused by bacterial etiology or administering an anti-viral drug when the subject is determined to have pharyngitis caused by viral etiology.
[0066] Generally speaking, a host analyte as disclosed herein may be classified as differentially expressed or aberrant when it has an increased or decreased amount relative to a reference value. Any suitable reference value known in the art may be used. For example, a suitable reference value may be the amount of a host analyte in a biological sample obtained from a subject, or group of subjects, of the same species that has no clinically detectable symptom of a pharyngitis. In another example, a suitable reference value may be the amount of a host analyte in a biological sample obtained from a subject, or group of subjects, of the same species that has no detectable pharyngitis infection pathology. In another example, a suitable reference value may be the background signal of the assay as determined by methods known in the art. In another example, a suitable reference value may be a measurement of the amount of a host analyte in a reference sample obtained from the same subject. The reference sample comprises the same type of biological sample as the test sample and may be obtained from a subject when the subject had no clinically detectable symptom of pharyngitis. A skilled artisan will appreciate that it is not always possible or desirable to obtain a reference sample from a subject when the subject is otherwise healthy. For example, when monitoring the effectiveness of a therapy or progression of disease, a reference sample may be a sample obtained from a subject before therapy or at an earlier point in the disease. In such an example, a subject may have a risk of infection but may not have other symptoms of an infection or the subject may have one or more other symptom of a pharyngitis. In an additional example, a suitable reference sample may be a biological sample from an individual or group of individuals that has been shown not to have pharyngitis. In an embodiment, the reference value may be a sample of the same type of biological sample obtained from one or more individuals that has not been administered therapy but has a respiratory infection.
[0067] In certain embodiments, to classify the amount of a host analyte as increased in a biological sample, the amount of the host analyte in the biological sample compared to the reference value is increased at least 1-fold. For example, the amount of the host analyte in the sample compared to the reference value is increased at least 1-fold, at least 1 .25-fold, at least 1 .5-fold, at least 1 .75-fold, at least 2-fold, at least 5- fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold. In a specific embodiment, the amount of the host analyte in the sample compared to the reference value is increased at least 10-fold.
[0068] In certain embodiments, to classify the amount of a host analyte as decreased in a biological sample, the amount of the host analyte in the biological sample compared to the reference value is decreased at least 1-fold. For example, the amount of the host analyte in the sample compared to the reference value is decreased at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30- fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold.
[0069] In another embodiment, the increase or decrease in the amount of a host analyte is measured using p-value. For instance, when using p-value, a RNA is identified as being differentially expressed between a biological sample and a reference value when the p-value is less than 0.1 , preferably less than 0.05, more preferably less than 0.01 , even more preferably less than 0.005, the most preferably less than 0.001 .
(e) Treatment
[0070] In another aspect, the disclosure provides a method of treating a subject exhibiting symptoms of a pharyngitis, the method comprising: a) measuring the expression levels of one or more modulated host analytes; optionally normalizing analyte expression level for the technology used to make said measurement; b) comparing the levels of one or more host analytes with a reference value; and, wherein if the normalized level of the one or more host analyte is above or below the reference level the subject is classified for the presence or absence of a GAS pharyngitis or viral pharyngitis. The method may further comprise, testing the subject for the presence of at least one virus or bacteria. Non-limiting examples of symptoms of pharyngitis include sore throat; dry, scratchy throat; pain when swallowing; and pain when speaking
[0071] In another aspect, the invention provides a method of treating a subject exhibiting symptoms of pharyngitis, the method comprising: a) measuring the expression levels of one or more modulated host analytes; optionally normalizing analyte expression level for the technology used to make said measurement; b) comparing the levels of one or more host analytes with a reference value; and, wherein if the normalized level of the one or more host analyte is above or below the reference level the subject is classified for the presence or absence of a GAS pharyngitis or viral pharyngitis, the subject receives an appropriate treatment regimen for pharyngitis. The method may further comprise, testing the subject for the presence of at least one respiratory virus or bacteria; or determining the number of white blood cells and/or neutrophils in a biological sample obtained from the subject.
[0072] In certain aspects, a therapeutically effective amount of a pharmaceutical composition may be administered to a subject. Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system. Peripheral administration includes but is not limited to oral, inhalation, intravenous, intraperitoneal, intra-articular, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation. The route of administration may be dictated by the disease or condition to be treated.
[0073] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners.
[0074] For therapeutic applications, a therapeutically effective amount of a composition of the invention is administered to a subject. A “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response. Actual dosage levels of active ingredients in a therapeutic composition of the invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, age, the symptoms, and the physical condition and prior medical history of the subject being treated. As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the severity, duration and/or progression of a disease or disorder or one or more symptoms thereof resulting from the administration of one or more therapies. Such terms refer to a reduction in the replication of a virus or bacteria, or a reduction in the spread of a virus or bacteria to other organs or tissues in a subject or to other subjects. The term "effective amount" refers to an amount of a therapeutic agent that is sufficient to exert a physiological effect in the subject.
[0075] The term "appropriate treatment regimen" refers to the standard of care needed to treat a specific disease or disorder. Often such regimens require the act of administering to a subject a therapeutic agent(s) capable of producing a curative effect in a disease state. For example, a therapeutic agent for treating a subject having bacteremia is an antibiotic which include, but are not limited to, penicillins, cephalosporins, fluroquinolones, tetracyclines, macrolides, and aminoglycosides. A therapeutic agent for treating a subject having a viral respiratory infection includes, but is not limited to, oseltamivir, RNAi antivirals, inhaled ribavirin, monoclonal antibody respigam, zanamivir, and neuraminidase blocking agents. The invention contemplates the use of the methods of the invention to determine treatments with antivirals or antibiotics that are not yet available.
[0076] Often such regimens require the act of administering to a subject a therapeutic agent(s) capable of producing reduction of symptoms associated with a disease state. Examples such therapeutic agents include, but are not limited to, NSAIDS, acetaminophen, anti-histamines, beta-agonists, anti-tussives or other medicaments that reduce the symptoms associated with the disease process. [0077] In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
[0078] The frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms. The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.
[0079] Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.
[0080] A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In still another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human.
[0081] Additionally, a subject in need thereof may be a subject suffering from, suspected of suffering from or at risk of pharyngitis.
(f) Kits
[0082] In still other aspects, the present invention provides articles of manufacture and kits containing materials useful for treating the conditions described herein. The article of manufacture may include a container of a composition as described herein with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition comprising reagents to detect a respiratory illness in a patient and instructions for the use thereof, wherein the instructions comprise: analyzing one or more host analytes to determine a level of at least one modulated host analyte; comparing the level of the at least one modulated host analyte with a predetermined reference level; and, wherein if the level of the at least one modulated host analyte is above or below the respective reference level, the patient is determined to have GAS pharyngitis, viral pharyngitis, or a GAS carrier.
DEFINITIONS
[0083] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
[0084] 'About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.
[0085] The terms "antimicrobial" or "antimicrobial agent" mean any compound with bactericidal or bacteriostatic activity which may be used for the treatment of bacterial infection. Non-limiting examples include antibiotics.
[0086] "Measuring" or "measurement," or alternatively "detecting" or "detection," means determining the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise determining the values or categorization of a subject's clinical parameters. [0087] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
[0088] "Platform" or "technology" as used herein refers to an apparatus (e.g., instrument and associated parts, computer, computer-readable media comprising one or more databases as taught herein, reagents, etc.) that may be used to measure a signature, e.g., gene expression levels, in accordance with the present disclosure. Examples of platforms include, but are not limited to, an array platform, a thermal cycler platform (e.g., multiplexed and/or real-time PCR platform), a nucleic acid sequencing platform, a hybridization and multi-signal coded (e.g., fluorescence) detector platform, etc., a nucleic acid mass spectrometry platform, a magnetic resonance platform, and combinations thereof.
[0089] In some embodiments, the platform is configured to measure gene expression levels semi-quantitatively, that is, rather than measuring in discrete or absolute expression, the expression levels are measured as an estimate and/or relative to each other or a specified marker or markers (e.g., expression of another, "standard" or "reference," gene).
[0090] In some embodiments, semi-quantitative measuring includes "real- time PCR" by performing PCR cycles until a signal indicating the specified mRNA is detected, and using the number of PCR cycles needed until detection to provide the estimated or relative expression levels of the genes within the signature.
[0091] A real-time PCR platform includes, for example, a TaqMan® Low Density Array (TLDA), in which samples undergo multiplexed reverse transcription, followed by real-time PCR on an array card with a collection of wells in which real-time PCR is performed. A real-time PCR platform also includes, for example, a Biocartis IdyllaTM sample-to-result technology, in which cells are lysed, DNA/RNA extracted and real-time PCR is performed and results detected. A real-time PCR platform also includes, for example, CyTOF analysis: CyTOF (Fludigm) is a recently introduced mass- cytometer capable of detecting up to 40 markers conjugated to heavy metals simultaneously on single cells.
[0092] A magnetic resonance platform includes, for example, T2 Biosystems® T2 Magnetic Resonance (T2MR®) technology, in which molecular targets may be identified in biological samples without the need for purification.
[0093] The terms "array," "microarray" and "micro array" are interchangeable and refer to an arrangement of a collection of nucleotide sequences presented on a substrate. Any type of array can be utilized in the methods provided herein. For example, arrays can be on a solid substrate (a solid phase array), such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane. Arrays can also be presented on beads, i.e. , a bead array. These beads are typically microscopic and may be made of, e.g., polystyrene. The array can also be presented on nanoparticles, which may be made of, e.g., particularly gold, but also silver, palladium, or platinum. See, e.g., Nanosphere Verigene® System, which uses gold nanoparticle probe technology. Magnetic nanoparticles may also be used. Other examples include nuclear magnetic resonance microcoils. The nucleotide sequences can be DNA, RNA, or any permutations thereof (e.g., nucleotide analogues, such as locked nucleic acids (LNAs), and the like). In some embodiments, the nucleotide sequences span exon/intron boundaries to detect gene expression of spliced or mature RNA species rather than genomic DNA. The nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences. The arrays may additionally comprise other compounds, such as antibodies, peptides, proteins, tissues, cells, chemicals, carbohydrates, and the like that specifically bind proteins or metabolites.
[0094] An array platform includes, for example, the TaqMan® Low Density Array (TLDA) mentioned above, and an Affymetrix® microarray platform.
[0095] A hybridization and multi-signal coded detector platform includes, for example, NanoString nCounter® technology, in which hybridization of a color-coded barcode attached to a target-specific probe (e.g., corresponding to a gene expression transcript of interest) is detected; and Luminex® xMAP® technology, in which microsphere beads are color coded and coated with a target-specific (e.g., gene expression transcript) probe for detection; and Illumina® BeadArray, in which microbeads are assembled onto fiber optic bundles or planar silica slides and coated with a target-specific (e.g., gene expression transcript) probe for detection.
[0096] A nucleic acid mass spectrometry platform includes, for example, the Ibis Biosciences Plex-ID® Detector, in which DNA mass spectrometry is used to detect amplified DNA using mass profiles.
[0097] A thermal cycler platform includes, for example, the FilmArray® multiplex PCR system, which extract and purifies nucleic acids from an unprocessed sample and performs nested multiplex PCR; the RainDrop Digital PCR System, which is a droplet-based PCR platform using microfluidic chips; and the GenMark eSensor or ePlex systems.
[0098] The term "genetic material" refers to a material used to store genetic information in the nuclei or mitochondria of an organism's cells. Examples of genetic material include, but are not limited to, double-stranded and single-stranded DNA, cDNA, RNA, and mRNA.
[0099] The term "plurality of nucleic acid oligomers" refers to two or more nucleic acid oligomers, which can be DNA or RNA.
[00100] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. General Techniques
[00101] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991 ); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.l. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).
[00102] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
[00103] As various changes could be made in the above-described materials and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.
EXAMPLES
[00104] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 : Host analyte analysis to improve the detection of group A streptococcal pharyngitis
[00105] Current detection methods used to evaluate patients with pharyngitis for the presence of group A Streptococcus (GAS) do not discriminate between acute infection and asymptomatic carriage, potentially resulting in overuse of antibiotics. Host response as measured by the transcriptom ic profile of peripheral blood leukocytes could make this distinction and could also distinguish between GAS and viral infection. The present example uses RNA sequencing to generate transcriptomes from 37 children, including 10 with acute GAS pharyngitis, 5 asymptomatic GAS carriers, 3 with adenoviral pharyngitis, 3 with pharyngitis of unknown etiology, and 16 asymptomatic children negative for GAS. Transcriptional profiles from each group were distinct. 1357 genes were upregulated in the children with symptomatic GAS compared to those with asymptomatic carriage. A panel of 13 genes distinguished between children with acute GAS and all others with 91 % accuracy. The gene encoding CD177, a marker of neutrophil activation, was markedly overexpressed in children with acute GAS. Thus, the present Example establishes that measurement of host response is highly useful to accurately detect GAS pharyngitis and limit unnecessary antibiotic use.
Methods
[00106] Subjects. Case subjects were children evaluated in the St. Louis Children’s Hospital Emergency Department between January, 2017 and February, 2018 for acute pharyngitis. Other enrollment criteria were age >3 years and a modified Centor score of 4 or 5. The components of the modified Centor score (R. M. Centor et al., Med Decis Making 1 , 239-246 (1981 ); W. J. Mclsaac et al., CMAJ 158, 75-83 (1998)) are temperature > 38°; tonsillar swelling or exudate; swollen, tender anterior cervical adenopathy; lack of a cough; and age < 15 years. One point is counted for each component. Children were excluded if they had an underlying medical condition, had received an antibiotic in the 7 days preceding enrollment, or were receiving immunosuppressive therapy. Children whose parents or guardian did not speak English were also not enrolled. Control subjects were children >3 years of age being evaluated in the SLCH Emergency Department for a non-infectious condition, with the same exclusion criteria applied as for the case subjects. The study was approved by the Washington University Human Research Protection Office and informed consent/assent were obtained from all subjects and their families or guardian before participation.
[00107] Samples. All subjects had a throat swab obtained for detection of group A Streptococcus (GAS) using standard methods. These swabs were collected by Emergency Department staff as part of routine care for case subjects and by study personnel for control subjects. The throat samples for standard GAS detection were processed according to the routine procedures of the Barnes-Jewish Microbiology Laboratory, which included a rapid strep antigen test followed by a culture if the rapid strep test was negative. After the first 21 subjects were enrolled, this procedure was modified so that a culture was performed on all subjects regardless of the results of the rapid test. [00108] Each subject also had the following research samples obtained: two throat swabs, a throat wash, a sample of oral secretions, a blood sample drawn into a Tempus blood RNA tube (Thermo Fisher, Waltham, MA) for RNA preservation, a complete blood count, and serum for C-reactive protein, procalcitonin, and IgM antibodies for Epstein-Barr virus. The throat swabs were collected using ESwabs (Copan Diagnostics, Murietta, CA). One throat swab was placed in Universal Transport Media (Copan), stored at -80°C and tested for respiratory viruses using the GenMark eSensor respiratory virus panel (Genmark, Carlsbad, CA). The eSensor testing was performed using a research protocol that detected the following viruses: influenza A and B, respiratory syncytial virus A and B, parainfluenza virus types 1-4, rhinovirus, human metapneumovirus, adenovirus groups B/E and C, coronaviruses OC43, 229E, NL63 and HKU1 . The throat wash was collected by asking subjects to gargle with a volume of 6 ml of normal saline, which was spit into a sterile container. The oral secretion sample was collected by asking the subject to spit 2.0 ml of oral secretions into an Oragene- RNA tube. RNA was extracted from blood samples using the Tempus Spin RNA Isolation Kit and processed for globin reduction using GLOBINclear Human Kit. All throat, oral secretion, and throat wash samples were tested for GAS nucleic acid using the Alethia Group A Streptococcus molecular assay (Meridian Bioscience, Cincinnati, OH).
[00109] RNA-seq Libraries and Sequencing. For RNA-seq, cDNA libraries were generated using 200 ng of globin-reduced total RNA from each sample. Library construction was performed using the TruSeq Stranded mRNA library kit (Illumina, San Diego, CA). cDNA quantity was determined with the Qubit Fluorometer (Life Technologies, Grand Island, NY) and quality assessed using the Agilent Bioanalyzer 2100 (Santa Clara, CA). Libraries were sequenced (single end reads) on the Illumina HiSeq 2500 (Illumina, San Diego, CA) to generate 20 million reads/sample.
[00110] Pre-processing of the Sequencing Reads. RNA-seq reads were aligned to the Ensembl release 76 primary assembly with STAR program (version 2.5.1a) (A. Dobin et al., Bioinformatics 29, 15-21 (2013)). Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount (version 1.4.6-p5) (Y. Liao et al., Bioinformatics 30, 923-930 (2014)). Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC (version 2.6.2) (L. Wang et al., Bioinformatics 28, 2184-2185 (2012)).
[00111] Differential Expression Analysis. All gene counts were then imported into the R package EdgeR (M. D. Robinson et al., Bioinformatics (Oxford, England) 26, 139-140 (2010)) and trimmed mean of M values (TMM) normalization size factors were calculated to adjust samples for differences in library size. Ribosomal genes and genes not expressed at a level greater than or equal to 1 count per million reads in the smallest group size were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into the R package limma.( M. E. Ritchie et al., Nucleic Acids Res 43, e47 (2015)) Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples with the voomWithQualityWeights. (R. Liu et al., Nucleic Acids Res 43, e97 (2015)). The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Differential expression analysis was then performed to analyze for differences between sample groups. Differentially expressed genes (DEG) were defined as those with at least 2-fold difference between two individual groups at P <0.05. To ensure quality RNA-seq data being used in the study, more stringent analyses were also performed with the DEG that were filtered with Benjamini-Hochberg false-discovery rate (FDR) adjusted P <0.05.
[00112] Classification of Sample Groups. In order to define a minimal number of genes as classifiers that distinguish individual subgroups (classes), Prediction Analysis of Microarrays (PAM) was utilized (R. Tibshirani et al., Proc Natl Acad Sci U S A 99, 6567-6572 (2002)), which uses nearest shrunken centroids to identify subsets of genes that best characterize each class based on gene expression data (R. Tibshirani et al., Proc Natl Acad Sci U S A 99, 6567-6572 (2002)). In addition, PAM cross-validates the classifiers with a 2-level nested leave-one-out cross validation procedure (L10-CV), with options for the number of folds of data division and number of model cut-offs to run. Classifiers were defined using the default of 30 cutoffs and 10-fold L10-CV.
[00113] Biological Pathway Enrichment. A web-based pathway enrichment tool “Enrichr” (27) was utilized to analyze Gene Ontology Biological Processes (http://amp.pharm.mssm.edu/Enrichr/). Enrichr determines the significance of genes in the gene-list compared to the background of random gene-lists. The resulting list of mapped terms was ranked by the Benjamini-Hochberg (BH) FDR-adjusted p-value.
[00114] Statistical Analysis. For the comparison of analytes between clinical subgroups, we used one-way analysis of variance for initial determination of differences in means between subgroups. We performed post hoc analysis of differences in means between pairs of subgroups using Tukey’s honest significance test. P values < 0.05 were considered significant. Analyses were carried out using IBM SPSS Statistics for Windows, version 24 (IBM Corp., Armonk, N.Y. USA.
Results
(i) Subjects
[00115] 40 subjects were enrolled, including 18 cases with pharyngitis and
22 asymptomatic controls. No samples were obtained from one control subject, leaving 18 cases and 21 controls for analysis. Demographic characteristics of cases and controls are shown in Table 1. Cases were younger (median years 9.8 vs 12.8), more likely to be female (83% vs 57%) and more likely to be Black than controls (83% vs 71 %).
Table 1. Demographic characteristics of subjects broken down by clinical subgroups
Figure imgf000044_0001
Figure imgf000045_0001
a A total of 40 subjects were enrolled in this study. However, one subject was excluded from analysis because no samples were obtained, leaving 39 subject for analysis. b RNA sequencing result not available on 1 subject
(ii) Microbial Testing
[00116] Results of microbial testing for GAS, adenovirus, other viruses, and other bacteria are shown in FIG. 1 and Table 2. Subjects were considered positive for GAS if GAS was detected by either culture or NAT. Using this definition, 11 (61 %) of the 18 cases and 5 (24%) of the 21 controls were positive for GAS. Throat culture and NAT results for each subject are shown in Tables 3 and 4.
Table 2. Demographic characteristics, Case/Control status, results of throat culture and nucleic acid
Figure imgf000045_0002
Figure imgf000046_0001
Figure imgf000047_0001
aYears at the last birthday. bAny species reported in clinical laboratory report. CGAS status is positive if culture or NAT from any site tested was positive. Detailed GAS culture and NAT results are shown in Table 3. dSubroup corresponds to the clinical subgroups shown in Figure 1. Abbreviations: F: female; M: male; B: Black or African-American; W: Black; ND: not done; GAS: group A Streptococcus; Pos: Positive; Neg: Negative; AdV: adenovirus; RV: rhinovirus; EBV: Epstein-Barr virus; HCoV: human coronavirus
Table 3. Results of GAS throat cultures and NAT for GAS performed on throat swab, throat wash, and saliva
Figure imgf000047_0002
Figure imgf000048_0001
Abbreviations: Pos: Positive; Neg: Negative; ND: not done; Inv: invalid (based on assay controls).
Table 4. Relationship between GAS throat culture and NAT results
Figure imgf000048_0002
[00117] AdV was detected in 4 cases and in 1 control. The 4 cases included 2 that were positive only for AdV, 1 that was positive for AdV and RV, and 1 that was positive for AdV and Streptococcus dysgalactiae.
[00118] Respiratory viruses other than AdV were detected in 2 cases and in 2 controls. Serologic evidence of current or recent Epstein-Barr Virus infection was found in 1 case and 1 control, both of whom were also positive for GAS. Thirteen controls were negative for viruses or bacteria.
[00119] For purposes of analysis, subjects were classified into 6 clinical subgroups shown with color codes in FIG. 1 . Subgroups of symptomatic subjects were as follows; symptomatic GAS (sGAS, n=11 ); symptomatic AdV (sAdV, n=3); and symptomatic “other pharyngitis” (sOP, n=4). A symptomatic subject who was positive for AdV and grew Streptococcus dysgalactiae on a throat culture was classified as sOP because of uncertainty about the relative roles of the two pathogens that are each recognized as causes of symptomatic pharyngitis. Subgroups of asymptomatic controls were as follows; asymptomatic GAS (asGAS, n=5); virus-positive controls (vCtrl, n=3); and virus-negative controls (nCtrl, n=13). Demographic characteristics of each of the subgroups are shown in Table 3. One subject with symptomatic GAS and one subject with symptomatic adenovirus infection were each also positive for RV. These subjects were classified as sGAS and sAdV respectively because RV was most likely a bystander since it is not recognized as a cause of severe pharyngitis. One subject with symptomatic GAS and one control subject who was a GAS carrier were each positive for EBV IgM. The symptomatic subject was classified as sGAS because the EBV was not clinically significant based on absence of lymphocytosis. The control subject was classified as asGAS since the asymptomatic child did not have a clinically significant EBV infection.
(iii) White Blood Cell Count, CRP, and Procalcitonin
[00120] Total white blood cell (WBC) counts, percent neutrophils, CRP, and procalcitonin for the sGAS, sAdV, asGAS, vCtrl and nCtrl subgroups are shown in FIG. 2. For this analysis, vCtrl and nCtrl were combined as “controls”. WBC counts, percent neutrophils, CRP, and PCT were higher in subjects with sGAS compared to the other groups. Differences between sGAS and other subgroups that were statistically significant were WBC, percent neutrophils, and CRP compared to subjects with asGAS; and WBC, percent neutrophils, CRP, and PCT compared to the controls. Only WBC was significantly higher in sGAS compared to sAdV.
(iv) Gene Expression Profiles Distinguishing Symptomatic Cases and Asymptomatic Controls
[00121] Blood samples yielded analyzable seguence data for 37 subjects including 16 cases and 21 controls (the 2 subjects with non-analyzable seguence data are designated in FIG. 1 ). 37 gene expression profiles were first examined to identify significant DEG that would provide accurate and unbiased classification of symptomatic versus asymptomatic subjects. For this analysis, DEG were defined as genes with a
Figure imgf000049_0001
2-fold change in expression level with a false discovery rate (Q) <0.05. Comparisons of sGAS, sAdV, and sOP each versus nCtrl yielded a total of 1464 DEG (FIG. 3A). Analysis of the DEG using unsupervised hierarchical clustering with Euclidean distance and Ward’s method separated the 37 subjects into 2 major clusters designated Cluster 1 and Cluster 2 (FIG. 3B). Cluster 1 consisted of all 16 symptomatic subjects plus 2 controls (1 asGAS and 1 nCtrl). Cluster 2 consisted of the remaining 19 asymptomatic subjects. Seven of 10 sGAS cases were clustered tightly within Cluster 1 . Clusters of genes that were generally upregulated in sGAS, upregulated in sAdV, and downregulated in sGAS were apparent (FIG. 3B). The effect of excluding 3 subjects who were positive for a respiratory virus or EBV in addition to GAS was also investigated (boxes B, C, J). As shown in Fig. 8, excluding these subjects had only a minor impact on the detection of DEG.
(v) Distinct Gene Expression Profiles of Clinical Subgroups
[00122] To begin a process of defining gene expression profiles characteristic of each clinical subgroup, genes of interest were identified by comparing each of the subgroups (other than sOP) to nCtrl, selecting DEG with fold change ^2.0 with P < 0.05. The number of DEG identified in each comparison is shown in FIG. 4A. The largest group of DEG came from the comparison of sGAS versus nCtrl (1334 DEG), followed by the comparison of sAdV versus nCtrl (508 DEG). Comparisons of asGAS and vCtrl versus nCtrl yielded much smaller numbers of DEG. In all, the union of the 4 sets of DEG defined a total of 1864 unique DEG. Gene expression profiles for the 6 clinical subgroups generated using supervised hierarchical clustering applied to the 1864 DEG are shown in FIG. 4B. Clear differences were evident between sGAS and sAdV, each of which also differed from the asymptomatic controls. Expression profiles of the 3 subgroups of asymptomatic controls were similar to one another, indicating that the presence of GAS or a virus in these asymptomatic control subjects did not have a strong impact on their gene expression profile. Within the control subgroups, 3 outlier profiles were evident: P021 whose true class was asGAS had a profile that resembled that of sGAS, and P003 whose true class was vCtrl (positive for AdV) had a profile that resembled that of sAdV. These results suggest that even though these subjects were asymptomatic, they were experiencing a host response to the presence of the respective organism. P024 whose true class was nCtrl had a profile resembling that of sGAS, suggesting that despite negative tests for GAS, the subject may have had a recent infection with GAS. CRP and PCT were not elevated in any of the 3 subjects, nor was WBC which was measured in 2 of the 3. Including or excluding these 3 subjects from the overall analysis of gene expression profiles had minimal effect on the overall pattern of results or on the clustering of subjects (FIG. 9).
(vi) Gene Expression Profiles of Subjects with Other Pharyngitis
[00123] Inspection of the expression profiles of the 3 subjects with sOP provided clues to their etiology which were not evident in their clinical evaluations. Subject P022 had a peritonsillar abscess without a specific microbiologic diagnosis. This patient had findings including a markedly elevated white blood cell count (19,900 per microliter) with 78% neutrophils and an elevated CRP of 214 mg/dL that strongly suggesting bacterial infection, although procalcitonin was not elevated. The gene expression profile resembled that of the subjects with sGAS (FIG. 3B and FIG. 4B and FIG. 4D). Subject P019 had findings suggestive of a viral infection, including a normal white blood cell count with 46% neutrophils, a modestly elevated CRP of 43.6 mg/dL, and a borderline procalcitonin value of 0.15 ng/mL. This subject’s gene expression profile strongly resembled those of subjects with sAdV (FIG. 2, suggesting that the subject may have had an unrecognized AdV infection. The gene expression profile of the third subject with sOP did not closely resemble that of any of the other clinical subgroups.
(vii) Gene Classifiers for GAS
[00124] Because the ultimate goal was to find gene classifiers to distinguish sGAS from all other subgroups, another round of analysis in which DEG resulting from comparisons of sGAS versus sAdV, asGAS, vCtrl, and nCtrl was carried out. The results are shown in FIG. 4C. From this set of comparisons, a set of 535 common DEG that distinguished sGAS from each of the other subgroups was identified. Expression profiles of these genes for each of the 6 clinical subgroups are shown in FIG. 4D. As shown, the expression profile of sGAS was clearly different from the other groups, with a large number of upregulated genes in the subjects with sGAS. Of note, the 2 asymptomatic subjects (P021 and P024) and one subject with sOP (P022) described previously with gene expression profiles similar to that of sGAS had corresponding profiles in this analysis that were even more strongly suggestive of sGAS.
(viii) Selection and Performance of Classifiers [00125] To identify gene classifiers, a shrunken centroid-based model system was applied (Prediction Analysis of Microarray [PAM])( R. Tibshiraniet al., Proc Natl Acad Sci II S A 99, 6567-6572 (2002)) to the set of 535 DEG that were uniquely up- or down-regulated in sGAS. This analysis yielded classifier panels comprising 5-14 genes with expression profiles that accurately distinguished sGAS from each of the other clinical subgroups (excluding sOP). ROC curves displaying performance of the panels in distinguishing sGAS from each of the other subgroups are shown in FIG. 5, and ROC curves for 8 other comparisons are included in FIG. 10. The genes included in each panel are listed in Table 5. In addition to defining panels that distinguished sGAS from the other individual subgroups, a panel of 13 genes that distinguished sGAS from all other subgroups was also identified. For this clinically important comparison, the 13- gene panel had an AUC of 0.93.
Table 5. Gene panels from Prediction Analysis of Microarrays (PAM)
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
(ix) Individual Analyte with Highest Performance
[00126] FIG. 6 shows fold-changes in gene expression and heat maps for the classifier genes making up the panels used to distinguish sGAS from other subgroups. In each of the 5 comparisons, CD177 had the highest fold-change of any of the classifier genes. For example, in the comparison of sGAS to nCtrl, CD177 was upregulated 217.5-fold, with the next most elevated gene (FCGR1A) upregulated 10.7- fold. Likewise, in the comparison of sGAS to asGAS, CD177 was upregulated 152.9- fold, with the next most upregulated gene (ARG1 ) upregulated 13.4-fold. CD177 is a cell surface glycoprotein that is involved in neutrophil activation and transmigration. Gene expression databases were retrieved from other studies of acute infections, and found evidence of upregulation of CD177 in acute bacterial infection in each study (FIG. 12)
(x) Enrichment of GO Biological Process Terms.
[00127] Enrichr (M. V. Kuleshov et al., Nucleic Acids Res 44, W90-97 (2016)) was used to analyze biological processes enriched among the upregulated DEG from the comparison of sGAS versus nCtrl. FIG. 7 shows the 20 most enriched GO Biological Process terms. The most significantly enriched terms related to various aspects of neutrophil activation. FIG. 7 shows neutrophil degranulation, two other neutrophil process terms were enriched to the similar level but are not shown because FIG. 7 shows only non-redundant terms. Most of the other enriched terms were related to cytokine signaling especially IL-1 beta, and components of the inflammatory response. No terms were significantly enriched among down-regulated genes.
Discussion
[00128] Accurate laboratory diagnosis of streptococcal pharyngitis, a common infectious disease of childhood, has been elusive. A vexing problem has been inability of microbiology-based tests to distinguish the asymptomatic carrier state from symptomatic infection, resulting in unnecessary use of antibiotics given to carriers with intercurrent viral infection. Because of the frequency of pharyngitis as a reason for outpatient medical visits and the fact that approximately 12% of young children may be carriers who do not require antibiotic treatment (N. Shaikh et al., Pediatrics 126, e557- 564 (2010)), the magnitude of overuse from treating carriers is substantial (G. P. DeMuri et al., J Pediatric Infect Dis Soc 3, 336-342 (2014)). In this example, it is established that host response as measured by the transcriptional profile of peripheral blood leukocytes (PBL) readily distinguishes symptomatic infection from carriage. Furthermore, the transcriptional profile also clearly distinguishes between streptococcal pharyngitis and pharyngitis caused by adenovirus, a notable cause of viral pharyngitis, which has clinical findings that may overlap those of streptococcal pharyngitis (O. Dominguez et al., Pediatr Infect Dis J 24, 733-734 (2005)). This is the first step in developing a new generation of diagnostic tests for GAS pharyngitis that will guide accurate targeting of antibiotic therapy.
[00129] In carrying out this example, the fact that the microbial agents causing pharyngitis can be accurately detected in readily available samples was taken advantage of. This contrasts with infection in other parts of the respiratory tract such as pneumonia or sinusitis in which an invasive procedure such as bronchoscopy or endoscopy is required to obtain diagnostic samples. Using throat swab samples, culture and molecular tests on cases and controls to determine the presence of GAS and respiratory viruses were performed. IgM serology was also used to look for evidence of current or recent EBV infection. This intensive microbiological profiling allowed well- defined groups of subjects with symptomatic GAS infection, symptomatic adenovirus infection, asymptomatic GAS carriers, and asymptomatic controls to be identified. Measurements of analytes including white blood cell count, CRP, and procalcitonin corroborated the clinical distinctions between symptomatic cases and asymptomatic controls. Clear delineation of subject subgroups classes is a key requirement of studies to define analytes intended to serve as classifiers. Thus, even though the number of subjects in our clinical subgroups was not large, the differences in gene expression profiles were dramatic and allowed identification of potentially useful transcripts that can serve as analytes. Further confidence in the results is provided using 2-level leave-one- out cross validation, which is part of the PAM procedure used to select and validate classifiers.
[00130] A consequence of the intensive microbiological characterization of subjects defined a number of dual and/or asymptomatic infections, including subjects who were GAS carriers, others who were asymptomatic but had a respiratory virus detected, and other symptomatic subjects whose illness was characteristic of GAS pharyngitis but also had a respiratory virus detected. Through complete analyses of gene expression profiles, only minimum impact of these dual or asymptomatic infections were observed on the detection of DEG and on the clustering of cases and controls (FIG. 8). These results indicated that the second pathogen, especially when asymptomatic, may not be an important confounding factor when defining distinguishable gene expression profiles. These results also suggest that it may not be necessary to exclude asymptomatic virus and GAS carriers from an asymptomatic control group in the expression profile classification analysis. A further implication is that as more experience accrues with gene expression profiles in specific infections, it may be possible to use those profiles to understand the relative roles of 2 or more pathogens that may be detected in the same sample, which is a common finding when sensitive molecular diagnostic tests are used.
[00131] In this example, RNA sequencing (RNA-seq) was used as a broad, unbiased approach to prove the hypothesis that differences in host response were present among children with symptomatic GAS, asymptomatic GAS, viral infection as exemplified by adenovirus, and pathogen-negative controls. A shrunken centroid methodology was then used (R. Tibshirani et al., Proc Natl Acad Sci II S A 99, 6567- 6572 (2002)), a machine learning technique, to identify panels consisting of limited numbers of transcripts that could distinguish among the clinical subgroups under analysis. A panel of 13 transcripts was identified that distinguished the subjects with symptomatic GAS from all other subjects.
[00132] To find small numbers of transcripts that could provide useful information in the detection and identification of pathological etiology, the gene encoding CD177 emerged as a uniquely discriminatory marker. Also known as human neutrophil antigen 2 (HNA-s), CD177 is a cell surface glycoprotein that is involved in neutrophil activation and neutrophil transmigration. Antibodies to HNA-2 are involved in transfusion-associated lung injury (TRALI). The gene is overexpressed in polycythemia vera, and mutations of the gene are associated with myeloproliferative diseases. CD177 has also been shown to be upregulated in severe bacterial infections, pneumococcal meningitis, tuberculosis, septic shock, and Kawasaki disease. Whether CD177 is uniquely responsive to GAS infection versus other bacterial species requires further investigation. However, because bacteria other than GAS are not common causes of pharyngitis, species specificity may not be required for it to be a useful marker in the setting of acute pharyngitis.
[00133] Although the main goal of this study was development of enhanced methods of detection, the availability of transcripts from children with acute GAS pharyngitis plus comparison groups provided the opportunity to analyze patterns of gene regulation that could provide insights into pathogenesis. To achieve this goal the enrichment of upregulated genes was analyzed for Gene Ontology (GO) Biologic Process terms using the enrichment analysis tool Enrichr. Consistent with the detection of intense upregulation of the neutrophil marker CD177, the 3 most enriched biologic processes were related to neutrophil degranulation and other aspects of neutrophil activity. The other most enriched pathways were related to cytokine signaling and other pathways related to inflammation. Interestingly, there was also activation of pathways not typically associated with gram-positive bacteria including responses to lipopolysaccharide and interferons.
[00134] In summary, the present example has shown that transcriptional analysis of peripheral blood leukocytes can provide valuable information that will improve the diagnosis of GAS pharyngitis and allow more appropriate targeting of antibiotic therapy than is possible using currently available tests. A key step will be to use analytes identified in this study in simple and rapid test formats that can be used at or near the point of care. In addition, it will be important to evaluate the possibility of measuring the transcriptional response in pharyngeal samples, which could allow microbial detection and transcriptional analysis to be performed on the same sample. Indeed, recent work reported that the nasal transcriptome was more informative than the transcriptome in peripheral blood leukocytes for diagnosing acute viral respiratory infection (J. Yu et al., J Infect Dis 219, 1151-1161 (2019)). The present study provides rapid progress along the translational pathway to provide detection methods to promote appropriately targeted antibiotic therapy for pharyngitis, an important component in the societal battle to curb unnecessary antibiotic use and prevent the further development of antibiotic resistance (Antibiotic Resistance Threats in the United States, 2019. (2019)).
EQUIVALENTS
[00135] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. [00136] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
[00137] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
[00138] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
[00139] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[00140] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[00141] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[00142] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. [00143] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

What is claimed is:
1 . A method for detecting an etiology of pharyngitis in a subject, the method comprising: a) measuring the level of expression of one or more host analytes in a biological sample obtained from a subject; and b) comparing the level of the at least one host analytes with a predetermined reference level, wherein if the level of the at least one host gene is above or below the respective reference value, the subject is determined to have a Group A Streptococcus (GAS) mediated pharyngitis or a viral mediated pharyngitis.
2. The method of claim 1 further comprising, testing the subject for the presence of at least one virus or bacteria.
3. The method of claim 1 or claim 2, wherein said subject is suspected of having a bacterial infection or a viral infection.
4. The method of any one of claims 1 -3, wherein the subject is suffering from symptoms of pharyngitis.
5. The method of claim 1 , wherein the one or more host analytes include one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, TLR5, FFAR3, SPATC1 , KCNH7, ADM, IL18RAP, LY6G6C, ARG1 , FCGR1 B, and TAS2R40.
6. The method of claim 1 , wherein the level of at least one host analyte is determined by measuring an amount of nucleic acid or protein in the biological sample.
7. The method of claim 1 , wherein the biological sample is a blood sample. The method of claim 1 , wherein the expression level of one or more host analytes selected from CD177, FAM20A, FCGR1A, and, CD274 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, FCGR1A, and, CD274 exhibit increased expression relative to a healthy control reference value. The method of claim 1 , wherein the expression level of one or more host analytes selected from CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a viral carrier reference value. The method of claim 1 , wherein the expression level of one or more host analytes selected from CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to an asymptomatic GAS carrier reference value. The method of claim 1 , wherein the expression level of one or more host analytes selected from CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a symptomatic viral infection reference value. The method of claim 1 , wherein the expression level of one or more host analytes selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 exhibit increased expression relative to reference value. A method of treating pharyngitis in a subject comprising: a) measuring the level of expression of one or more host analyte in a biological sample obtained from a subject; b) comparing the level of the at least one host analyte with a predetermined reference level, wherein if the level of the at least one host analyte is above or below the respective reference value, the subject is determined to have a symptomatic Group A Streptococcus (GAS) mediated pharyngitis or a symptomatic viral mediated pharyngitis; and c) administering to said subject an appropriate treatment regimen based on the type of infection.
14. The method of claim 13 further comprising, testing the subject for the presence of at least one virus or bacteria.
15. The method of claim 13, wherein said subject is suspected of having a bacterial infection or a viral infection.
16. The method of claim 13, wherein the subject is suffering from symptoms of pharyngitis.
17. The method of claim 13, wherein the one or more host analytes include one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, TLR5, FFAR3, SPATC1 , KCNH7, ADM, IL18RAP, LY6G6C, ARG1 , FCGR1 B, and TAS2R40.
18. The method of claim 13, wherein the level of at least one host analyte is determined by measuring an amount of nucleic acid or protein in the biological sample.
19. The method according to claim 13, wherein the appropriate treatment regimen comprises an antibacterial therapy when the etiology is determined to comprise a GAS bacteria.
20. The method according to claim 13, wherein the appropriate treatment regimen comprises an antiviral therapy when the etiology is determined to comprise a virus.
21 . The method of claim 13, wherein the biological sample is a blood sample.
22. The method of claim 13, wherein the expression level of one or more host analytes selected from CD177, FAM20A, FCGR1A, and, CD274 is detected, and the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, FCGR1A, and, CD274 exhibit increased expression relative to a healthy control reference value. The method of claim 13, wherein the expression level of one or more host analytes selected from CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 is detected, and the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, ANXA3, FAM20A, ADM, TASR40, and KCNH7 exhibit increased expression relative to a viral carrier reference value. The method of claim 13, wherein the expression level of one or more host analytes selected from CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 is detected, and the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of when one or more of CD177, FAM20A, ANXA3, CASP5, MGAM2, ADM, IL18RAP, FCGR1A, CD274, CACNA1 E, LY6G6C, ARG1 , FCGR1 B, and TLR5 exhibit increased expression relative to an asymptomatic GAS carrier reference value. The method of claim 13, wherein the expression level of one or more host analytes selected from CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 is detected, and the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a healthy control reference value or determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FFAR3, ANXA3, SPATC1 , MCEMP1 , and KCNH7 exhibit increased expression relative to a symptomatic viral infection reference value. The method of claim 13, wherein the expression level of one or more host analytes selected from CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 is detected; and the method further comprises c) determining that the subject with pharyngitis has pharyngitis caused by GAS etiology when one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, and TLR5 exhibit increased expression relative to reference value. A method of classifying a subject with a symptomatic infection versus an asymptomatic carrier, the method comprising: a) measuring the level of expression of one or more host analytes in a biological sample obtained from a subject; and b) comparing the level of the at least one host analytes with a predetermined reference level, wherein if the level of the at least one host gene is above or below the respective reference value, the subject is determined to have a symptomatic GAS or viral infection. The method of claim 27 further comprising, testing the subject for the presence of at least one virus or bacteria. The method of claim 27, wherein said subject is suspected of having a bacterial infection or a viral infection. The method of claim 27, wherein the one or more host analytes include one or more of CD177, FAM20A, CASP5, ZDHHC19, FCGR1A, ITGA7, SOCS3, MGAM2, CACNA1 E, MCEMP1 , ANXA3, CD274, TLR5, FFAR3, SPATC1 , KCNH7, ADM, IL18RAP, LY6G6C, ARG1 , FCGR1 B, and TAS2R40. The method of claim 27, wherein the level of at least one host analyte is determined by measuring an amount of nucleic acid or protein in the biological sample. The method of claim 27, wherein if the subject is classified with a symptomatic GAS infection, the subject is treated with an antibiotic composition. The method of claim 27, wherein if the subject is classified with an asymptomatic GAS infection, a symptomatic viral infection, or asymptomatic viral infection, the subject is not treated with an antibiotic composition.
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