US20230045305A1 - Rna determinants for distinguishing between bacterial and viral infections - Google Patents

Rna determinants for distinguishing between bacterial and viral infections Download PDF

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US20230045305A1
US20230045305A1 US17/955,599 US202217955599A US2023045305A1 US 20230045305 A1 US20230045305 A1 US 20230045305A1 US 202217955599 A US202217955599 A US 202217955599A US 2023045305 A1 US2023045305 A1 US 2023045305A1
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xloc
tcons
rna
determinants
infection
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Kfir Oved
Eran Eden
Olga BOICO
Gali KRONENFELD
Roy NAVON
Assaf COHEN-DOTAN
Einav SIMON
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Memed Diagnostics Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
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    • 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
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • 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
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
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Definitions

  • the present invention in some embodiments thereof, relates to the identification of signatures and determinants associated with bacterial and viral infections. More specifically it was discovered that certain RNA determinants are differentially expressed in a statistically significant manner in subjects with bacterial and viral infections.
  • Antibiotics are the world's most prescribed class of drugs with a 25-30 billion $US global market. Antibiotics are also the world's most misused drug with a significant fraction of all drugs (40-70%) being wrongly prescribed (Linder and Stafford 2001; Scott and Cohen 2001; Davey, P. and E. Brown, et al 2006; Cadieux, G. and R. Tamblyn, et al. 2007; Pulcini, C. and E. Cua, et al. 2007) (“CDC—Get Smart: Fast Facts About Antibiotic Resistance” 2011).
  • antibiotics misuse is when the drug is administered in case of a non-bacterial disease, such as a viral infection, for which antibiotics is ineffective.
  • a non-bacterial disease such as a viral infection
  • antibiotics is ineffective.
  • the health-care and economic consequences of the antibiotics over-prescription include: (i) the cost of antibiotics that are unnecessarily prescribed globally, estimated at >$10 billion annually; (ii) side effects resulting from unnecessary antibiotics treatment are reducing quality of healthcare, causing complications and prolonged hospitalization (e.g. allergic reactions, Antibiotics-associated diarrhea, intestinal yeast etc.) and (iii) the emergence of resistant strains of bacteria as a result of the overuse.
  • CDC Get Smart: Fast Facts About Antibiotic Resistance” 2013; “European Surveillance of Antimicrobial Consumption Network (ESAC-Net)” 2014; “CDC—About Antimicrobial Resistance” 2013; “Threat Report 2013 I Antimicrobial Resistance I CDC” 2013), with a concomitant increase in morbidity and mortality associated with infections caused by antibiotic resistant pathogens (“Threat Report 2013 I Antimicrobial Resistance I CDC” 2013). At least 2 million people are infected with antibiotic resistant bacteria each year in the US alone, and at least 23,000 people die as a direct result of these infections (“Threat Report 2013 I Antimicrobial Resistance I CDC” 2013).
  • Antibiotics under-prescription is not uncommon either. For example up to 15% of adult bacterial pneumonia hospitalized patients in the US receive delayed or no Abx treatment, even though in these instances early treatment can save lives and reduce complications (Houck, P. M. and D. W. Bratzler, et al 2002).
  • Such a technology should: (i) accurately differentiate between a bacterial and viral infections; (ii) be rapid (within minutes); (iii) be able to differentiate between pathogenic and non-pathogenic bacteria that are part of the body's natural flora; (iv) differentiate between mixed co-infections and pure viral infections and (v) be applicable in cases where the pathogen is inaccessible (e.g. sinusitis, pneumonia, otitis-media, bronchitis, etc).
  • the pathogen is inaccessible (e.g. sinusitis, pneumonia, otitis-media, bronchitis, etc).
  • Oved et al. 2015 has developed an immune signature, combining both bacterial- and viral-induced circulating host-proteins, which can aid in the correct diagnosis of patients with acute infections.
  • a method of determining an infection type in a subject comprising measuring the amount of a determinant which is set forth in Tables 1 or 2 in a sample derived from the subject, wherein the amount is indicative of the infection type.
  • a method of distinguishing between an infectious and non-infectious subject comprising measuring the amount of a determinant which is set forth in Table 16E in a sample derived from the subject, wherein the amount is indicative whether the subject is infectious or non-infectious.
  • a method of determining whether a subject has a bacterial or non-bacterial infection comprising measuring the amount of a determinant which is set forth in Table 16D in a sample derived from the subject, wherein the amount is indicative whether the subject has a bacterial or non-bacterial infection.
  • a method of determining an infection type in a subject comprising measuring at least two RNAs in a sample derived from the subject, wherein pairs of the at least two RNAs are set forth in Tables 9, 11, 12, 17 or 18 wherein the amount of the at least two RNAs is indicative of the infection type.
  • a method of determining an infection type in a subject comprising measuring at least three RNAs in a sample derived from the subject, wherein triplets of the at least three RNAs are set forth in Tables 13, 14, 19 or 20 wherein amount of the at least three RNAs is indicative of the infection type.
  • a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 3 or Table 4, in a sample derived from the subject, wherein no more than 20 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 5 or Table 6A, in a sample derived from the subject, wherein no more than 5 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • kits for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in Table 1 or Table 2, and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6A or Tables 15A-B.
  • a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two RNA detection reagents specifically detects a first RNA which is set forth in Table 3 or Table 4, and a second of the at least two RNA detection reagents specifically detects a second RNA which is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in any one of Tables 1-6A or Tables 15A-B and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the kit comprises detection reagents that specifically detect no more than 5 determinants.
  • composition of matter comprising a sample derived from a subject suspected of having an infection and a first agent which binds specifically to at least one determinant of the sample which is set forth in Table 1 or Table 2.
  • the amount of the determinant set forth in Table 1 is above a predetermined level, the infection is a viral infection.
  • the infection when the amount of the determinant set forth in Table 2 is above a predetermined level, the infection is a bacterial infection.
  • the determinant is an RNA.
  • the RNA is a protein-coding RNA.
  • the RNA is a non protein-coding
  • the determinant is a polypeptide.
  • the method further comprises measuring the amount of at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • the at least one additional RNA is set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C, wherein the amount of the at least two RNAs is indicative of the infection type.
  • the pairs of the at least two RNAs are set forth in Tables 9, 11, 17 or 18.
  • the determinant is selected from the group consisting of OTOF, PI3, CYBRD1, EIF2AK2, CMPK2, CR1, CYP1B1, DDX60, DGAT2, PARP12, PNPT1, PYGL, SULT1B1, TRIB2, USP41, ZCCHC2 and uc003hr1.1.
  • the determinant is selected from the group consisting of TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_00072, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 and TCONS_12_00002367-XLOC_12_000720.
  • the method further comprises analyzing at least two additional RNAs so as to measure the amount of at least three RNAs, wherein the at least two additional RNAs are set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least three RNAs is indicative of the infection type.
  • the at least two additional RNAs are set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • the triplets of the at least three RNAs are set forth in Table 13, Table 19 or Table 20.
  • the infection is a viral infection, a bacterial infection or a mixed infection.
  • the method further comprises analyzing the expression level of CRP polypeptide or TRAIL polypeptide in the sample.
  • the determinant is set forth in Table 21.
  • RNAs are measured.
  • RNAs are measured.
  • the at least one RNA is set forth in Table 3.
  • the infection when the RNA set forth in Table 3 is above a predetermined level, the infection is a viral infection.
  • the method further comprises measuring at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • the at least one additional RNA is set forth in Tables 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • the at least one additional RNA set forth in Table 1 is OTOF, PI3, EIF2AK2, CMPK2, PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2 or TCONS_00003184-XLOC_001966.
  • the at least one additional RNA set forth in Table 2 is CYBRD1, CR1, DGAT2, PYGL, SULT1B1, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 or TCONS_12_00002367-XLOC_12_000720.
  • the at least one RNA is set forth in Table 5.
  • the infection is a viral infection.
  • the method further comprises measuring at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • the at least one additional RNA is set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • the at least one additional RNA set forth in Table 1 is selected from the group consisting of OTOF, PI3, EIF2AK2, CMPK2,
  • the at least one additional RNA set forth in Table 2 is CYBRD1, CR1, DGAT2, PYGL, SULT1B1, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 or TCONS_12_00002367-XLOC_12_000720.
  • the sample is whole blood or a fraction thereof.
  • the infection is a viral infection, a bacterial infection or a mixed infection.
  • the blood fraction sample comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.
  • the blood fraction sample comprises serum or plasma.
  • the at least two determinant detection reagents comprise RNA detection reagents.
  • the at least two RNA detection reagents are attached to a detectable moiety.
  • the first RNA detection reagent comprises a first polynucleotide that hybridizes to the first RNA and the second RNA detection reagent comprises a polynucleotide that hybridizes to the second RNA.
  • the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • the kit comprises detection reagents that specifically detect no more than 10 RNAs.
  • the kit comprises detection reagents that specifically detect no more than 5 RNAs.
  • each of the 5 RNAs are set forth in any one of Tables 1-6A or Tables 15A-B.
  • the kit comprises detection reagents that specifically detect no more than 5 RNAs.
  • the kit comprises detection reagents that specifically detect no more than 3 RNAs.
  • the determinant is an RNA.
  • the determinant is a polypeptide.
  • the composition further comprises an additional agent which binds specifically to an additional determinant which is set forth in any one of Tables 1-6A or Tables 15A-B.
  • the infectious subject has sepsis and the non-infectious subject has SIRS without sepsis.
  • FIG. 1 is a flow-chart of the clinical study design.
  • FIG. 2 illustrates distinctive gene expression profiles of 6 bacterial and 9 viral patients. Hierarchical clustering of 65 genes using Euclidean distance metric and average linkage. Transformed expression levels are indicated by color scale, with red representing high expression and green indicating low expression.
  • FIG. 3 A is a heat map illustrating the classification accuracy in terms of MCC of 9 viral versus 6 bacterial infected patients attained for pairs of the 65 RNA determinants described in Table 8 (according to the serial numbers) using a logistic regression model.
  • FIG. 3 B is a heat map illustrating the change in classification accuracy (dMCC) for the 65 RNA determinants described in Table 8 (according to the serial numbers).
  • the change is computed as follows: MCCi,j ⁇ max(MCCi, MCCj), where MCCi and MCCj correspond to the MCC obtained for determinant i and j individually and MCCi,j is obtained for the pair.
  • Hot and cold colors indicate pairs of RNA determinants whose combined classification accuracy compared to the individual determinant accuracy is higher and lower.
  • FIG. 4 A is a heat map illustrating the classification accuracy in terms of AUC of 9 viral versus 6 bacterial infected patients attained for pairs of the 65 RNA determinants described in Table 8 (according to the serial numbers) using a logistic regression model.
  • FIG. 4 B is a heat map illustrating the change in classification accuracy (dAUC) for the 65 RNA determinants described in Table 8 (according to the serial numbers) is computed as follows: AUCi,j ⁇ max(AUCi, AUCj), where AUCi and AUCj correspond to the AUC obtained for determinant i and j individually and AUCi,j is obtained for the pair. Hot and cold colors indicate pairs of RNA determinants whose combined classification accuracy compared to the individual determinant accuracy is higher and lower.
  • FIGS. 5 A- 5 K Differential expression levels of the RNA determinants described in Tables 16A-C in bacterial, viral and non-infectious (including healthy) patients. Red line and circle correspond to group median and average respectively; Wilcoxon rank sum (RS) p-values between bacterial and viral groups and between infectious (bacterial and viral) vs non-infectious (including healthy subjects) are depicted. Gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) are included.
  • FIG. 6 Volcano plot (scatter-plot) presenting the fold change as well as t-test p-value of the evaluated RNA determinants. Viral- and bacterial-induced RNAs that are described in Tables 16A-C are marked in red and blue “x” (respectively).
  • FIGS. 7 A- 7 I Expression levels (arbitrary units) of selected RNAs in non-infectious (including healthy) patients as well as in patients with bacterial or various viral infections as indicated.
  • RSV Respiratory syncytial virus
  • hMPV human Metapneumovirus. Red line corresponds to group median.
  • the present invention in some embodiments thereof, relates to the identification of signatures and determinants associated with bacterial and viral infections. More specifically it was discovered that certain RNA determinants are differentially expressed in a statistically significant manner in subjects with bacterial and viral infections.
  • the present inventors studied the gene expression profiles of blood leukocytes obtained from patients with acute infections. The results indicate there is a differential response of the immune system to bacterial and viral infections, which can potentially be used to classify patients with acute infections.
  • FIG. 2 presents distinctive gene expression profiles of the identified genes after applying a hierarchical clustering that uses Euclidean distance metric and average linkage. Based on these results the present inventors developed a classifier for distinguishing between bacterial and viral patients using logistic regression.
  • RNA markers that could serve as determinants for distinguishing between bacterial and viral infections.
  • Tables 15A-B Further analysis identified which of these RNAs are the most differentially expressed between bacterial and viral patients (Tables 16A-C). Particular RNAs were shown to be highly accurate at diagnosing infection type at disease onset (Table 24). Additional RNAs were shown to be accurate at indicating a specific type of viral strain (Table 25).
  • a method of determining an infection type in a subject comprising measuring the amount of a determinant which is set forth in Tables 1 and 2 in a sample derived from the subject, wherein the amount is indicative of the infection type.
  • determinant refers to a polypeptide or a polynucleotide (e.g. RNA).
  • the infection type may be a bacterial infection, a viral infection or a mixed infection (a combination of bacterial and viral infection).
  • the bacterial infection may be the result of gram-positive, gram-negative bacteria or atypical bacteria.
  • Gram-positive bacteria refers to bacteria that are stained dark blue by Gram staining. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall.
  • Gram-negative bacteria refers to bacteria that do not retain the crystal violet dye in the Gram staining protocol.
  • Atypical bacteria are bacteria that do not fall into one of the classical “Gram” groups. They are usually, though not always, intracellular bacterial pathogens. They include, without limitations, Mycoplasmas spp., Legionella spp. Rickettsiae spp., and Chlamydiae spp.
  • the infection may be an acute or chronic infection.
  • a chronic infection is an infection that develops slowly and lasts a long time. Viruses that may cause a chronic infection include Hepatitis C and HIV.
  • One difference between acute and chronic infection is that during acute infection the immune system often produces IgM+antibodies against the infectious agent, whereas the chronic phase of the infection is usually characteristic of IgM ⁇ /IgG+antibodies.
  • acute infections cause immune mediated necrotic processes while chronic infections often cause inflammatory mediated fibrotic processes and scaring (e.g. Hepatitis C in the liver). Thus, acute and chronic infections may elicit different underlying immunological mechanisms.
  • the level of the determinant may be used to rule in an infection type. In another embodiment, the level of the determinant may be used to rule out an infection type.
  • Measurement or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of the determinant within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such determinants.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, saliva, mucus, breath, urine, CSF, sputum, sweat, stool, hair, seminal fluid, biopsy, rhinorrhea, tissue biopsy, cytological sample, platelets, reticulocytes, leukocytes, epithelial cells, or whole blood cells.
  • the sample is a blood sample—e.g. serum or a sample comprising white blood cells (which is depleted of red blood cells).
  • a blood sample e.g. serum or a sample comprising white blood cells (which is depleted of red blood cells).
  • the sample is a blood sample comprising white blood cells such as lymphocytes, monocytes and granulocytes (which is depleted of red blood cells).
  • the sample is not a serum sample.
  • red blood cells include for example hemolysis, centrifugation, sedimentation, filtration or combinations thereof.
  • the determinant to be measured is a protein
  • a protein sample is generated.
  • RNA sample is generated.
  • the RNA sample may comprise RNA from a heterogeneous population of cells or from a single population of cells.
  • the RNA may comprise total RNA, mRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, viral RNA, cell free RNA, and mixtures thereof.
  • the RNA sample is devoid of DNA.
  • the sample may be fresh or frozen.
  • Isolation, extraction or derivation of RNA may be carried out by any suitable method.
  • Isolating RNA from a biological sample generally includes treating a biological sample in such a manner that the RNA present in the sample is extracted and made available for analysis. Any isolation method that results in extracted RNA may be used in the practice of the present invention. It will be understood that the particular method used to extract RNA will depend on the nature of the source.
  • Phenol based extraction methods These single-step RNA isolation methods based on Guanidine isothiocyanate (GITC)/phenol/chloroform extraction require much less time than traditional methods (e.g. CsCl 2 ultracentrifugation). Many commercial reagents (e.g. Trizol, RNAzol, RNAWIZ) are based on this principle. The entire procedure can be completed within an hour to produce high yields of total RNA.
  • GITC Guanidine isothiocyanate
  • RNAWIZ RNAWIZ
  • Silica gel-based purification methods RNeasy is a purification kit marketed by Qiagen. It uses a silica gel-based membrane in a spin-column to selectively bind RNA larger than 200 bases. The method is quick and does not involve the use of phenol.
  • Oligo-dT based affinity purification of mRNA Due to the low abundance of mRNA in the total pool of cellular RNA, reducing the amount of rRNA and tRNA in a total RNA preparation greatly increases the relative amount of mRNA.
  • the use of oligo-dT affinity chromatography to selectively enrich poly (A)+RNA has been practiced for over 20 years. The result of the preparation is an enriched mRNA population that has minimal rRNA or other small RNA contamination. mRNA enrichment is essential for construction of cDNA libraries and other applications where intact mRNA is highly desirable.
  • the original method utilized oligo-dT conjugated resin column chromatography and can be time consuming. Recently more convenient formats such as spin-column and magnetic bead based reagent kits have become available.
  • the sample may also be processed prior to carrying out the diagnostic methods of the present invention. Processing of the sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like.
  • cDNA may be generated therefrom.
  • template mRNA may be obtained directly from lysed cells or may be purified from a total RNA or mRNA sample.
  • the total RNA sample may be subjected to a force to encourage shearing of the RNA molecules such that the average size of each of the RNA molecules is between 100-300 nucleotides, e.g. about 200 nucleotides.
  • various technologies may be used which are based on the use of oligo(dT) oligonucleotides attached to a solid support.
  • oligo(dT) oligonucleotides examples include: oligo(dT) cellulose/spin columns, oligo(dT)/magnetic beads, and oligo(dT) oligonucleotide coated plates.
  • RNA-DNA hybrid For this, a primer is required that hybridizes to the 3′ end of the RNA. Annealing temperature and timing are determined both by the efficiency with which the primer is expected to anneal to a template and the degree of mismatch that is to be tolerated.
  • the annealing temperature is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 50° C. to about 80° C., usually from about 55° C. to about 70° C., and more usually from about 60° C. to about 68° C. Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.
  • the primer comprises a polydT oligonucleotide sequence.
  • the polydT sequence comprises at least 5 nucleotides. According to another is between about 5 to 50 nucleotides, more preferably between about 5-25 nucleotides, and even more preferably between about 12 to 14 nucleotides.
  • RNA-DNA hybrid is synthesized by reverse transcription using an RNA-dependent DNA polymerase.
  • RNA-dependent DNA polymerases for use in the methods and compositions of the invention include reverse transcriptases (RTs).
  • RTs include, but are not limited to, Moloney murine leukemia virus (M-MLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, rous associated virus (RAV) reverse transcriptase, and myeloblastosis associated virus (MAV) reverse transcriptase or other avian sarcoma-leukosis virus (ASLV) reverse transcriptases, and modified RTs derived therefrom. See e.g. U.S. Pat. No. 7,056,716.
  • AMV-RT avian myeloblastosis virus
  • MMLV-RT Moloney murine leukemia virus
  • AMV-RT avian myeloblastosis virus
  • MMLV-RT Moloney murine leukemia virus
  • dNTPS dATP, dCTP, dGTP and dTTP
  • DTT Dithiothreitol
  • MnCl 2 MnCl 2
  • At least one determinant e.g. RNA determinant of Table 1 or Table 2 is measured.
  • the RNA determinant is an RNA that codes for a protein.
  • the RNA determinant does not code for a protein (non-coding RNA).
  • the level of determinant in Table 1 when the level of determinant in Table 1 is above a predetermined level (e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a bacterial infection, as further described herein below), it is indicative that the subject has a viral infection (i.e. a viral infection may be ruled in).
  • a predetermined level e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a bacterial infection, as further described herein below.
  • the level of determinant in Table 1 is above a predetermined level, it is indicative that the subject does not have a bacterial infection (i.e. a bacterial infection is ruled out).
  • the level of determinant in Table 2 when the level of determinant in Table 2 is above a predetermined level (e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a viral infection, as further described herein below), it is indicative that the subject has a bacterial infection (i.e. a bacterial infection is ruled in).
  • a predetermined level e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a viral infection, as further described herein below.
  • the level of determinant in Table 2 is above a predetermined level, it is indicative that the subject does not have a viral infection (i.e. a viral infection is ruled out).
  • the predetermined level of any of the aspects of the present invention may be a reference value derived from population studies, including without limitation, such subjects having a known infection, subject having the same or similar age range, subjects in the same or similar ethnic group, or relative to the starting sample of a subject undergoing treatment for an infection.
  • Such reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of infection.
  • Reference determinant indices can also be constructed and used using algorithms and other methods of statistical and structural classification.
  • the predetermined level is the amount (i.e. level) of determinants in a control sample derived from one or more subjects who do not have an infection (i.e., healthy, and or non-infectious individuals).
  • such subjects are monitored and/or periodically retested for a diagnostically relevant period of time (“longitudinal studies”) following such test to verify continued absence of infection.
  • Such period of time may be one day, two days, two to five days, five days, five to ten days, ten days, or ten or more days from the initial testing date for determination of the reference value.
  • retrospective measurement of determinants in properly banked historical subject samples may be used in establishing these reference values, thus shortening the study time required.
  • a reference value can also comprise the amounts of determinants derived from subjects who show an improvement as a result of treatments and/or therapies for the infection.
  • a reference value can also comprise the amounts of determinants derived from subjects who have confirmed infection by known techniques.
  • An example of a bacterially infected reference value is the mean or median concentrations of that determinant in a statistically significant number of subjects having been diagnosed as having a bacterial infection.
  • the present inventors contemplate measuring at least one additional (non-identical) determinant (so as to measure at least two determinants), wherein the at least one additional determinant is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two determinants is indicative of the infection type.
  • the present inventors contemplate measuring at least one additional (non-identical) determinant (so as to measure at least two determinants), wherein the at least one additional determinant is set forth in Table 6B.
  • RNA determinants is a coding RNA e.g. OTOF, PI3, CYBRD1, EIF2AK2 or CMPK2, PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2.
  • a coding RNA e.g. OTOF, PI3, CYBRD1, EIF2AK2 or CMPK2, PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2.
  • At least one of the RNA determinants is a non-coding RNA e.g, TCONS_00003184-XLOC_001966.
  • CMPK2+TCONS_00013664-XLOC_006324 CMPK2+TCONS_12_00028242-XLOC_12_014551, CMPK2+TCONS_12_00001926-XLOC_12_000004, CMPK2+TCONS_12_00002386-XLOC_12_000726, CMPK2+TCONS_12_00002811-XLOC_12_001398. CMPK2+TCONS_12_00003949-XLOC_12_001561.
  • CMPK2+TCONS_00019559-XLOC_009354 CMPK2+TCONS_12_00010440-XLOC_12_005352, CMPK2+TCONS_12_00016828-XLOC_12_008724, CMPK2+TCONS_12_00021682-XLOC_12_010810, CMPK2+TCONS_12_00002367-XLOC_12_000720, CMPK2+FAM89A, CMPK2+MX1.
  • CMPK2+RSAD2 CMPK2+IFI44L, CMPK2+USP18, CMPK2+IFI27, CR1+CYP1B1, CR1+DDX60.
  • CR1+DGAT2 CR1+PARP12, CR1+PNPT1, CR1+PYGL, CR1+SULT1B1, CR1+TRIB2, CR1+uc003hr1.1, CR1+USP41, CR1+ZCCHC2, CR1+n332762, CR1+n407780, CR1+n332510.
  • CYP1B1+TCONS_00019559-XLOC_009354 CYP1B1+TCONS_12_00010440-XLOC_12_005352, CYP1B1+TCONS_12_00016828-XLOC_12_008724, CYP1B1+TCONS_12_00021682-XLOC_12_010810, CYP1B1+TCONS_12_00002367-XLOC_12_000720, CYP1B1+FAM89A, CYP1B1+MX1, CYP1B1+RSAD2, CYP1B1+IFI44L, CYP1B1+USP18, CYP1B1+IFI27, DDX60+DGAT2, DDX60+PARP12, DDX60+PNPT1, DDX60+PYGL, DDX60+SULT1B1, DDX60+TRIB2, DDX60+uc
  • FAM89A+USP18 FAM89A+USP18. FAM89A+IFI27, MX1+RSAD2. MX1+IFI44L, MX1+USP18, MX1+IFI27, RSAD2+IFI44L, RSAD2+USP18, RSAD2+IFI27, IFI44L+USP18, IFI44L+IFI27, USP18+IFI27
  • At least three determinants are measured, wherein the at least two additional determinant are set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least three determinants is indicative of the infection type.
  • At least three non-identical RNAs are measured, the first from Tables 1 or 2 and the second and third from any one of Tables 1-6A or 16A-C.
  • the second and third RNA is set forth in Tables 1, 2, 3, 5 or 16A-C and even more preferably, the second RNA is set forth in Tables 1 or 2.
  • CMPK2+CR1+CYP1B1 CMPK2+CR1+DDX60, CMPK2+CR1+DGAT2, CMPK2+CR1+PARP12, CMPK2+CR1+PNPT1, CMPK2+CR1+PYGL, CMPK2+CR1+SULT1B1, CMPK2+CR1+TRIB2, CMPK2+CR1+uc003hr1.1, CMPK2+CR1+USP41, CMPK2+CR1+ZCCHC2, CMPK2+CYP1B1+DDX60, CMPK2+CYP1B1+DGAT2, CMPK2+CYP1B1+PARP12, CMPK2+CYP1B1+PNPT1, CMPK2+CYP1B1+PYGL, CMPK2+CYP1B1+SULT1B1, CMPK2+CYP1B1+TRIB2, CMPK2+CYP1B1+uc003h
  • no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • NC_000022.11 apolipoprotein B mRNA editing NC_018933.2 enzyme catalytic polypeptide NT_011520.13 like 3C BST2 NC_000019.10 bone marrow stromal cell NC_018930.2 antigen 2 NT_011295.12 C1orf29 NC_000001.11 interferon induced protein 44 NT_032977.10 like NC_018912.2 CD44 NC_000011.10 CD44 molecule (Indian blood NT_009237.19 group) NC_018922.2 dJ507I15.1 NC_000023.11 ribosomal protein L36a NC_018934.2 pseudogene NT_011786.17 DNAPTP6 NC_000002.12 spermatogenesis associated, NT_005403.18 serine rich 2 like NC_018913.2 EEF1G NC_000011.10 eukaryotic translation NC_018922.2 elongation factor 1 gamma
  • a method of determining an infection type in a subject comprising measuring at least two RNAs in a sample derived from the subject, wherein pairs of the at least two RNAs are set forth in Tables 9, 11, 12, 18 or 19, wherein the amount of the at least two RNAs is indicative of the infection type.
  • no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • a method of determining an infection type in a subject comprising measuring at least three RNAs in a sample derived from the subject, wherein triplets of the at least three RNAs are set forth in Tables 13, 14, 19 or 20 wherein amount of the at least three RNAs is indicative of the infection type.
  • no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, or even no more than 3 determinants are measured.
  • a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 3 or Table 4, in a sample derived from the subject, wherein no more than 20 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • the at least one RNA is set forth in Table 3.
  • the level of RNA set forth in Table 3 is above a predetermined level, it is indicative of a viral infection.
  • the additional determinant is set forth in any one of Tables 1-6A or Tables 15A-B.
  • pathogen bacterial or viral
  • RNA determinants that can distinguish between influenza and parainfluenza are described in Table 25 of the Examples section, herein below. This may be carried out in order to aid in identification of a specific pathogen.
  • the measurements may be effected simultaneously with the above described measurements or consecutively.
  • the measurement may be performed on the biological sample used to determine the patient immune response (as described herein above) or on a different biological patient-derived sample (e.g., blood sample; serum sample; saliva; nasopharyngeal sample; etc.).
  • the host immune RNA determinants are measured in a patient derived serum sample and analysis of the pathogen specific RNA determinants is performed on a nasopharyngeal sample.
  • the additional RNA is set forth in Table 1 (e.g. OTOF, PI3, EIF2AK2 and CMPK2), Table 2 (e.g. CYBRD1), Table 3, Table 5 or Tables 16A-C (protein-coding RNAs such as CR1, CYP1B1, DDX60, DGAT2,
  • Non-protein coding RNAs such as TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_00072, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 and
  • a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 5 or Table 6A, in a sample derived from the subject, wherein no more than 5 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • the at least one RNA is set forth in Table 5.
  • the infection may be classified as a viral infection.
  • additional RNAs may be measured so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6, wherein the amount of the at least two RNAs is indicative of the infection type.
  • the at least one additional RNA is set forth in Table 1 (e.g., PI3, EIF2AK2 and CMPK2), Table 2 (e.g. CYBRD1), Table 3,Table 5 or Tables 16A-C(e.g.
  • coding RNAs such as CR1, CYP1B1, DDX60, DGAT2, PARP12, PNPT1, PYGL, SULT1B1, TRIB2, USP41, ZCCHC2, uc003hr1.1; or non-coding RNAs such as TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_
  • Northern Blot analysis This method involves the detection of a particular RNA in a mixture of RNAs.
  • An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation.
  • the individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere.
  • the membrane is then exposed to labeled DNA probes.
  • Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
  • RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine.
  • a reverse transcriptase enzyme such as an MMLV-RT
  • primers such as, oligo dT, random hexamers or gene specific primers.
  • a PCR amplification reaction is carried out in a PCR machine.
  • Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification
  • RNA in situ hybridization stain DNA or RNA probes are attached to the RNA molecules present in the cells.
  • the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe.
  • the hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe.
  • formamide and salts e.g., sodium chloride and sodium citrate
  • any unbound probe is washed off and the bound probe is detected using known methods.
  • a radio-labeled probe is used, then the slide is subjected to a photographic emulsion which reveals signals generated using radio-labeled probes; if the probe was labeled with an enzyme then the enzyme-specific substrate is added for the formation of a colorimetric reaction; if the probe is labeled using a fluorescent label, then the bound probe is revealed using a fluorescent microscope; if the probe is labeled using a tag (e.g., digoxigenin, biotin, and the like) then the bound probe can be detected following interaction with a tag-specific antibody which can be detected using known methods.
  • a tag e.g., digoxigenin, biotin, and the like
  • DNA microarrays consist of thousands of individual gene sequences attached to closely packed areas on the surface of a support such as a glass microscope slide.
  • Various methods have been developed for preparing DNA microarrays. In one method, an approximately 1 kilobase segment of the coding region of each gene for analysis is individually PCR amplified.
  • a robotic apparatus is employed to apply each amplified DNA sample to closely spaced zones on the surface of a glass microscope slide, which is subsequently processed by thermal and chemical treatment to bind the DNA sequences to the surface of the support and denature them.
  • such arrays are about 2 ⁇ 2 cm and contain about individual nucleic acids 6000 spots.
  • multiple DNA oligonucleotides usually 20 nucleotides in length, are synthesized from an initial nucleotide that is covalently bound to the surface of a support, such that tens of thousands of identical oligonucleotides are synthesized in a small square zone on the surface of the support.
  • Multiple oligonucleotide sequences from a single gene are synthesized in neighboring regions of the slide for analysis of expression of that gene. Hence, thousands of genes can be represented on one glass slide.
  • Such arrays of synthetic oligonucleotides may be referred to in the art as “DNA chips”, as opposed to “DNA microarrays”, as described above [Lodish et al. (eds.). Chapter 7.8: DNA Microarrays: Analyzing Genome-Wide Expression. In: Molecular Cell Biology, 4th ed., W. H. Freeman, New York. (2000)].
  • Oligonucleotide microarray In this method oligonucleotide probes capable of specifically hybridizing with the polynucleotides of some embodiments of the invention are attached to a solid surface (e.g., a glass wafer). Each oligonucleotide probe is of approximately 20-25 nucleic acids in length.
  • a specific cell sample e.g., blood cells
  • RNA is extracted from the cell sample using methods known in the art (using e.g., a TRIZOL solution, Gibco BRL, USA).
  • Hybridization can take place using either labeled oligonucleotide probes (e.g., 5′-biotinylated probes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA).
  • labeled oligonucleotide probes e.g., 5′-biotinylated probes
  • cDNA complementary DNA
  • cRNA RNA
  • double stranded cDNA is prepared from the RNA using reverse transcriptase (RT) (e.g., Superscript II RT), DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, Md., USA).
  • RT reverse transcriptase
  • DNA ligase DNA polymerase I
  • the double stranded cDNA is subjected to an in vitro transcription reaction in the presence of biotinylated nucleotides using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, Affymetrix Santa Clara Calif.).
  • the labeled cRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate for 35 minutes at 94° C.
  • the microarray is washed and the hybridization signal is scanned using a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • each gene on the array is represented by a series of different oligonucleotide probes, of which, each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. While the perfect match probe has a sequence exactly complimentary to the particular gene, thus enabling the measurement of the level of expression of the particular gene, the mismatch probe differs from the perfect match probe by a single base substitution at the center base position.
  • the hybridization signal is scanned using the Agilent scanner, and the Microarray Suite software subtracts the non-specific signal resulting from the mismatch probe from the signal resulting from the perfect match probe.
  • RNA sequencing Methods for RNA sequence determination are generally known to the person skilled in the art. Preferred sequencing methods are next generation sequencing methods or parallel high throughput sequencing methods.
  • An example of an envisaged sequence method is pyrosequencing, in particular 454 pyrosequencing, e.g. based on the Roche 454 Genome Sequencer. This method amplifies DNA inside water droplets in an oil solution with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.
  • Yet another envisaged example is Illumina or Solexa sequencing, e.g. by using the Illumina Genome
  • Analyzer technology which is based on reversible dye-terminators. DNA molecules are typically attached to primers on a slide and amplified so that local clonal colonies are formed. Subsequently one type of nucleotide at a time may be added, and non-incorporated nucleotides are washed away. Subsequently, images of the fluorescently labeled nucleotides may be taken and the dye is chemically removed from the DNA, allowing a next cycle. Yet another example is the use of Applied Biosystems' SOLiD technology, which employs sequencing by ligation. This method is based on the use of a pool of all possible oligonucleotides of a fixed length, which are labeled according to the sequenced position.
  • Such oligonucleotides are annealed and ligated. Subsequently, the preferential ligation by DNA ligase for matching sequences typically results in a signal informative of the nucleotide at that position. Since the DNA is typically amplified by emulsion PCR, the resulting bead, each containing only copies of the same DNA molecule, can be deposited on a glass slide resulting in sequences of quantities and lengths comparable to Illumina sequencing.
  • a further method is based on Helicos' Heliscope technology, wherein fragments are captured by polyT oligomers tethered to an array. At each sequencing cycle, polymerase and single fluorescently labeled nucleotides are added and the array is imaged.
  • sequencing techniques encompassed within the methods of the present invention are sequencing by hybridization, sequencing by use of nanopores, microscopy-based sequencing techniques, microfluidic Sanger sequencing, or microchip-based sequencing methods.
  • the present invention also envisages further developments of these techniques, e.g. further improvements of the accuracy of the sequence determination, or the time needed for the determination of the genomic sequence of an organism etc.
  • the sequencing method comprises deep sequencing.
  • deep sequencing refers to a sequencing method wherein the target sequence is read multiple times in the single test.
  • a single deep sequencing run is composed of a multitude of sequencing reactions run on the same target sequence and each, generating independent sequence readout.
  • oligonucleotides may be used that are capable of hybridizing thereto or to cDNA generated therefrom.
  • a single oligonucleotide is used to determine the presence of a particular determinant, at least two oligonucleotides are used to determine the presence of a particular determinant, at least five oligonucleotides are used to determine the presence of a particular determinant, at least four oligonucleotides are used to determine the presence of a particular determinant, at least five or more oligonucleotides are used to determine the presence of a particular determinant.
  • the method of this aspect of the present invention is carried out using an isolated oligonucleotide which hybridizes to the RNA or cDNA of any of the determinants listed in Tables 1-6 by complementary base-pairing in a sequence specific manner, and discriminates the determinant sequence from other nucleic acid sequence in the sample.
  • Oligonucleotides e.g. DNA or RNA oligonucleotides
  • Oligonucleotides typically comprises a region of complementary nucleotide sequence that hybridizes under stringent conditions to at least about 8, 10, 13, 16, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule.
  • the consecutive nucleotides include the determinant nucleic acid sequence.
  • isolated means an oligonucleotide, which by virtue of its origin or manipulation, is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained.
  • isolated it is alternatively or additionally meant that the oligonucleotide of interest is produced or synthesized by the hand of man.
  • the gene/transcript of interest is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence.
  • Typical algorithms will then identify oligonucleotides of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, lack predicted secondary structure that may interfere with hybridization, and/or possess other desired characteristics or that lack other undesired characteristics.
  • the sequence of the oligonucleotide may be analyzed by computer analysis to see if it is homologous (or is capable of hybridizing to) other known sequences.
  • a BLAST 2.2.10 (Basic Local Alignment Search Tool) analysis may be performed on the chosen oligonucleotide (worldwidewebdotncbidotnlmdotnihdotgov/blast/).
  • the BLAST program finds regions of local similarity between sequences. It compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches thereby providing valuable information about the possible identity and integrity of the ‘query’ sequences.
  • the oligonucleotide is a probe.
  • probe refers to an oligonucleotide which hybridizes to the determinant specific nucleic acid sequence to provide a detectable signal under experimental conditions and which does not hybridize to additional determinant sequences to provide a detectable signal under identical experimental conditions.
  • the probes of this embodiment of this aspect of the present invention may be, for example, affixed to a solid support (e.g., arrays or beads).
  • Solid supports are solid-state substrates or supports onto which the nucleic acid molecules of the present invention may be associated.
  • the nucleic acids may be associated directly or indirectly.
  • Solid-state substrates for use in solid supports can include any solid material with which components can be associated, directly or indirectly.
  • Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination.
  • Solid-state substrates and solid supports can be porous or non-porous.
  • a chip is a rectangular or square small piece of material.
  • Preferred forms for solid-state substrates are thin films, beads, or chips.
  • a useful form for a solid-state substrate is a microtiter dish. In some embodiments, a multiwell glass slide can be employed.
  • the solid support is an array which comprises a plurality of nucleic acids of the present invention immobilized at identified or predefined locations on the solid support.
  • Each predefined location on the solid support generally has one type of component (that is, all the components at that location are the same).
  • multiple types of components can be immobilized in the same predefined location on a solid support.
  • Each location will have multiple copies of the given components.
  • the spatial separation of different components on the solid support allows separate detection and identification.
  • the array does not comprise nucleic acids that specifically bind to more than 50 determinants, more than 40 determinants, 30 determinants, 20 determinants, 15 determinants, 10 determinants, 5 determinants or even 3 determinants.
  • the oligonucleotide is a primer of a primer pair.
  • the term “primer” refers to an oligonucleotide which acts as a point of initiation of a template-directed synthesis using methods such as PCR (polymerase chain reaction) or LCR (ligase chain reaction) under appropriate conditions (e.g., in the presence of four different nucleotide triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse-transcriptase, DNA ligase, etc, in an appropriate buffer solution containing any necessary co-factors and at suitable temperature(s)).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Such a template directed synthesis is also called “primer extension”.
  • a primer pair may be designed to amplify a region of DNA using PCR.
  • Such a pair will include a “forward primer” and a “reverse primer” that hybridize to complementary strands of a DNA molecule and that delimit a region to be synthesized/amplified.
  • a primer of this aspect of the present invention is capable of amplifying, together with its pair (e.g. by PCR) a determinant specific nucleic acid sequence to provide a detectable signal under experimental conditions and which does not amplify other determinant nucleic acid sequence to provide a detectable signal under identical experimental conditions.
  • the oligonucleotide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. While the maximal length of a probe can be as long as the target sequence to be detected, depending on the type of assay in which it is employed, it is typically less than about 50, 60, 65, or 70 nucleotides in length. In the case of a primer, it is typically less than about 30 nucleotides in length. In a specific preferred embodiment of the invention, a primer or a probe is within the length of about 18 and about 28 nucleotides. It will be appreciated that when attached to a solid support, the probe may be of about 30-70, 75, 80, 90, 100, or more nucleotides in length.
  • the oligonucleotide of this aspect of the present invention need not reflect the exact sequence of the determinant nucleic acid sequence (i.e. need not be fully complementary), but must be sufficiently complementary to hybridize with the determinant nucleic acid sequence under the particular experimental conditions. Accordingly, the sequence of the oligonucleotide typically has at least 70% homology, preferably at least 80%, 90%, 95%, 97%, 99% or 100% homology, for example over a region of at least 13 or more contiguous nucleotides with the target determinant nucleic acid sequence. The conditions are selected such that hybridization of the oligonucleotide to the determinant nucleic acid sequence is favored and hybridization to other determinant nucleic acid sequences is minimized.
  • hybridization of short nucleic acids can be effected by the following hybridization protocols depending on the desired stringency; (i) hybridization solution of 6 ⁇ SSC and 1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 ⁇ g/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C.
  • hybridization solution 6 ⁇ SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 ⁇ g/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature at 2.5-3° C. below the Tm and final wash solution of 6 ⁇ SSC at 22° C. (moderate hybridization solution).
  • Oligonucleotides of the invention may be prepared by any of a variety of methods (see, for example, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, M. A. Innis (Ed.), Academic Press: New York, N.Y.; P. Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, M. A.
  • oligonucleotides may be prepared using any of a variety of chemical techniques well-known in the art, including, for example, chemical synthesis and polymerization based on a template as described, for example, in S. A. Narang et al., Meth. Enzymol. 1979, 68: 90-98; E. L. Brown et al., Meth. Enzymol. 1979, 68: 109-151; E. S.
  • oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach.
  • each nucleotide is individually added to the 5′-end of the growing oligonucleotide chain, which is attached at the 3′-end to a solid support.
  • the added nucleotides are in the form of trivalent 3′-phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5′-position.
  • DMT dimethoxytriyl
  • oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide.
  • These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.).
  • oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc. (Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.
  • Purification of the oligonucleotides of the invention may be carried out by any of a variety of methods well-known in the art. Purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC as described, for example, by J. D. Pearson and F. E. Regnier (J. Chrom., 1983, 255: 137-149) or by reverse phase HPLC (G. D. McFarland and P. N. Borer, Nucleic Acids Res., 1979, 7: 1067-1080).
  • sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation (A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles et al., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (H. Wu and H. Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like.
  • chemical degradation A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560
  • MALDI-TOF matrix-assisted laser desorption ionization time-of-flight
  • mass spectrometry U. Pieles et al., Nucleic Acid
  • modified oligonucleotides may be prepared using any of several means known in the art.
  • Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc).
  • Oligonucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc), intercalators (e.g., acridine, psoralen, etc), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc), and alkylators.
  • the oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • the oligonucleotide sequences of the present invention may also be modified with a label.
  • the detection probes or amplification primers or both probes and primers are labeled with a detectable agent or moiety before being used in amplification/detection assays.
  • the detection probes are labeled with a detectable agent.
  • a detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.
  • Labeled detection probes can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g., through a linker). Linkers or spacer arms of various lengths are known in the art and are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules (see, for example, E. S. Mansfield et al., Mol. Cell. Probes, 1995, 9: 145-156).
  • Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (L. M. Smith et al., Nucl. Acids Res., 1985, 13: 2399-2412) or of enzymes (B.
  • nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of mono-reactive cisplatin derivatives with the N7 position of guanine moieties in DNA (R. J. Heetebrij et al., Cytogenet. Cell. Genet. 1999, 87: 47-52), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (C. Levenson et al., Methods Enzymol. 1990, 184: 577-583; and C. Pfannschmidt et al., Nucleic Acids Res.
  • ULS Universal Linkage System
  • detectable agents include, but are not limited to, various ligands, radionuclides (such as, for example, 32 P, 35 S, 3 H 14 C, 125 I, 131 I, and the like); fluorescent dyes (for specific exemplary fluorescent dyes, see below); chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper and platinum) or nanoclusters; enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (such as, for example, dyes, colloidal gold, and
  • the inventive detection probes are fluorescently labeled.
  • fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6 carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B,
  • fluorescein and fluorescein dyes e.g., fluorescein isothiocyanine or FITC, naph
  • fluorescent dyes and methods for linking or incorporating fluorescent dyes to nucleic acid molecules see, for example, “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg. Fluorescent dyes as well as labeling kits are commercially available from, for example, Amersham Biosciences, Inc. (Piscataway, N.J.), Molecular Probes Inc. (Eugene, Oreg.), and New England Biolabs Inc. (Berverly, Mass.).
  • identification of the determinant may be carried out using an amplification reaction.
  • amplification refers to a process that increases the representation of a population of specific nucleic acid sequences in a sample by producing multiple (i.e., at least 2) copies of the desired sequences.
  • Methods for nucleic acid amplification include, but are not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • a nucleic acid sequence of interest is often amplified at least fifty thousand fold in amount over its amount in the starting sample.
  • a “copy” or “amplicon” does not necessarily mean perfect sequence complementarity or identity to the template sequence.
  • copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
  • nucleotide analogs such as deoxyinosine
  • intentional sequence alterations such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template
  • sequence errors that occur during amplification.
  • a typical amplification reaction is carried out by contacting a forward and reverse primer (a primer pair) to the sample DNA together with any additional amplification reaction reagents under conditions which allow amplification of the target sequence.
  • forward primer and “forward amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the target (template strand).
  • reverse primer and “reverse amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the complementary target strand. The forward primer hybridizes with the target sequence 5′ with respect to the reverse primer.
  • amplification conditions refers to conditions that promote annealing and/or extension of primer sequences. Such conditions are well-known in the art and depend on the amplification method selected. Thus, for example, in a PCR reaction, amplification conditions generally comprise thermal cycling, i.e., cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, amplification occurs without thermal cycling although an initial temperature increase may be required to initiate the reaction. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and temperature cycling, buffer, salt, ionic strength, and pH, and the like.
  • amplification reaction reagents refers to reagents used in nucleic acid amplification reactions and may include, but are not limited to, buffers, reagents, enzymes having reverse transcriptase and/or polymerase activity or exonuclease activity, enzyme cofactors such as magnesium or manganese, salts, nicotinamide adenine dinuclease (NAD) and deoxynucleoside triphosphates (dNTPs), such as deoxyadenosine triphospate, deoxyguano sine triphosphate, deoxycytidine triphosphate and thymidine triphosphate.
  • Amplification reaction reagents may readily be selected by one skilled in the art depending on the amplification method used.
  • the amplifying may be effected using techniques such as polymerase chain reaction (PCR), which includes, but is not limited to Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence-specific PCR (ISSR), Inverse PCR, Ligation-mediated PCR, Methylation-specific PCR (MSP), Miniprimer PCR, Multiplex Ligation-dependent Probe Amplification, Multiplex-PCR, Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR), Reverse Transcription PCR (RT-PCR), Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous
  • PCR polymerase chain reaction
  • K. B. Mullis and F. A. Faloona Methods Enzymol., 1987, 155: 350-355 and U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,800,159 (each of which is incorporated herein by reference in its entirety).
  • PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA.
  • a plurality of reaction cycles results in the exponential accumulation of a specific DNA fragment
  • PCR Protocols A Guide to Methods and Applications”, M. A. Innis (Ed.), 1990, Academic Press: New York; “PCR Strategies”, M. A. Innis (Ed.), 1995, Academic Press: New York; “Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach”, McPherson et al. (Eds.), 1991, IRL Press: Oxford; R. K. Saiki et al., Nature, 1986, 324: 163-166).
  • the termini of the amplified fragments are defined as the 5′ ends of the primers.
  • DNA polymerases capable of producing amplification products in PCR reactions include, but are not limited to: E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcus litoralis (“Vent” polymerase, New England Biolabs).
  • RNA target sequences may be amplified by reverse transcribing the mRNA into cDNA, and then performing PCR (RT-PCR), as described above.
  • RT-PCR PCR
  • a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770.
  • the duration and temperature of each step of a PCR cycle, as well as the number of cycles, are generally adjusted according to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated. The ability to optimize the reaction cycle conditions is well within the knowledge of one of ordinary skill in the art.
  • the number of reaction cycles may vary depending on the detection analysis being performed, it usually is at least 15, more usually at least 20, and may be as high as 60 or higher. However, in many situations, the number of reaction cycles typically ranges from about 20 to about 40.
  • the denaturation step of a PCR cycle generally comprises heating the reaction mixture to an elevated temperature and maintaining the mixture at the elevated temperature for a period of time sufficient for any double-stranded or hybridized nucleic acid present in the reaction mixture to dissociate.
  • the temperature of the reaction mixture is usually raised to, and maintained at, a temperature ranging from about 85° C. to about 100° C., usually from about 90° C. to about 98° C., and more usually from about 93° C. to about 96° C. for a period of time ranging from about 3 to about 120 seconds, usually from about 5 to about 30 seconds.
  • the reaction mixture is subjected to conditions sufficient for primer annealing to template DNA present in the mixture.
  • the temperature to which the reaction mixture is lowered to achieve these conditions is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 50° C. to about ° C., usually from about 55° C. to about 70° C., and more usually from about 60° C. to about 68° C.
  • Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.
  • the reaction mixture is subjected to conditions sufficient to provide for polymerization of nucleotides to the primer's end in a such manner that the primer is extended in a 5′ to 3′ direction using the DNA to which it is hybridized as a template, (i.e., conditions sufficient for enzymatic production of primer extension product).
  • conditions sufficient for enzymatic production of primer extension product i.e., conditions sufficient for enzymatic production of primer extension product.
  • the temperature of the reaction mixture is typically raised to a temperature ranging from about 65° C. to about 75° C., usually from about 67° C. to about 73° C., and maintained at that temperature for a period of time ranging from about 15 seconds to about 20 minutes, usually from about 30 seconds to about 5 minutes.
  • thermal cyclers that may be employed are described in U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610 (each of which is incorporated herein by reference in its entirety). Thermal cyclers are commercially available, for example, from Perkin Elmer-Applied Biosystems (Norwalk, Conn.), BioRad (Hercules, Calif.), Roche Applied Science (Indianapolis, Ind.), and Stratagene (La Jolla, Calif.).
  • Amplification products obtained using primers of the present invention may be detected using agarose gel electrophoresis and visualization by ethidium bromide staining and exposure to ultraviolet (UV) light or by sequence analysis of the amplification product.
  • UV ultraviolet
  • the amplification and quantification of the amplification product may be effected in real-time (qRT-PCR).
  • qRT-PCR real-time
  • QRT-PCR methods use double stranded DNA detecting molecules to measure the amount of amplified product in real time.
  • double stranded DNA detecting molecule refers to a double stranded DNA interacting molecule that produces a quantifiable signal (e.g., fluorescent signal).
  • a double stranded DNA detecting molecule can be a fluorescent dye that (1) interacts with a fragment of DNA or an amplicon and (2) emits at a different wavelength in the presence of an amplicon in duplex formation than in the presence of the amplicon in separation.
  • a double stranded DNA detecting molecule can be a double stranded DNA intercalating detecting molecule or a primer-based double stranded DNA detecting molecule.
  • a double stranded DNA intercalating detecting molecule is not covalently linked to a primer, an amplicon or a nucleic acid template.
  • the detecting molecule increases its emission in the presence of double stranded DNA and decreases its emission when duplex DNA unwinds. Examples include, but are not limited to, ethidium bromide, YO-PRO-1, Hoechst 33258, SYBR Gold, and SYBR Green I.
  • Ethidium bromide is a fluorescent chemical that intercalates between base pairs in a double stranded DNA fragment and is commonly used to detect DNA following gel electrophoresis. When excited by ultraviolet light between 254 nm and 366 nm, it emits fluorescent light at 590 nm.
  • the DNA-ethidium bromide complex produces about 50 times more fluorescence than ethidium bromide in the presence of single stranded DNA.
  • SYBR Green I is excited at 497 nm and emits at 520 nm. The fluorescence intensity of SYBR Green I increases over 100 fold upon binding to double stranded DNA against single stranded DNA.
  • SYBR Gold introduced by Molecular Probes Inc. Similar to SYBR Green I, the fluorescence emission of SYBR Gold enhances in the presence of DNA in duplex and decreases when double stranded DNA unwinds. However, SYBR Gold's excitation peak is at 495 nm and the emission peak is at 537 nm.
  • Hoechst 33258 is a known bisbenzimide double stranded DNA detecting molecule that binds to the AT rich regions of DNA in duplex. Hoechst 33258 excites at 350 nm and emits at 450 nm. YO-PRO-1, exciting at 450 nm and emitting at 550 nm, has been reported to be a double stranded DNA specific detecting molecule.
  • the double stranded DNA detecting molecule is SYBR Green I.
  • a primer-based double stranded DNA detecting molecule is covalently linked to a primer and either increases or decreases fluorescence emission when amplicons form a duplex structure. Increased fluorescence emission is observed when a primer-based double stranded DNA detecting molecule is attached close to the 3′ end of a primer and the primer terminal base is either dG or dC.
  • the detecting molecule is quenched in the proximity of terminal dC-dG and dG-dC base pairs and dequenched as a result of duplex formation of the amplicon when the detecting molecule is located internally at least 6 nucleotides away from the ends of the primer. The dequenching results in a substantial increase in fluorescence emission.
  • Examples of these type of detecting molecules include but are not limited to fluorescein (exciting at 488 nm and emitting at 530 nm), FAM (exciting at 494 nm and emitting at 518 nm), JOE (exciting at 527 and emitting at 548), HEX (exciting at 535 nm and emitting at 556 nm), TET (exciting at 521 nm and emitting at 536 nm), Alexa Fluor 594 (exciting at 590 nm and emitting at 615 nm), ROX (exciting at 575 nm and emitting at 602 nm), and TAMRA (exciting at 555 nm and emitting at 580 nm).
  • fluorescein exciting at 488 nm and emitting at 530 nm
  • FAM exciting at 494 nm and emitting at 518 nm
  • JOE exciting at 527 and emitting at 548
  • HEX exciting at 535
  • primer-based double stranded DNA detecting molecules decrease their emission in the presence of double stranded DNA against single stranded DNA.
  • examples include, but are not limited to, rhodamine, and BODIPY-FI (exciting at 504 nm and emitting at 513 nm).
  • These detecting molecules are usually covalently conjugated to a primer at the 5′ terminal dC or dG and emit less fluorescence when amplicons are in duplex. It is believed that the decrease of fluorescence upon the formation of duplex is due to the quenching of guanosine in the complementary strand in close proximity to the detecting molecule or the quenching of the terminal dC-dG base pairs.
  • the primer-based double stranded DNA detecting molecule is a 5′ nuclease probe.
  • Such probes incorporate a fluorescent reporter molecule at either the 5′ or 3′ end of an oligonucleotide and a quencher at the opposite end.
  • the first step of the amplification process involves heating to denature the double stranded DNA target molecule into a single stranded DNA.
  • a forward primer anneals to the target strand of the DNA and is extended by Taq polymerase.
  • a reverse primer and a 5′ nuclease probe then anneal to this newly replicated strand.
  • At least one of the primer pairs or 5′ nuclease probe should hybridize with a unique determinant sequence.
  • the polymerase extends and cleaves the probe from the target strand. Upon cleavage, the reporter is no longer quenched by its proximity to the quencher and fluorescence is released. Each replication will result in the cleavage of a probe. As a result, the fluorescent signal will increase proportionally to the amount of amplification product.
  • the determinants described in this application may be used to distinguish between an infectious (bacterial or viral) and non-infectious subject.
  • the determinant is one which appears in Table 16E.
  • a determinant in Table 16E which is characterized as increasing during an infection, when that determinant is above a predetermined threshold it is indicative that the subject has an infection.
  • a determinant in Table 16E which is characterized as increasing not due to an infection, then when the determinant is above a predetermined threshold it is indicative that the subject does not have an infection.
  • the non-infectious subject of this aspect of the present invention has SIRS and the infectious subject has sepsis.
  • SIRS is a serious condition related to systemic inflammation, organ dysfunction, and organ failure. It is defined as 2 or more of the following variables: fever of more than 38° C. (100.4° F.) or less than 36° C. (96.8° F.); heart rate of more than 90 beats per minute; respiratory rate of more than 20 breaths per minute or arterial carbon dioxide tension (PaCO 2 ) of less than 32 mm Hg; abnormal white blood cell count (>12,000/ ⁇ L or ⁇ 4,000/ ⁇ L or >10% immature [band] forms). SIRS is nonspecific and can be caused by ischemia, inflammation, trauma, infection, or several insults combined. Thus, SIRS is not always related to infection.
  • Sepsis is a life-threatening condition that is caused by inflammatory response to an infection. It is currently diagnosed as the presence of SIRS criteria in the presence of a known infection. The early diagnosis of sepsis is essential for clinical intervention before the disease rapidly progresses beyond initial stages to the more severe stages, such as severe sepsis or septic shock, which are associated with high mortality.
  • non-infectious SIRS associated with acute tissue injury and innate immune activation can induce clinical syndromes analogous to sepsis, including multiple trauma, pancreatitis, burns, and autoimmune diseases.
  • Current diagnostics are limited in their ability to distinguish between SIRS and sepsis. Therefore, there is a need for new biomarkers or combinations of biomarkers that can provide added value in the accurate and timely diagnosis of sepsis.
  • the method of this aspect of the present invention may be used to distinguish between a subject who has SIRS (and no infection) and a subject who has sepsis. If the subject has sepsis then at least one of the RNAs from Table 16E is above a predetermined level (e.g. above the amount in a subject who does not have an infection, such as a healthy subject).
  • a predetermined level e.g. above the amount in a subject who does not have an infection, such as a healthy subject.
  • RNA determinants that may be used to distinguish between non-infectious SIRS and infectious sepsis include any pair or triplet of determinants that appears in Table 16E.
  • RNA determinants that may be used to distinguish between non-infectious SIRS and infectious sepsis include but are not limited to:
  • the determinants described in this application may be used to distinguish between a bacterial infection and a non-bacterial infection.
  • the determinant is one which appears in Table 16D.
  • a determinant in Table 16D which is characterized as increasing during a bacterial infection, when that determinant is above a predetermined threshold it is indicative that the subject has a bacterial infection.
  • a determinant in Table 16D which is characterized as increasing due to a non-bacterial infection, then when the determinant is above a predetermined threshold it is indicative that the subject does not have a bacterial infection.
  • RNA determinants are the biological precursor of proteins and changes in its expression levels are expected to precede those of its protein counterparts. Consequently, protein and RNA biomarkers may differ in their temporal dynamics patterns and can complement each other to detect infection prior to symptom onset or following convalescence. Therefore, it will be appreciated that as well as determining the level of the RNA determinants described herein, the present inventors also contemplate combining these measurements with measurements of polypeptide determinants that are known to be indicative of infection type. Examples of polypeptide determinants that are contemplated by the present invention include those that are described in WO 2013/117746, WO 2011/132086, PCT Application IL 2015/051024 and PCT Application IL 2015/051201, the contents of each are incorporated herein by reference. Other polypeptide determinants contemplated by the present inventors are the polypeptide counterparts of the RNA determinants described herein.
  • polypeptides contemplated by the present inventors are those set forth in Table 15 herein below.
  • Other examples include, but are not limited to: TRAIL, CRP, IP-10, MX1, RSAD2, PCT, OTOF, PI3, CYBRD1, EIF2AK2 and CMPK2.
  • polypeptide combinations contemplated by the present inventors include, but are not limited to: TRAIL+CRP+IP-10; MX1+CRP; MX1+RSAD2; CRP+RSAD2; MX1+CRP+RSAD2; MX1+CRP+PCT; MX1+CRP+TRAIL; TRAIL+CRP+RSAD2; and OTOF, PI3, CYBRD1, EIF2AK2 and CMPK2.
  • RNA determinants and polypeptide determinants include but are not limited to:
  • RNA determinant selected from Tables 1-6 or 15A-B and analyzing a polypeptide determinant indicative of infection type for example CRP or TRAIL.
  • exemplary contemplated combinations of polypeptide determinants and RNA determinants are provided in Table 21 of the Examples section herein below.
  • RNA and protein determinants may be combined to provide a single predictive score.
  • the level of the polypeptide determinant is used for initial screening of subjects with suspected infection and the level of the RNA determinant is used as a follow-up test only in cases where the polypeptide test is inconclusive (e.g. higher/lower than a predetermined threshold).
  • the level of the RNA determinant is used for initial screening of subjects with suspected infection and the level of the polypeptide determinant is used as a follow-up test only in cases where the polypeptide test is inconclusive (e.g. higher/lower than a predetermined threshold).
  • RNA signatures comprised of the RNA determinants set forth in Tables 1-6 are combined with their protein counterparts in order to improve diagnostic accuracy.
  • Methods of measuring the levels of polypeptides include, e.g., immunoassays based on antibodies to proteins, aptamers or molecular imprints.
  • the polypeptide determinants can be detected in any suitable manner, but are typically detected by contacting a sample from the subject with an antibody, which binds the determinant and then detecting the presence or absence of a reaction product.
  • the antibody may be monoclonal, polyclonal, chimeric, or a fragment of the foregoing, as discussed in detail above, and the step of detecting the reaction product may be carried out with any suitable immunoassay.
  • the sample from the subject is typically a biological sample as described above, and may be the same sample of biological sample used to conduct the method described above.
  • the antibody which specifically binds the determinant is attached (either directly or indirectly) to a signal producing label, including but not limited to a radioactive label, an enzymatic label, a hapten, a reporter dye or a fluorescent label.
  • Immunoassays carried out in accordance with some embodiments of the present invention may be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody (e.g., anti-determinant antibody), a labeled analyte, and the sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte.
  • Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution.
  • Immunochemical labels which may be employed, include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.
  • the reagents are usually the sample, the antibody, and means for producing a detectable signal.
  • Samples as described above may be used.
  • the antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the sample.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, or enzyme labels.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are oligonucleotides, immunoblotting, immunofluorescence methods, immunoprecipitation, chemiluminescence methods, electrochemiluminescence (ECL) or enzyme-linked immunoassays.
  • Antibodies can be conjugated to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding.
  • Antibodies as described herein may likewise be conjugated to detectable labels or groups such as radiolabels (e.g., 35 S, 125 I, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques.
  • a diagnostic assay e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene
  • Antibodies as described herein may likewise be conjugated to detectable labels or groups such as radiolabel
  • Antibodies can also be useful for detecting post-translational modifications of determinant proteins, polypeptides, mutations, and polymorphisms, such as tyrosine phosphorylation, threonine phosphorylation, serine phosphorylation, glycosylation (e.g., O-GlcNAc).
  • Such antibodies specifically detect the phosphorylated amino acids in a protein or proteins of interest, and can be used in immunoblotting, immunofluorescence, and ELISA assays described herein. These antibodies are well-known to those skilled in the art, and commercially available.
  • Post-translational modifications can also be determined using metastable ions in reflector matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Wirth U. and Muller D. 2002).
  • MALDI-TOF reflector matrix-assisted laser desorption ionization-time of flight mass spectrometry
  • the activities can be determined in vitro using enzyme assays known in the art.
  • enzyme assays include, without limitation, kinase assays, phosphatase assays, reductase assays, among many others.
  • Modulation of the kinetics of enzyme activities can be determined by measuring the rate constant K M using known algorithms, such as the Hill plot, Michaelis-Menten equation, linear regression plots such as Lineweaver-Burk analysis, and Scatchard plot.
  • Suitable sources for antibodies for the detection of determinants include commercially available sources such as, for example, Abazyme, Abnova, AssayPro, Affinity Biologicals, AntibodyShop, Aviva bioscience, Biogenesis, Biosense Laboratories, Calbiochem, Cell Sciences, Chemicon International, Chemokine, Clontech, Cytolab, DAKO, Diagnostic BioSystems, eBioscience, Endocrine Technologies, Enzo Biochem, Eurogentec, Fusion Antibodies, Genesis Biotech, GloboZymes, Haematologic Technologies, Immunodetect, Immunodiagnostik, Immunometrics, Immunostar, Immunovision, Biogenex, Invitrogen, Jackson ImmunoResearch Laboratory, KMI Diagnostics, Koma Biotech, LabFrontier Life Science Institute, Lee Laboratories, Lifescreen, Maine Biotechnology Services, Mediclone, MicroPharm Ltd., ModiQuest, Molecular Innovations, Molecular Probes, Neoclone, Neuromics, New England Biolabs, Novocastra,
  • a “subject” in the context of the present invention may be a mammal (e.g. human, dog, cat, horse, cow, sheep, pig, goat).
  • the subject is a bird (e.g. chicken, turkey, duck or goose).
  • the subject is a human.
  • the subject can be male or female.
  • the subject may be an adult (e.g. older than 18, 21, or 22 years or a child (e.g. younger than 18, 21 or 22 years).
  • the subject is an adolescent (between 12 and 21 years), an infant (29 days to less than 2 years of age) or a neonate (birth through the first 28 days of life).
  • the subject can be one who has been previously diagnosed or identified as having an infection, and optionally has already undergone, or is undergoing, a therapeutic intervention for the infection.
  • a subject can also be one who has not been previously diagnosed as having an infection.
  • a subject can be one who exhibits one or more risk factors for having an infection.
  • the marker which is used to diagnose the infection may be selected according to the particular subject.
  • RNA determinants for ruling in infection type in children include but are not limited to SIGLEC1, IFI27, n334829, LY6E, USP41, USP18, uc003hr1.1, n332456, n332510, GAS7, TCONS_00003184-XLOC_001966, OAS1, n333319, OASL, IFI44L, SULT1B1.
  • RNA determinants for ruling in infection type in adults include but are not limited to IFI44, DGAT2, PYGL, n407780, ZCCHC2, PARP12, TRIB2.
  • RNA determinants for ruling in infection type in male subjects include, but are not limited to DDX60, ZCCHC2, LY6E, IFI44, n332510, IFIT1, OAS1, TCONS_00003184-XLOC_001966, PNPT1.
  • RNA determinants for ruling in infection type in female subjects include but are not limited to n407780, CYP1B1, IMPA2, KREMEN1, n332456, CR1, GAS7, PARP12, n333319, PPM1K, n334829, IFI27, uc003hr1.1, SULT1B1, LTA4H.
  • the subject presents with symptoms of pneumonia and the test is carried out to determine if the source of the pneumonia is from a bacterial or viral infection.
  • Particular recommended determinants for this use are at least one of those listed in Table 23 of the Examples section herein below.
  • RNA determinants for ruling in influenza include SIGLEC1, n332456, DDX60, HER6 and TCONS_00003184-XLOC_001966.
  • Particular RNA determinants for ruling in parainfluenza include CYP1B1, GAS7 and TCONS_00003184-XLOC_001966.
  • CYP1B1, GAS7 are below a predetermined level (e.g. below the level found in an identical sample type of a healthy or non-infected subject), a parainfluenza infection is ruled in.
  • TCONS_00003184-XLOC_001966 is above a predetermined level (e.g.
  • the RNA determinant is used for ruling in CMV, EBV or Bocavirus.
  • a predetermined level e.g. below the level found in an identical sample type of a healthy or non-infected subject
  • a CMV, EBV or Bocavirus infection is ruled in.
  • RNA determinants have also been shown to be highly accurate in distinguishing between viral infections and bacterial infections of a gram negative bacteria. Such RNA determinants are set forth in Table 22 of the Examples section herein below.
  • kits may contain in separate containers oligonucleotides or antibodies (either already bound to a solid matrix or packaged separately with reagents for binding them to the matrix), control formulations (positive and/or negative), and/or a detectable label such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among others.
  • the detectable label may be attached to a secondary antibody which binds to the Fc portion of the antibody which recognizes the determinant.
  • the detectable label may also be attached to the oligonucleotides. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may be included in the kit.
  • kits of this aspect of the present invention may comprise additional components that aid in the detection of the determinants such as enzymes, salts, buffers etc. necessary to carry out the detection reactions.
  • kits for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in Table 1 or Table 2, and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6 or Tables 15A-B.
  • the at least two determinant detection reagents comprise RNA detection reagents (e.g. oligonucleotides, as described herein above) or protein detection reagents (antibodies, as described herein above).
  • RNA detection reagents e.g. oligonucleotides, as described herein above
  • protein detection reagents antibodies, as described herein above.
  • the detection reagents may be attached to detectable moieties as described herein above.
  • the kit contains a number of detection reagents such that no more than 20 determinants (e.g. RNAs) can be detected.
  • determinants e.g. RNAs
  • the kit contains a number of detection reagents such that no more than 10 determinants (e.g. RNAs) can be detected.
  • determinants e.g. RNAs
  • the kit contains a number of detection reagents such that no more than 5 determinants (e.g. RNAs) can be detected.
  • determinants e.g. RNAs
  • the kit contains a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • the kit contains a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-6 or Tables 15A-B.
  • the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-6 or Tables 16A.
  • the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-2.
  • kits of the present invention comprise detection reagents such that pairs of determinants set forth in Tables 9, 11, 12, 17 or 18 may be analyzed.
  • kits of the present invention comprise detection reagents such that triplets of determinants set forth in Tables 13,14, 19 or 20 may be analyzed.
  • kits for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two RNA detection reagents specifically detects a first RNA which is set forth in Table 3 or Table 4, and a second of the at least two RNA detection reagents specifically detects a second RNA which is set forth in any one of
  • kits 1-6 or Tables 15A-B wherein the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • the kit of this aspect of the present invention contains a number of detection reagents such that no more than 10 determinants (e.g. RNAs) can be detected.
  • determinants e.g. RNAs
  • the kit of this aspect of the present invention contains a number of detection reagents such that no more than 5 determinants (e.g. RNAs) can be detected.
  • determinants e.g. RNAs
  • the kit of this aspect of the present invention contains a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • the kit of this aspect of the present invention contains a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in any one of Tables 1-6 or 16A and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6 or 16A, wherein the kit comprises detection reagents that specifically detect no more than 5 determinants.
  • Some aspects of the present invention can also be used to screen patient or subject populations in any number of settings.
  • a health maintenance organization, public health entity or school health program can screen a group of subjects to identify those requiring interventions, as described above, or for the collection of epidemiological data.
  • Insurance companies e.g., health, life or disability
  • Data collected in such population screens, particularly when tied to any clinical progression to conditions like infection, will be of value in the operations of, for example, health maintenance organizations, public health programs and insurance companies.
  • Such data arrays or collections can be stored in machine-readable media and used in any number of health-related data management systems to provide improved healthcare services, cost effective healthcare, improved insurance operation, etc. See, for example, U.S.
  • Such systems can access the data directly from internal data storage or remotely from one or more data storage sites as further detailed herein.
  • a machine-readable storage medium can comprise a data storage material encoded with machine readable data or data arrays which, when using a machine programmed with instructions for using the data, is capable of use for a variety of purposes.
  • Measurements of effective amounts of the biomarkers of the invention and/or the resulting evaluation of risk from those biomarkers can be implemented in computer programs executing on programmable computers, comprising, inter alia, a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code can be applied to input data to perform the functions described above and generate output information.
  • the output information can be applied to one or more output devices, according to methods known in the art.
  • the computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. The language can be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette or others as defined elsewhere in this disclosure) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • a storage media or device e.g., ROM or magnetic diskette or others as defined elsewhere in this disclosure
  • the health-related data management system used in some aspects of the invention may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform various functions described herein.
  • the determinants of the present invention in some embodiments thereof, can be used to generate a “reference determinant profile” of those subjects who do not have an infection.
  • the determinants disclosed herein can also be used to generate a “subject determinant profile” taken from subjects who have an infection.
  • the subject determinant profiles can be compared to a reference determinant profile to diagnose or identify subjects with an infection.
  • the subject determinant profile of different infection types can be compared to diagnose or identify the type of infection.
  • the reference and subject determinant profiles of the present invention in some embodiments thereof, can be contained in a machine-readable medium, such as but not limited to, analog tapes like those readable by a VCR, CD-ROM, DVD-ROM, USB flash media, among others.
  • Such machine-readable media can also contain additional test results, such as, without limitation, measurements of clinical parameters and traditional laboratory risk factors.
  • the machine-readable media can also comprise subject information such as medical history and any relevant family history.
  • the machine-readable media can also contain information relating to other disease-risk algorithms and computed indices such as those described herein.
  • the effectiveness of a treatment regimen can be monitored by detecting a determinant in an effective amount (which may be one or more) of samples obtained from a subject over time and comparing the amount of determinants detected. For example, a first sample can be obtained prior to the subject receiving treatment and one or more subsequent samples are taken after or during treatment of the subject.
  • the methods of the invention can be used to discriminate between bacterial, viral and mixed infections (i.e. bacterial and viral co-infections.) This will allow patients to be stratified and treated accordingly.
  • a treatment recommendation i.e., selecting a treatment regimen for a subject is provided by identifying the type infection (i.e., bacterial, viral, mixed infection or no infection) in the subject according to the method of any of the disclosed methods and recommending that the subject receive an antibiotic treatment if the subject is identified as having bacterial infection or a mixed infection; or an anti-viral treatment is if the subject is identified as having a viral infection.
  • type infection i.e., bacterial, viral, mixed infection or no infection
  • the methods of the invention can be used to prompt additional targeted diagnosis such as pathogen specific PCRs, chest-X-ray, cultures etc.
  • additional targeted diagnosis such as pathogen specific PCRs, chest-X-ray, cultures etc.
  • a diagnosis that indicates a viral infection according to embodiments of this invention may prompt the usage of additional viral specific multiplex-PCRs
  • a diagnosis that indicates a bacterial infection according to embodiments of this invention may prompt the usage of a bacterial specific multiplex-PCR.
  • a diagnostic test recommendation for a subject is provided by identifying the infection type (i.e., bacterial, viral, mixed infection or no infection) in the subject according to any of the disclosed methods and recommending a test to determine the source of the bacterial infection if the subject is identified as having a bacterial infection or a mixed infection; or a test to determine the source of the viral infection if the subject is identified as having a viral infection.
  • the infection type i.e., bacterial, viral, mixed infection or no infection
  • the performance and thus absolute and relative clinical usefulness of the invention may be assessed in multiple ways as noted above.
  • some aspects of the invention are intended to provide accuracy in clinical diagnosis and prognosis.
  • the accuracy of a diagnostic or prognostic test, assay, or method concerns the ability of the test, assay, or method to distinguish between subjects having an infection is based on whether the subjects have, a “significant alteration” (e.g., clinically significant and diagnostically significant) in the levels of a determinant.
  • a “significant alteration” e.g., clinically significant and diagnostically significant
  • effective amount it is meant that the measurement of an appropriate number of determinants (which may be one or more) to produce a “significant alteration” (e.g.
  • the difference in the level of determinant is preferably statistically significant. As noted below, and without any limitation of the invention, achieving statistical significance, and thus the preferred analytical, diagnostic, and clinical accuracy, may require that combinations of several determinants be used together in panels and combined with mathematical algorithms in order to achieve a statistically significant determinant index.
  • AUC area under the ROC curve
  • an “acceptable degree of diagnostic accuracy” is herein defined as a test or assay (such as the test used in some aspects of the invention for determining the clinically significant presence of determinants, which thereby indicates the presence an infection type) in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.
  • the methods predict the presence or absence of an infection or response to therapy with at least 75% total accuracy, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater total accuracy.
  • the methods predict the presence of a bacterial infection or response to therapy with at least 75% sensitivity, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater sensitivity.
  • the methods predict the presence of a viral infection or response to therapy with at least 75% specificity, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater specificity.
  • the methods predict the presence or absence of an infection or response to therapy with an MCC larger than 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.
  • diagnostic accuracy In general, alternative methods of determining diagnostic accuracy are commonly used for continuous measures, when a disease category has not yet been clearly defined by the relevant medical societies and practice of medicine, where thresholds for therapeutic use are not yet established, or where there is no existing gold standard for diagnosis of the pre-disease.
  • measures of diagnostic accuracy for a calculated index are typically based on curve fit and calibration between the predicted continuous value and the actual observed values (or a historical index calculated value) and utilize measures such as R squared, Hosmer-Lemeshow P-value statistics and confidence intervals.
  • the degree of diagnostic accuracy i.e., cut points on a ROC curve
  • defining an acceptable AUC value determining the acceptable ranges in relative concentration of what constitutes an effective amount of the determinants of the invention allows for one of skill in the art to use the determinants to identify, diagnose, or prognose subjects with a pre-determined level of predictability and performance.
  • biomarkers will be very highly correlated with the determinants (for the purpose of this application, any two variables will be considered to be “very highly correlated” when they have a Coefficient of Determination (R 2 ) of 0.5 or greater).
  • R 2 Coefficient of Determination
  • Some aspects of the present invention encompass such functional and statistical equivalents to the aforementioned determinants.
  • the statistical utility of such additional determinants is substantially dependent on the cross-correlation between multiple biomarkers and any new biomarkers will often be required to operate within a panel in order to elaborate the meaning of the underlying biology.
  • One or more of the listed determinants can be detected in the practice of the present invention, in some embodiments thereof. For example, two (2), three (3), four (4), five (5), ten (10), fifteen (15), twenty (20), forty (40), or more determinants can be detected.
  • all determinants listed herein can be detected.
  • Preferred ranges from which the number of determinants can be detected include ranges bounded by any minimum selected from between one and, particularly two, three, four, five, six, seven, eight, nine ten, twenty, or forty. Particularly preferred ranges include two to five (2-5), two to ten (2-10), two to twenty (2-20), or two to forty (2-40).
  • a “panel” within the context of the present invention means a group of biomarkers (whether they are determinants, clinical parameters, or traditional laboratory risk factors) that includes one or more determinants.
  • a panel can also comprise additional biomarkers, e.g., clinical parameters, traditional laboratory risk factors, known to be present or associated with infection, in combination with a selected group of the determinants listed herein.
  • a common measure of statistical significance is the p-value, which indicates the probability that an observation has arisen by chance alone; preferably, such p-values are 0.05 or less, representing a 5% or less chance that the observation of interest arose by chance. Such p-values depend significantly on the power of the study performed.
  • biomarkers can yield significant improvement in performance compared to the individual components when proper mathematical and clinical algorithms are used; this is often evident in both sensitivity and specificity, and results in a greater AUC or MCC.
  • Significant improvement in performance could mean an increase of 1%, 2%, 3%, 4%, 5%, 8%, 10% or higher than 10% in different measures of accuracy such as total accuracy, AUC, MCC, sensitivity, specificity, PPV or NPV.
  • RNA or protein biomarkers it is often useful to restrict the number of measured diagnostic determinants (e.g., RNA or protein biomarkers), as this allows significant cost reduction and reduces required sample volume and assay complexity. Accordingly, even when two signatures have similar diagnostic performance (e.g., similar AUC or sensitivity), one which incorporates less RNAs could have significant utility and ability to reduce to practice. For example, a signature that includes 5 genes compared to 10 genes and performs similarly has many advantages in real world clinical setting and thus is desirable. Therefore, there is value and invention in being able to reduce the number of genes incorporated in a signature while retaining similar levels of accuracy.
  • diagnostic determinants e.g., RNA or protein biomarkers
  • a significant reduction in the number of genes of a signature includes reducing the number of genes by 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 genes.
  • formula such as statistical classification algorithms can be directly used to both select determinants and to generate and train the optimal formula necessary to combine the results from multiple determinants into a single index.
  • techniques such as forward (from zero potential explanatory parameters) and backwards selection (from all available potential explanatory parameters) are used, and information criteria, such as AIC or BIC, are used to quantify the tradeoff between the performance and diagnostic accuracy of the panel and the number of determinants used.
  • information criteria such as AIC or BIC
  • any formula may be used to combine determinant results into indices useful in the practice of the invention.
  • indices may indicate, among the various other indications, the probability, likelihood, absolute or relative risk, time to or rate of conversion from one to another disease states, or make predictions of future biomarker measurements of infection. This may be for a specific time period or horizon, or for remaining lifetime risk, or simply be provided as an index relative to another reference subject population.
  • model and formula types beyond those mentioned herein and in the definitions above are well known to one skilled in the art.
  • the actual model type or formula used may itself be selected from the field of potential models based on the performance and diagnostic accuracy characteristics of its results in a training population.
  • the specifics of the formula itself may commonly be derived from determinant results in the relevant training population.
  • such formula may be intended to map the feature space derived from one or more determinant inputs to a set of subject classes (e.g.
  • Preferred formulas include the broad class of statistical classification algorithms, and in particular the use of discriminant analysis.
  • the goal of discriminant analysis is to predict class membership from a previously identified set of features.
  • LDA linear discriminant analysis
  • features can be identified for LDA using an eigengene based approach with different thresholds (ELDA) or a stepping algorithm based on a multivariate analysis of variance (MANOVA). Forward, backward, and stepwise algorithms can be performed that minimize the probability of no separation based on the Hotelling-Lawley statistic.
  • Eigengene-based Linear Discriminant Analysis is a feature selection technique developed by Shen et al. (2006). The formula selects features (e.g. biomarkers) in a multivariate framework using a modified eigen analysis to identify features associated with the most important eigenvectors. “Important” is defined as those eigenvectors that explain the most variance in the differences among samples that are trying to be classified relative to some threshold.
  • a support vector machine is a classification formula that attempts to find a hyperplane that separates two classes.
  • This hyperplane contains support vectors, data points that are exactly the margin distance away from the hyperplane.
  • the dimensionality is expanded greatly by projecting the data into larger dimensions by taking non-linear functions of the original variables (Venables and Ripley, 2002).
  • filtering of features for SVM often improves prediction.
  • Features e.g., biomarkers
  • KW non-parametric Kruskal-Wallis
  • a random forest (RF, Breiman, 2001) or recursive partitioning (RPART, Breiman et al., 1984) can also be used separately or in combination to identify biomarker combinations that are most important. Both KW and RF require that a number of features be selected from the total. RPART creates a single classification tree using a subset of available biomarkers.
  • an overall predictive formula for all subjects, or any known class of subjects may itself be recalibrated or otherwise adjusted based on adjustment for a population's expected prevalence and mean biomarker parameter values, according to the technique outlined in D'Agostino et al., (2001) JAMA 286:180-187, or other similar normalization and recalibration techniques.
  • Such epidemiological adjustment statistics may be captured, confirmed, improved and updated continuously through a registry of past data presented to the model, which may be machine readable or otherwise, or occasionally through the retrospective query of stored samples or reference to historical studies of such parameters and statistics. Additional examples that may be the subject of formula recalibration or other adjustments include statistics used in studies by Pepe, M. S.
  • numeric result of a classifier formula itself may be transformed post-processing by its reference to an actual clinical population and study results and observed endpoints, in order to calibrate to absolute risk and provide confidence intervals for varying numeric results of the classifier or risk formula.
  • Some determinants may exhibit trends that depends on the patient age (e.g. the population baseline may rise or fall as a function of age).
  • the patient gender may influence the diagnostic accuracy of an RNA based diagnostic signature.
  • the RNA determinants EIF1AY and UTY which are located on the Y chromosome
  • the RNA determinant BMX which is located on the X chromosome
  • the sex of the subject is taken into account.
  • the RNA determinants EIF1AY and UTY are used to diagnose male subjects.
  • TP is true positive, means positive test result that accurately reflects the tested-for activity.
  • a TP is for example but not limited to, truly classifying a bacterial infection as such.
  • TN is true negative, means negative test result that accurately reflects the tested-for activity.
  • a TN is for example but not limited to, truly classifying a viral infection as such.
  • FN is false negative, means a result that appears negative but fails to reveal a situation.
  • a FN is for example but not limited to, falsely classifying a bacterial infection as a viral infection.
  • FP is false positive, means test result that is erroneously classified in a positive category.
  • a FP is for example but not limited to, falsely classifying a viral infection as a bacterial infection.
  • Specificity is calculated by TN/(TN+FP) or the true negative fraction of non-disease or normal subjects.
  • Total accuracy is calculated by (TN+TP)/(TN+FP+TP+FN).
  • PSV Positive predictive value
  • ROC Receiver Operating Characteristics
  • “Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), Mathews correlation coefficient (MCC), or as a likelihood, odds ratio, Receiver Operating Characteristic (ROC) curve, Area Under the Curve (AUC) among other measures.
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an “index” or “index value”.
  • “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical-determinants, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations.
  • determinants Of particular use in combining determinants are linear and non-linear equations and statistical classification analyses to determine the relationship between levels of determinants detected in a subject sample and the subject's probability of having an infection or a certain type of infection.
  • structural and syntactic statistical classification algorithms, and methods of index construction utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, and Hidden Markov Models, among others.
  • PCA Principal Components Analysis
  • LogReg Logistic Regression
  • LDA Linear Discriminant Analysis
  • ELDA Eigen
  • AIC Akaike's Information Criterion
  • BIC Bayes Information Criterion
  • the resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV).
  • LEO Leave-One-Out
  • 10-Fold cross-validation 10-Fold CV.
  • false discovery rates may be estimated by value permutation according to techniques known in the art.
  • a “health economic utility function” is a formula that is derived from a combination of the expected probability of a range of clinical outcomes in an idealized applicable patient population, both before and after the introduction of a diagnostic or therapeutic intervention into the standard of care.
  • a cost and/or value measurement associated with each outcome, which may be derived from actual health system costs of care (services, supplies, devices and drugs, etc.) and/or as an estimated acceptable value per quality adjusted life year (QALY) resulting in each outcome.
  • the sum, across all predicted outcomes, of the product of the predicted population size for an outcome multiplied by the respective outcome's expected utility is the total health economic utility of a given standard of care.
  • the difference between (i) the total health economic utility calculated for the standard of care with the intervention versus (ii) the total health economic utility for the standard of care without the intervention results in an overall measure of the health economic cost or value of the intervention.
  • This may itself be divided amongst the entire patient group being analyzed (or solely amongst the intervention group) to arrive at a cost per unit intervention, and to guide such decisions as market positioning, pricing, and assumptions of health system acceptance.
  • Such health economic utility functions are commonly used to compare the cost-effectiveness of the intervention, but may also be transformed to estimate the acceptable value per QALY the health care system is willing to pay, or the acceptable cost-effective clinical performance characteristics required of a new intervention.
  • a health economic utility function may preferentially favor sensitivity over specificity, or PPV over NPV based on the clinical situation and individual outcome costs and value, and thus provides another measure of health economic performance and value which may be different from more direct clinical or analytical performance measures.
  • “Analytical accuracy” refers to the reproducibility and predictability of the measurement process itself, and may be summarized in such measurements as coefficients of variation (CV), Pearson correlation, and tests of concordance and calibration of the same samples or controls with different times, users, equipment and/or reagents. These and other considerations in evaluating new biomarkers are also summarized in Vasan, 2006.
  • “Performance” is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g., stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate “performance metrics,” such as AUC and MCC, time to result, shelf life, etc. as relevant.
  • Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is often considered highly significant at a p-value of 0.05 or less.
  • Abx Antibiotics
  • AE Adverse Event
  • A.U. Arbitrary Units
  • CBC Complete Blood Count
  • CXR Case Report Form
  • eCRF Electronic Case Report Form
  • FDA Food and Drug Administration
  • GCP Good Clinical Practice
  • GCP Gastrointestinal
  • GE Gastroenteritis
  • ICH International Conference on Harmonization
  • ID Infectious Disease
  • IVD In vitro diagnostics
  • LRTI Lower Respiratory Tract Infection
  • MI Myocardial infarction
  • MI Polymerase chain reaction
  • PCR Per-oss
  • P.R Per-rectum
  • SoC Standard Operating Procedure
  • SOP Standard Operating Procedure
  • Urinary Tract Infection UMI
  • URI Upper Respiratory Tract Infection
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • 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, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Inclusion criteria for the infectious disease cohort included: clinical suspicion of an acute infectious disease, peak fever >37.5° C. since symptoms onset, and duration of symptoms ⁇ 12 days.
  • Inclusion criteria for the control group included: clinical impression of a non-infectious disease (e.g. trauma, stroke and myocardial infarction), or healthy subjects.
  • Exclusion criteria included: evidence of any episode of acute infectious disease in the two weeks preceding enrollment; diagnosed congenital immune deficiency; current treatment with immunosuppressive or immunomodulatory therapy; active malignancy, proven or suspected human immunodeficiency virus (HIV)-1, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection.
  • HAV human immunodeficiency virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • Enrollment process and data collection For each patient, the following baseline variables were recorded: demographics, physical examination, medical history (e.g. main complaints, underlying diseases, chronically-administered medications, comorbidities, time of symptom onset, and peak temperature), complete blood count (CBC) obtained at enrollment, and chemistry panel (e.g. creatinine, urea, electrolytes, and liver enzymes).
  • CBC complete blood count
  • chemistry panel e.g. creatinine, urea, electrolytes, and liver enzymes.
  • a nasal swab was obtained from each patient for further microbiological investigation, and a blood sample was obtained for protein screening and validation. Additional samples were obtained as deemed appropriate by the physician (e.g. urine and stool samples in cases of suspected urinary tract infection [UTI], and gastroenteritis [GI] respectively). Radiological tests were obtained at the discretion of the physician (e.g. chest X-ray for suspected lower respiratory tract infection [LRTI]). Thirty days after enrollment, disease course and response
  • Multiplex-PCR assays were performed by a certified service laboratory. Patients were also tested for additional pathogens according to their suspected clinical syndrome, including: blood culture, urine culture and stool culture for Shigella spp., Campylobacter spp. and Salmonella spp.; serological testing (IgM and/or IgG) for cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma Pneumonia, and Coxiella burnetii (Q-Fever).
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • Mycoplasma Pneumonia and Coxiella burnetii
  • RNA purification Nasal swabs and stool samples were stored at 4° C. for up to 72 hours and subsequently transported to a certified service laboratory for multiplex PCR-based assay. Venous blood samples were collected in EDTA contained CBC tube and stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma and cell pellet. Red cells were lysed using Red Cell Lysis Buffer (RCLB) at room temperature (RT) and total RNA was purified using RNeasy plus Mini Kit (QIAGEN, Cat. 74134) according to manufacturer recommended protocols.
  • RCLB Red Cell Lysis Buffer
  • RT room temperature
  • Microarray experiments A total of 10 ⁇ l of 200 ng/3 ⁇ l (66.67 ng/ ⁇ l) RNA were transferred to microarray chip hybridization.
  • Amplified cRNA was prepared from 200 ng total RNA using the WT cDNA Synthesis and WT cDNA Amplification Kits (900672, AFFYMETRIXTM).
  • Biotinylated single-stranded cDNA was generated from the amplified cRNA and then fragmented and labeled with the WT Terminal Labeling Kit (AFFYMETRIXTM), following manufacturer protocol.
  • P, N, TP, FP, TN, FN are positives, negatives, true-positives, false-positives, true-negatives, and false-negatives, respectively.
  • positives and negatives refer to patients with bacterial and viral infections, respectively.
  • RNA determinants Single and combinations of RNA determinants can distinguish between bacterial and viral patients
  • the gene expression profiles of blood leukocytes obtained from the described acute infection patients using the Human Gene 1.0 ST Array (Affymetrix) were studied. The results suggest a differential response of the immune system to bacterial and viral infections, which can potentially be used classify acute infection patients.
  • 65 RNA determinants were identified that were differentially expressed in the bacterial and viral patients tested (Table 8; log 2 -fold change was calculated compared to bacterial patients baseline).
  • FIG. 2 presents distinctive gene expression profiles of the identified genes after applying a hierarchical clustering that uses Euclidean distance metric and average linkage. Based on these results, a classifier was developed for distinguishing between bacterial and viral patients using logistic regression. The present inventors further calculated for these RNA determinants the measures of accuracy in distinguishing between bacterial and viral patients including AUC, MCC, total accuracy, sensitivity, specificity and Wilcoxon ranksum P-value (Table 8).
  • RNA determinants and their measures of accuracy in differentiating between bacterial or mixed versus viral infected subjects Changes in expression levels were calculated as log2 (fold change bacterial)-log2 (fold change viral).
  • AUC MCC accuracy Sensitivity Specificity value 1 AIM2 Viral ⁇ 1.56 0.722 0.167 0.667 0.667 1.81E ⁇ 01 2
  • ANKRD2 Viral ⁇ 2.13 0.648 0.444 0.8 0.667 0.889 3.88E ⁇ 01 3 BMX Viral ⁇ 1.53 0.667 0.444 0.733 0.667 0.778 3.28E ⁇ 01 4 C19orf59 Viral ⁇ 1.51 0.611 0.167 0.533 0.556 5.29E ⁇ 01 5 CCL2 Viral ⁇ 0.95 0.87 0.764 0.867 1 0.778 1.76E ⁇ 02 6 CD177 Vi
  • Table 9 summarizes the 50 RNA pairs that demonstrated the highest accuracy improvement as calculated by the difference in AUC of the pair compared to the AUC of the single RNA (out of the same pair) with the highest AUC (delta AUC).
  • pairs of RNA determinants improved diagnostic accuracy up to 53% (when comparing AUC of single vs. pairs of RNA determinants), to generate highly discriminative combinations (AUCs between 0.83-1.0, average AUC 0.95, when testing 50 pairs with highest accuracy; Table 9, FIGS. 3 A- 3 B and 4 A- 4 B ).
  • AUC_1 and AUC_2 are the respective AUCs of RNA #1 and RNA #2.
  • AUC pair (RNA#1 and RNA #1 RNA #2 AUC_1 AUC_2 RNA#2) AUC_delta BMX CYP1B1 0.667 0.63 1 0.333 C19orf59 CYP1B1 0.611 0.63 0.926 0.296 CLEC4D CYP1B1 0.685 0.63 0.963 0.278 CYP1B1 PLSCR1 0.63 0.704 0.981 0.277 CYP1B1 FFAR3 0.63 0.741 1 0.259 CYP1B1 PSTPIP2 0.63 0.611 0.87 0.24 FFAR3 LOC100132244 0.741 0.685 0.981 0.24 PLSCR1 TMEM176A 0.704 0.667 0.926 0.222 ANKRD22 CYP1B1 0.648 0.63 0.87 0.222 BMX TMEM176A 0.667 0.667 0.889 0.222 CD177 CYP1B1 0.63 0.63 0.852 0.222 CD177 FFAR3 0.63
  • RNA determinant combinations exhibit an improved diagnostic accuracy compared to that of the corresponding individual determinants, whereas other combinations exhibit a reduced accuracy.
  • RNA determinants performed a rigorous bioinformatics analysis of RNA determinants using an external set generated from a cohort of 91 pediatric patients with acute infectious diseases caused by influenza A virus, Escherichia coli, Staphylococcus aureus or Streptococcus pneumoniae , which were tested using AFFYMETRIXTM HGU133A GENECHIPSTM (Ramilo et al. 2007).
  • RNA determinant For each RNA determinant they calculated the log 2 fold change between bacterial and viral patients and created a logistic regression classifier to calculate its AUC, MCC, total accuracy, sensitivity and specificity. They also created logistic regression classifiers for the pairs of RNA determinants and calculated the same measures of accuracy (Tables 10-12).
  • RNA DETERMINANTS TABLE 11 Pairs of RNA DETERMINANTS and their measures of accuracy in differentiating between bacterial versus viral infected subjects as tested using an external data set (measured using AFFYMETRIX TM HGU133A GENECHIPS TM on 73 bacterial and 18 viral patients).
  • RNA DETERMINANTS TABLE 12 Additional pairs of RNA DETERMINANTS and their measures of accuracy in differentiating between bacterial versus viral infected subjects as tested using an external data set (measured using AFFYMETRIX TM HGU133A GENECHIPS TM on 73 bacterial and 18 viral patients).
  • Inclusion criteria for the infectious disease cohort included: clinical suspicion of an acute infectious disease, peak fever >37.5° C. since symptoms onset, and duration of symptoms ⁇ 12 days.
  • Inclusion criteria for the control group included: clinical impression of a non-infectious disease (e.g. trauma, stroke and myocardial infarction), or healthy subjects.
  • Exclusion criteria included: evidence of any episode of acute infectious disease in the two weeks preceding enrollment; diagnosed congenital immune deficiency; current treatment with immunosuppressive or immunomodulatory therapy; active malignancy, proven or suspected human immunodeficiency virus (HIV)-1, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection.
  • HAV human immunodeficiency virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • RNA samples were collected in EDTA contained CBC tube and stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma and cell pellet.
  • Red cells were lysed using EL buffer (QIAGEN, Cat 79217) at room temperature (RT).
  • Leukocytes were lysed in RLT buffer (QIAGEN, Cat 79216) and Homogenized via QIAshredder homogenizer (QIAGEN, Cat 79654).
  • Total RNA was purified from 400 ⁇ l lysed Leukocytes using RNeasyTM Micro Kit (QIAGEN, Cat. 74004) according to manufacturer recommended protocols.
  • Microarray experiments A total of 30 of 255 ng/30 (85 ng/ ⁇ l) RNA were transferred and prepare for whole transcriptome expression analysis with GeneChipTM Whole Transcript (WT) Expression Arrays.
  • Amplified ss-cDNA was prepared from 255 ng total RNA using GeneChipTM WT PLUS Reagent Kit (902310, AFFYMETRIXTM), following manufacturer protocol.
  • Samples were hybridized to GENECHIPTM Human Transcriptome Arrays 2.0 (HTA-AFFYMETRIXTM) which is the highest resolution microarray for gene expression profiling of all transcript isoforms.
  • HTA display approximately ten probes per exon and four probes per exon-exon splice junction. This array interrogates >245,000 coding transcripts, >40,000 non coding transcripts and >339,000 probe sets covering exon-exon junctions.
  • Arrays were scanned using the AFFYMETRIXTM GENECHIPTM Scanner 3000 7G.
  • the studied group of patients included 71 children and 59 adults and was gender balanced (65 females and 65 males).
  • the cohort included 51 patients with bacterial infection and 79 patients with viral infections as determined by the expert panel as described above. Additionally, we studied 13 non-infectious patients (as controls).
  • RNA determinants can distinguish between bacterial and viral patients.
  • the gene expression profiles of blood leukocytes obtained from the described acute infection patients using the GeneChip Human Transcriptome Arrays 2.0 (HTA-Affymetrix) were studied. The results suggest a differential response of the immune system to bacterial and viral infections, which can potentially be used classify acute infection patients. 1089 RNA determinants, out of which 828 are coding determinants (Table 15A) and 261 are non-coding determinants (Table 15B), were identified as differentially expressed in the bacterial and viral patients tested.
  • RNA determinants out of which 34 are coding determinants (Tables 16A-B) and 7 are non-coding determinants (Table 16C), with the highest ability to differentiate between bacterial and viral patients according to the following criteria: ttest p-value ⁇ 10 ⁇ 14 AND absolute linear fold change >3.5.
  • the present inventors further calculated for these RNA determinants the measures of accuracy in distinguishing between bacterial and viral patients including AUC, sensitivity, specificity and ttest P-value (Tables 15-16).
  • CD 177 NM_020406 14.7 2.57E ⁇ 07 B 0.76 0.73 0.61 hg.1 TC17000383.
  • CCL2 NM_002982 ⁇ 14.3 2.76E ⁇ 11 V 0.86 0.94 0.67 hg.1 TC19001585.
  • CD177P1 ENST00000378007 14.1 1.98E ⁇ 07 B 0.77 0.73 0.58 hg.1 TC22000519.
  • USP41 ENST00000454608 ⁇ 13.8 3.33E ⁇ 16 V 0.91 0.92 0.82 hg.1 TC02005020.
  • CMPK2 NM_207315 ⁇ 13.6 ⁇ 10-17 V 0.89 0.82 0.82 hg.1 TC04001305.
  • PARP12 NM_022750 ⁇ 6.4 ⁇ 10-17 V 0.89 0.78 0.79 hg.1 TC06000983.
  • ARG1 NM_000045 6.4 2.25E ⁇ 08 B 0.78 0.69 0.72 hg.1 TC12000884.
  • OAS1 NM_001032409 ⁇ 6.4 1.11E ⁇ 16 V 0.88 0.78 0.84 hg.1 TC01000272.
  • CYP1B1 OTTHUMT00000325647 6.3 1.11E ⁇ 16 B 0.88 0.82 0.81 hg.1 TC02004979.
  • IGKV1D-27 ENST00000558152 ⁇ 6.2 0.000015 V 0.71 0.69 0.67 hg.1 TC02004982.
  • IGKV1D-16 ENST00000492446 ⁇ 6.2 0.000036 V 0.71 0.73 0.63 hg.1 TC02004978.
  • IGKV2D-28 ENST00000453166 ⁇ 6.1 0.000005 V 0.73 0.73 0.66 hg.1 TC02004976.
  • IGKV1D-33 ENST00000390265 ⁇ 6.1 0.000023 V 0.72 0.73 0.65 hg.1 TC22000191.
  • IGKV2-24 ENST00000484817 ⁇ 5.2 0.000016 V 0.73 0.73 0.65 hg.1 TC10000640.
  • IGHD3-10 ENST00000390583 ⁇ 4.9 0.014346 V 0.63 0.65 0.51 hg.1 TC02000553.
  • IGKV2D-29 ENST00000491977 ⁇ 4.9 0.000049 V 0.71 0.75 0.63 hg.1 TC14002257.
  • IGHV3-23 ENST00000390609 ⁇ 4.9 0.001561 V 0.67 0.63 0.66 hg.1 TC16000387. 0 uc002ecv.1 ⁇ 4.9 0.001933 V 0.66 0.65 0.65 hg.1 TC22000119.
  • IGLV2-18 ENST00000390310 ⁇ 4.8 0.000062 V 0.70 0.67 0.66 hg.1 TC11000467.
  • IGKV2D-26 ENST00000390268 ⁇ 4.7 0.000015 V 0.72 0.75 0.63 hg.1 TC02004947.
  • IGKV2-29 ENST00000558710 ⁇ 4.7 0.000264 V 0.70 0.73 0.58 hg.1 TC02004945.
  • IGKV1-27 ENST00000498435 ⁇ 4.7 0.00001 V 0.73 0.73 0.63 hg.1 TC14002213.
  • IGHM ENST00000390559 ⁇ 4.7 0.004526 V 0.66 0.63 0.63 hg.1 TC11000812.
  • DGAT2 NM_001253891 4.6 7.77E ⁇ 16 B 0.89 0.88 0.80 hg.1 TC14002260.
  • IGKV1-17 ENST00000490686 ⁇ 4.2 0.00006 V 0.71 0.75 0.63 hg.1 TC10000636.
  • IGKC IGKJ5; ENST00000390237 ⁇ 4.2 0.000009 V 0.72 0.73 0.68 hg.1 IGKJ4; IGKJ3; IGKJ2; IGKJ1; IGKV3-15; IGKV2-30; IGKV1-33; IGKV1-16; IGKV1-12; IGKV3-20; IGKV1-39; IGK; OTTHUMG00000151686; AC096579.7; OTTHUMG00000151694; AC096579.13 TC22001445.
  • IGLV3-1 ENST00000390319 ⁇ 4.1 0.000276 V 0.69 0.71 0.58 hg.1 TC02004942.
  • IGKV3-11 ENST00000483158 ⁇ 4.1 0.000019 V 0.72 0.73 0.63 hg.1 TC14002261.
  • IGHV3-30 ENST00000390613 ⁇ 4.1 0.000883 V 0.67 0.65 0.66 hg.1 TC22000117.
  • MT1CP ENST00000379816 ⁇ 3.1 9.9E ⁇ 12 V 0.84 0.86 0.68 hg.1 TC17000545.
  • IFI35 NM_005533 ⁇ 3.1 8.5E ⁇ 08 V 0.76 0.67 0.71 hg.1 TC06001204.
  • TESPA1 NM_001136030 ⁇ 3.1 1.11E ⁇ 07 V 0.78 0.65 0.72 hg.1 TC17000736.
  • PGAP1 NM_024989 ⁇ 2.9 6.21E ⁇ 12 V 0.88 0.82 0.67 hg.1 TC10001555.
  • BLNK NM_001114094 ⁇ 2.9 2.38E ⁇ 07 V 0.76 0.73 0.66 hg.1 TC0X000922.
  • FAM129A NM_052966 2.8 1.49E ⁇ 14 B 0.89 0.88 0.80 hg.1 TC01003878.
  • 0 ENST00000390855 2.8 8.25E ⁇ 07 B 0.75 0.71 0.71 hg.1 TC01004037.
  • 0 ENST00000390928 2.8 8.25E ⁇ 07 B 0.75 0.71 0.71 hg.1 TC07000284.
  • 0 ENST00000391148 2.8 8.25E ⁇ 07 B 0.75 0.71 0.71 hg.1 TC06001668.
  • SRPK1 NM_003137 2.8 1.55E ⁇ 15 B 0.88 0.82 0.79 hg.1 TC12000160.
  • 0 uc021tmw.l 2.4 1.19E ⁇ 09 B 0.81 0.75 0.68 hg.1 TC07001920.
  • SLC37A3 NM_032295 2.4 1.26E ⁇ 10 B 0.82 0.69 0.76 hg.1 TC09000565.
  • HSDL2 NM_001195822 2.4 1.86E ⁇ 13 B 0.85 0.84 0.79 hg.1 TC10000259. OTTHUMG00000017996; ENST00000428915 2.4 5.8E ⁇ 12 B 0.82 0.71 0.77 hg.1 RP11- 291L22.4 TC15000930.
  • MCTP2 NM_001159643 2.4 1.6E ⁇ 10 B 0.82 0.77 0.73 hg.1 TC15001079.
  • RALGAPA2 NM_020343 2.4 6.71E ⁇ 09 B 0.78 0.78 0.75 hg.1 TC05001449.
  • 0 uc003jyj.1 2.4 1.3E ⁇ 07 B 0.78 0.75 0.67 hg.1 TC12001772.
  • LIN7A NM_004664 2.4 2E ⁇ 09 B 0.81 0.77 0.73 hg.1 TC01000127.
  • KIF1B NM_015074 2.4 1.89E ⁇ 10 B 0.82 0.73 0.75 hg.1 TC01001881.
  • 0 uc001hrn.2 2.4 1.16E ⁇ 09 B 0.82 0.80 0.73 hg.1 TC01002954.
  • TECPR2 NM_014844 2.3 2.89E ⁇ 09 B 0.81 0.77 0.76 hg.1 TC15000796.
  • TM6SF1 NM_001144903 2.3 6.81E ⁇ 11 B 0.83 0.77 0.73 hg.1 TC17001890.
  • ACOX1 NM_001185039 2.3 3.46E ⁇ 14 B 0.86 0.82 0.80 hg.1 TC20001000.
  • PPP1R3D NM_006242 2.3 2.09E ⁇ 10 B 0.81 0.73 0.77 hg.1 TC05001766.
  • OTTHUMG00000059494; ENST00000422204 2.3 1.08E ⁇ 08 B 0.79 0.75 0.73 hg.1 AC116366.5 TC13000425.
  • TMCO3 NM_017905 2.3 ⁇ 10-17 B 0.91 0.84 0.86 hg.1 TC15000844.
  • ABHD2 NM_007011 2.3 4.43E ⁇ 13 B 0.86 0.84 0.79 hg.1 TC16000098.
  • MMP25 NM_022468 2.3 1.42E ⁇ 09 B 0.82 0.80 0.76 hg.1 TC20000552.
  • 0 ENST00000516294 2.3 3.57E ⁇ 12 B 0.82 0.73 0.75 hg.1 TC20000944.
  • ATP9A NM_006045 2.3 3.92E ⁇ 10 B 0.84 0.69 0.89 hg.1 TC02002705.
  • ENTPD7 NM_020354 2.3 2.73E ⁇ 09 B 0.83 0.67 0.86 hg.1 TC12001981.
  • NDC80 NM_006101 ⁇ 2.3 7.22E ⁇ 15 V 0.90 0.84 0.79 hg.1 TC20000402. LOC284751; NR_034124 2.3 0.000001 B 0.76 0.73 0.72 hg.1 OTTHUMG00000032719; RP11- 290F20.1 TC11001414.
  • RNF141 NM_016422 2.3 1.82E ⁇ 12 B 0.84 0.77 0.76 hg.1 TC12001027.
  • GLT1D1 NM_144669 2.3 8.17E ⁇ 10 B 0.83 0.78 0.76 hg.1 TC14000630.
  • SAMD3 NM_001017373 ⁇ 2.2 1.38E ⁇ 07 V 0.78 0.71 0.72 hg.1 TC11000854.
  • APOBEC3D NM_152426 ⁇ 2.1 1.89E ⁇ 11 V 0.84 0.67 0.80 hg.1 TC01001315.
  • SLC25A44 NM_014655 2.1 7.65E ⁇ 11 B 0.83 0.86 0.80 hg.1 TC02001083.
  • ITGA4 NM_000885 ⁇ 2.1 1.83E ⁇ 08 V 0.78 0.65 0.75 hg.1 TC03002182.
  • UTRN NM_007124 ⁇ 2.1 1.11E ⁇ 16 V 0.89 0.82 0.79 hg.1 TC11001341.
  • DRAPI NM_006442 ⁇ 2.1 1.11E ⁇ 16 V 0.90 0.82 0.81 hg.1 TC22000430.
  • KLHDC7B NM_138433 ⁇ 2.1 5.97E ⁇ 11 V 0.84 0.84 0.79 hg.1 TC6_apd_ FLOT1 NM_005803 2.1 4.7E ⁇ 09 B 0.80 0.75 0.72 hap1000078.
  • TNFRSF9 NM_001561 2.1 2.17E ⁇ 12 B 0.83 0.69 0.76 hg.1 TC07000634.
  • SLC12A9 NM_020246 2.1 1.11E ⁇ 16 B 0.88 0.80 0.84 hg.1 TC17001312.
  • DHRS13 NM_144683 2.1 1.11E ⁇ 09 B 0.79 0.73 0.73 hg.1 TC19001163.
  • ICAM3 NM_002162 2.1 7.78E ⁇ 14 B 0.87 0.80 0.82 hg.1 TC01002260.
  • RASGRP3 NM_001139488 ⁇ 2.1 3.13E ⁇ 11 V 0.86 0.80 0.77 hg.1 TC04002922.
  • RASGEF1B NM_152545 ⁇ 2.1 4.13E ⁇ 10 V 0.82 0.75 0.77 hg.1 TC07001842.
  • LRRC4 NM_022143 2.1 4.14E ⁇ 09 B 0.80 0.77 0.75 hg.1 TC11000332.
  • CD44 NM_001001391 2.1 7.77E ⁇ 16 B 0.90 0.84 0.81 hg.1 TC22000213.
  • SIRPAP1 ENST00000422796 2.1 4.66E ⁇ 15 B 0.88 0.80 0.81 hg.1 TC03001188.
  • NUP210 NM_024923 ⁇ 2.0 0.000001 V 0.76 0.63 0.72 hg.1 TC05001188.
  • RNA5SP180 ENST00000516181 2.0 4.31E ⁇ 11 B 0.82 0.69 0.79 hg.1 TC07001102.
  • CARD11 NM_032415 ⁇ 2.0 5.67E ⁇ 11 V 0.82 0.73 0.80 hg.1 TC11000198.
  • MICAL2 NM_014632 2.0 3.18E ⁇ 08 B 0.77 0.75 0.76 hg.1 TC16000185.
  • 0 uc021tdw.1 ⁇ 2.0 1.26E ⁇ 09 V 0.80 0.71 0.77 hg.1 TC20000206.
  • ST3GAL4-AS1 NR_033839 2.0 2.43E ⁇ 12 B 0.83 0.77 0.79 hg.1 TC17000613.
  • TBX21 NM_013351 ⁇ 2.0 0.000001 V 0.77 0.78 0.65 hg.1 TC19001500.
  • RASGRP4 NM_001146202 2.0 4.26E ⁇ 11 B 0.85 0.78 0.75 hg.1 TC20001743.
  • ST6GALNAC3 NM_001160011 2.0 9.49E ⁇ 08 B 0.79 0.69 0.75 hg.1 TC04000075.
  • TBC1D14 NM_001113361 2.0 4.44E ⁇ 15 B 0.88 0.82 0.77 hg.1 TC05001455.
  • NAIP NM_004536 2.0 2.83E ⁇ 07 B 0.77 0.71 0.67 hg.1 LOC101060527 TC06001508.
  • FLOT1 NM_005803 2.0 9.42E ⁇ 12 B 0.83 0.80 0.77 hg.1 TC11000575.
  • RTN3 NM_006054 2.0 1.11E ⁇ 16 B 0.90 0.88 0.80 hg.1 TC11001540.
  • CD59; NM_000611 2.0 1.21E ⁇ 07 B 0.76 0.69 0.75 hg.1 C11orf91 TC14001436.
  • RAD50 NM_005732 ⁇ 2.0 0.000001 V 0.76 0.69 0.72 hg.1 TC08000591.
  • CPQ NM_016134 2.0 5.25E ⁇ 07 B 0.76 0.73 0.65 hg.1 TC15000945.
  • ARRDC4 NM_183376 2.0 8.38E ⁇ 10 B 0.79 0.67 0.73 hg.1 TC19000340.
  • PLCG1 NM_002660 ⁇ 2.0 2.31E ⁇ 07 V 0.77 0.67 0.72 hg.1 TC01002882.
  • GCLM NM_002061 2.0 4.01E ⁇ 12 B 0.85 0.69 0.84 hg.1 TC02001667.
  • OTOF NM_194248 ⁇ 2.0 1.36E ⁇ 10 V 0.88 0.92 0.73 hg.1 TC10000189.
  • MASTL NM_032844 ⁇ 2.0 3.66E ⁇ 11 V 0.86 0.80 0.73 hg.1 TC12000730.
  • PLXNC1 NM_005761 2.0 2.9E ⁇ 11 B 0.85 0.84 0.76 hg.1 TC12000947.
  • RNF10 NM_014868 2.0 4.3E ⁇ 10 B 0.79 0.69 0.70 hg.1 TC12001155.
  • 0 ENST00000549744 1.9 5.25E ⁇ 07 B 0.76 0.67 0.72 hg.1 TC06001715.
  • TREML3P NR_027256 1.9 8.62E ⁇ 08 B 0.77 0.61 0.75 hg.1 TC0X000006.
  • CSF2RA NM_001161530 1.9 5.7E ⁇ 10 B 0.82 0.75 0.73 hg.1 TC10002943.
  • ENTPD1; NM_001164182 1.9 4.33E ⁇ 07 B 0.76 0.71 0.67 hg.1 OTTHUMG00000018817; RP11- 429G19.3 TC11000824.
  • ACER3 NM_018367 1.9 3.89E ⁇ 15 B 0.87 0.75 0.82 hg.1 TC11000981.
  • DDX10 NM_004398 ⁇ 1.9 6.32E ⁇ 09 V 0.80 0.69 0.80 hg.1 TC19000168.
  • FAM160A2 NM_001098794 1.9 1.11E ⁇ 16 B 0.89 0.78 0.85 hg.1 TC11002344.
  • AMICA1 NM_001098526 1.9 4E ⁇ 13 B 0.87 0.82 0.73 hg.1 TC12001754.
  • OSBPL8 NM_001003712 1.9 ⁇ 10-17 B 0.94 0.84 0.86 hg.1 TC16002096.
  • WIPI1 NM_017983 1.9 4.09E ⁇ 10 B 0.82 0.75 0.77 hg.1 TC19001159.
  • DNMT1 NM_001130823 ⁇ 1.9 1.98E ⁇ 11 V 0.83 0.77 0.80 hg.1 TC07000252.
  • 0 ENST00000516671 1.9 3.98E ⁇ 07 B 0.75 0.69 0.67 hg.1 TC07002021.
  • GIMAP6 NM_001244071 ⁇ 1.9 1.78E ⁇ 08 V 0.78 0.65 0.73 hg.1 TC10001562.
  • FRAT2 NM_012083 1.9 1.45E ⁇ 07 B 0.78 0.77 0.70 hg.1 TC10001726.
  • OAT NM_001171814 1.9 1.11E ⁇ 16 B 0.89 0.78 0.85 hg.1 TC11000027.
  • TALDO1 NM_006755 1.9 3.33E ⁇ 16 B 0.89 0.88 0.82 hg.1 TC12001544.
  • ITGB7 NM_000889 ⁇ 1.9 1.46E ⁇ 07 V 0.76 0.65 0.75 hg.1 TC14001343.
  • TMED8 NM_213601 1.9 2.06E ⁇ 10 B 0.83 0.73 0.79 hg.1 TC21000128.
  • IFNAR1 NM_000629 1.9 ⁇ 10-17 B 0.90 0.84 0.81 hg.1 TC02001379.
  • ALOX5 NM_000698 1.9 7.51E ⁇ 13 B 0.86 0.84 0.77 hg.1 TC13000111.
  • BRCA2 NM_000059 ⁇ 1.9 9.05E ⁇ 11 V 0.87 0.80 0.79 hg.1 TC19000417.
  • URI1 NM_001252641 ⁇ 1.9 3.9E ⁇ 07 V 0.76 0.65 0.73 hg.1 TC22001467.
  • APOBEC3F NM_001006666 ⁇ 1.9 1.88E ⁇ 14 V 0.86 0.71 0.79 hg.1 TC01001572.
  • CACNA1E NM_000721 1.9 7.53E ⁇ 07 B 0.74 0.65 0.68 hg.1 TC01003677.
  • ACSL4 NM_004458 1.9 1.35E ⁇ 07 B 0.76 0.69 0.72 hg.1 TC14000417.
  • KIAA0247 NM_014734 1.9 1.18E ⁇ 09 B 0.83 0.77 0.77 hg.1 TC14001116.
  • SOS2 NM_006939 1.9 6.21E ⁇ 14 B 0.87 0.82 0.75 hg.1 TC15001552.
  • OAZ2 NM_002537 1.9 1.13E ⁇ 10 B 0.83 0.78 0.79 hg.1 TC19000993.
  • FCGRT NM_001136019 1.8 6.99E ⁇ 11 B 0.82 0.78 0.80 hg.1 TC20000554.
  • SIRPB1; NM_001083910 1.8 1.28E ⁇ 09 B 0.82 0.80 0.75 hg.1 LOC100653231; LOC100653194 TC22000722.
  • ATP2B4 NM_001001396 1.8 1.01E ⁇ 10 B 0.81 0.75 0.77 hg.1 TC02001781.
  • OXER1 NM_148962 1.8 3.25E ⁇ 07 B 0.77 0.61 0.76 hg.1 TC04000181.
  • RBPJ NM_005349 1.8 4.93E ⁇ 13 B 0.84 0.77 0.80 hg.1 TC10001312.
  • IPMK NM_152230 1.8 1.46E ⁇ 07 B 0.77 0.73 0.68 hg.1 TC12001846.
  • CXCL16 NM_022059 1.8 2.92E ⁇ 07 B 0.80 0.77 0.70 hg.1 TC19001242.
  • EMR2 NM_013447 1.8 1.55E ⁇ 08 B 0.79 0.71 0.71 hg.1 TC22000270.
  • NCF4 NM_000631 1.8 3.31E ⁇ 09 B 0.82 0.77 0.75 hg.1 TC02002398.
  • GTDC1 NM_001006636 1.8 2.47E ⁇ 10 B 0.85 0.78 0.84 hg.1 TC03000263.
  • NBEAL2 NM_015175 1.8 6.81E ⁇ 09 B 0.79 0.75 0.71 hg.1 TC03001389.
  • PRKAR2A NM_004157 1.8 2.44E ⁇ 15 B 0.89 0.80 0.84 hg.1 TC04001171.
  • NFXL1 NM_152995 1.8 3.05E ⁇ 08 B 0.78 0.65 0.82 hg.1 TC10000967.
  • IDI1 NM_004508 1.8 1.25E ⁇ 08 B 0.80 0.67 0.79 hg.1 TC11000184.
  • AMPD3 NM_001172431 1.8 3.21E ⁇ 13 B 0.85 0.69 0.85 hg.1 TC12000822.
  • TCP11L2 NM_152772 1.8 5.29E ⁇ 08 B 0.77 0.73 0.70 hg.1 TC15000311.
  • CYB5R4 NM_016230 1.8 0.000001 B 0.76 0.78 0.68 hg.1 TC06002122.
  • SLC18B1 NM_052831 ⁇ 1.8 1E ⁇ 07 V 0.79 0.73 0.70 hg.1 TC08000172.
  • PPP3CC NM_001243974 ⁇ 1.8 2.35E ⁇ 08 V 0.79 0.69 0.76 hg.1 TC08001541.
  • TRPS1 NM_014112 1.8 3.27E ⁇ 11 B 0.84 0.69 0.77 hg.1 TC09000637.
  • LOC100129034 NR_027406 1.8 7.34E ⁇ 08 B 0.76 0.67 0.67 hg.1 TC12001933.
  • CMKLR1 ENST00000397688 ⁇ 1.8 1.64E ⁇ 07 V 0.76 0.69 0.70 hg.1 TC19001653.
  • PRKD2 NM_001079880 ⁇ 1.8 8.62E ⁇ 09 V 0.78 0.69 0.67 hg.1 TC01000826.
  • SEMA4A NM_001193300 5.27E ⁇ 13 B 0.85 0.80 0.81 hg.1 TC01002421.
  • PTAFR NM_001164721 1.8 1.06E ⁇ 12 B 0.85 0.82 0.81 hg.1 TC02002477. 0 ENST00000435109 1.8 3.15E ⁇ 10 B 0.82 0.75 0.76 hg.1 TC04000466. ARHGAP24 NM_001025616 1.8 9.03E ⁇ 09 B 0.80 0.73 0.73 hg.1 TC04001673.
  • PDGFC NM_016205 1.8 1.36E ⁇ 10 B 0.85 0.65 0.87 hg.1 TC06002152.
  • IFNGR1 NM_000416 1.8 3.16E ⁇ 10 B 0.83 0.73 0.76 hg.1 TC07001562.
  • HGF NM_000601 1.8 1.77E ⁇ 07 B 0.79 0.63 0.81 hg.1 TC13000825.
  • VAPA NM_003574 1.8 1.01E ⁇ 14 B 0.87 0.80 0.81 hg.1 TC20000389.
  • SNORD12 NR_003030 1.8 0.000001 B 0.75 0.69 0.70 hg.1 TC20000469.
  • CD93 NM_012072 1.8 5.05E ⁇ 07 B 0.75 0.67 0.65 hg.1 TC22001468.
  • NPL NM_001200050 1.8 1.76E ⁇ 07 B 0.76 0.75 0.65 hg.1 TC01003868.
  • AIDA NM_022831 ⁇ 1.8 1.13E ⁇ 11 V 0.82 0.75 0.76 hg.1 TC03001466.
  • TKT NM_001064 1.8 1.11E ⁇ 16 B 0.90 0.78 0.80 hg.1 TC04000174.
  • TSPO NM_000714 1.8 ⁇ 10-17 B 0.90 0.86 0.82 hg.1 TC01001414.
  • ATF6 NM_007348 1.8 2.38E ⁇ 10 B 0.83 0.75 0.82 hg.1 TC01003970.
  • PAPSS1 NM_005443 1.8 9.03E ⁇ 10 B 0.81 0.65 0.81 hg.1 TC05000084.
  • FAM105A NM_019018 1.91E ⁇ 11 B 0.85 0.75 0.84 hg.1 TC06001990.
  • PRKCE NM_005400 ⁇ 1.7 1.26E ⁇ 13 V 0.92 0.88 0.75 hg.1 TC04000320.
  • SGMS2 NM_001136257 1.7 1.72E ⁇ 08 B 0.79 0.69 0.77 hg.1 TC06000697.
  • PTP4A1 NM_003463 ⁇ 1.7 2.97E ⁇ 07 V 0.75 0.78 0.66 hg.1 TC06001217.
  • MTMR3 NM_021090 1.7 1.6E ⁇ 07 B 0.77 0.75 0.71 hg.1 TC01001348.
  • IFI16 NM_001206567 ⁇ 1.7 1.18E ⁇ 09 V 0.80 0.67 0.75 hg.1 TC01004036.
  • LOC731275; NR_029401 1.7 9.71E ⁇ 07 B 0.75 0.71 0.62 hg.1 LOC100506479; LOC100288102; OTTHUMG00000039861; RP11- 261C10.3 TC05003427.
  • HIPK3 NM_001048200 1.7 4.19E ⁇ 12 B 0.84 0.80 0.82 hg.1 TC15000871.
  • FES NM_001143783 1.7 2.61E ⁇ 14 B 0.85 0.75 0.82 hg.1 TC16000472.
  • CLIP1 NM_002956 1.6 ⁇ 10-17 B 0.90 0.86 0.80 hg.1 TC15000864.
  • IQGAP1 NM_003870 1.6 1.11E ⁇ 1 B 0.88 0.84 0.82 hg.1 6 TC08001218.
  • LYPLA1 NM_006330 1.6 ⁇ 10-17 B 0.89 0.78 0.81 hg.1 TC11001915.
  • PRKACA NM_002730 1.6 3.33E ⁇ 16 B 0.87 0.73 0.81 hg.1 TC17000804.
  • PRKAR1A NM_002734 1.6 ⁇ 10-17 B 0.91 0.82 0.85 hg.1 TC05000664.
  • ACTR1A NM_005736 1.6 ⁇ 10-17 B 0.90 0.80 0.82 hg.1 TC03001074.
  • LINC00884; NR_033929 1.6 5.55E ⁇ 16 B 0.90 0.78 0.86 hg.1 OTTHUMG00000156037; AC046143.7 TC01003712.
  • TMEM167A NM_174909 1.4 5.55E ⁇ 16 B 0.89 0.75 0.82 hg.1 TC12001797.
  • ARPC1B NM_005720 1.4 ⁇ 10-17 B 0.89 0.78 0.87 hg.1 TC12000379.
  • TMBIM6 NM_001098576 1.3 ⁇ 10-17 B 0.89 0.77 0.80 hg.1 TC13000421.
  • LAMP1 NM_005561 1.3 3.33E ⁇ 16 B 0.88 0.82 0.81 hg.1 TC15000840.
  • ARF3 NM_001659 1.3 3.33E ⁇ 16 B 0.89 0.82 0.81 hg.1 TC17001755.
  • TBC1D3P1- NR_002924 1.3 3.33E ⁇ 16 B 0.88 0.77 0.80 hg.1 DHX40P1 TC02002826.
  • CUL3 NM_003590 1.3 ⁇ 10-17 B 0.90 0.88 0.79 hg.1 TC08000486.
  • TMEM70 NM_001040613 1.3 1.11E ⁇ 16 B 0.89 0.78 0.82 hg.1 TC09000550.
  • CLEC4D NM_080387 1.67 0.003066 B 0.65 0.63 0.63 hg.1 TC06001246.
  • F13A1 NM_000129 1.68 0.000269 B 0.68 0.73 0.58 hg.1 TC07000004.
  • FFAR3 NM_005304 1.03 0.740673 B 0.51 0.45 0.66 hg.1 TC6_cox_ HLA-DQA1 uc011fnq.1 ⁇ 1.96 0.010798 V 0.62 0.71 0.49 hap2000091. hg.1 TC11001230. IFITM3 NM_021034 ⁇ 1.18 0.001761 V 0.70 0.82 0.58 hg.1 TC0Y000185. KDM5D NM_001146705 2.64 0.847674 B 0.54 0.53 0.67 hg.1 TC17001661. PHOSPHO1 NM_001143804 1.2 0.131061 B 0.57 0.53 0.54 hg.1 TC03001869.
  • SH3BGRL2 NM_031469 1.36 0.04134 B 0.60 0.57 0.62 hg.1 TC02001159.
  • TNFAIP6 NM_007115 ⁇ 1.45 0.086888 V 0.60 0.67 0.51 hg.1 TC06000565.
  • Table 17 summarizes the 50 RNA pairs with the highest sensitivity in distinguishing between patients with bacterial and viral infections.
  • Table 18 presents the 50 RNA pairs that demonstrated the highest accuracy improvement as calculated by the difference in AUC of the pair compared to the AUC of the single RNA (out of the same pair) with the highest AUC (delta AUC).
  • AUCs between 0.93-0.96, average AUC 0.94, sensitivity between 0.92-0.96, sensitivity 0.93 Tables 17-18 were used to generate highly discriminative combinations.
  • RNA pairs that presented the highest sensitivity in distinguishing between patients with bacterial or viral infections.
  • the columns of Feature #1 and Feature #2 include the probe set ID and either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in brackets.
  • Logistic regression model parameters constant and single RNAs coefficient are depicted.
  • AUC_1 and AUC_2 are the respective AUCs of RNA #1 and RNA #2.
  • the columns of Feature #1 and Feature #2 include the probe set ID and either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in brackets.
  • CMPK2 0.88 0.89 0.94 0.06 TC02000082.hg.1
  • TAB2 TC12002059.hg.1
  • OASL 0.88 0.87 0.94 0.06 TC02001750.hg.1
  • CYP1B1 TC02003047.hg.1
  • n407780 0.88 0.88 0.94 0.06
  • TC02000034.hg.1 RSAD2
  • the evaluated cohort of patients provided the required statistical power to accurately evaluating of triplets of RNA determinants.
  • a linear logistic regression classifier was developed for each gene triplets (of the RNAs included in Tables 16A-C; 11480 combinations) and their diagnostic accuracy was evaluated.
  • Tables 18 and 19 present different RNA triplets with best sensitivity and the highest accuracy improvement respectively.
  • RNA triplets that presented the highest sensitivity in distinguishing between patients with bacterial or viral infections.
  • the columns of Feature #1, Feature #2 and Feature #3 include the probe set ID and either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in brackets.
  • Logistic regression model parameters constant and single RNAs coefficient are depicted.
  • AUC_1, AUC_2 and AUC_3 are the respective AUCs of RNA #1, RNA #2 and RNA #3.
  • the columns of Feature #1 and Feature #2 include the probe set ID and either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in brackets.
  • CRP C-reactive protein
  • TRAIL TNF-related apoptosis-inducing ligand
  • Feature #1 and Feature #2 include the probe set ID and either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in brackets. Logistic regression model parameters (constant and single RNAs coefficient) are depicted.
  • the inventors evaluated the accuracy measures of various RNAs in distinguishing between patients presenting with viral and gram negative bacterial infections (Table 22), and between Pneumonia caused by viral or bacterial infections (Table 23).
  • RNAs that are highly accurate at disease onset and thus might be used as biomarkers for early diagnosis of bacterial infections (for example CYP1B land HERC6; Table 24).
  • RNAs that are highly accurate at disease onset Gene AUC 0-1 AUC 3-10 Delta Probe Set ID mRNA Accession Symbol days days AUC TC02004017.hg.1 TCONS_00003184- — 0.94 0.86 0.08 XLOC_001966 TC02001750.hg.1 NM_000104 CYP1B1 0.92 0.85 0.08 TC02001749.hg.1 OTTHUMT00000325647 CYP1B1 0.91 0.84 0.07 TC04000484.hg.1 NM_001165136 HERC6 0.97 0.90 0.06 TC02005020.hg.1 NM_207315 CMPK2 0.94 0.87 0.06 TC18000223.hg.1 NM_017742 ZCCHC2 0.94 0.88 0.06 TC12002059.hg.1 NM_003733 OASL 0.92 0.87 0.05 TC04001718.hg.1 NM_017631
  • RNAs/genes were more differently expressed in certain types of viruses, and thus might server as specific indicators for this type of virus as part of a diagnostic assay. Examples of specific RNAs and the viral strain/s in which it is mostly differentially expressed are included in Table 25.
  • RNAs that might serve as potential indicators for a specific type of viral strain.
  • Gene symbol/mRNA accession Virus type differentiation CYP1B1 Parainfluenza SIGLEC1 Influenza GAS7 Parainfluenza, Influenza n332456 Influenza DDX60 Influenza SLUT1B1 CMV/EBV HER6 Influenza TCGNS_00003184-XLOC_001966 Parainfluenza, Influenza, RSV
  • RNA determinants that were found to be discriminative for distinguishing between viral and bacterial infections are summarized in Table 26 herein below.

Abstract

Methods of determining infection type are disclosed. In one embodiment, the method comprises measuring the amount of a determinant which is set forth in Tables 1 or 2 in a sample derived from the subject, wherein said amount is indicative of the infection type.

Description

    RELATED APPLICATIONS
  • This application is a continuation of U.S. patent application Ser. No. 16/081,906 filed on Sep. 2, 2018 which is a National Phase of PCT Patent Application No. PCT/IL2017/050271 having International Filing Date of Mar. 2, 2017, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/302,849, filed on Mar. 3, 2016. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
    • SEQUENCE LISTING STATEMENT
  • The XML file, entitled 93566SequenceListing.xml, created on Sep. 29, 2022, comprising 12,780 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
    • FIELD AND BACKGROUND OF THE INVENTION
  • The present invention, in some embodiments thereof, relates to the identification of signatures and determinants associated with bacterial and viral infections. More specifically it was discovered that certain RNA determinants are differentially expressed in a statistically significant manner in subjects with bacterial and viral infections.
  • Antibiotics are the world's most prescribed class of drugs with a 25-30 billion $US global market. Antibiotics are also the world's most misused drug with a significant fraction of all drugs (40-70%) being wrongly prescribed (Linder and Stafford 2001; Scott and Cohen 2001; Davey, P. and E. Brown, et al 2006; Cadieux, G. and R. Tamblyn, et al. 2007; Pulcini, C. and E. Cua, et al. 2007) (“CDC—Get Smart: Fast Facts About Antibiotic Resistance” 2011).
  • One type of antibiotics misuse is when the drug is administered in case of a non-bacterial disease, such as a viral infection, for which antibiotics is ineffective. For example, according to the USA center for disease control and prevention CDC, over 60 Million wrong antibiotics prescriptions are given annually to treat flu in the US. The health-care and economic consequences of the antibiotics over-prescription include: (i) the cost of antibiotics that are unnecessarily prescribed globally, estimated at >$10 billion annually; (ii) side effects resulting from unnecessary antibiotics treatment are reducing quality of healthcare, causing complications and prolonged hospitalization (e.g. allergic reactions, Antibiotics-associated diarrhea, intestinal yeast etc.) and (iii) the emergence of resistant strains of bacteria as a result of the overuse.
  • Resistance of microbial pathogens to antibiotics is increasing world-wide at an accelerating rate (“CDC—Get Smart: Fast Facts About Antibiotic Resistance” 2013; “European Surveillance of Antimicrobial Consumption Network (ESAC-Net)” 2014; “CDC—About Antimicrobial Resistance” 2013; “Threat Report 2013 I Antimicrobial Resistance I CDC” 2013), with a concomitant increase in morbidity and mortality associated with infections caused by antibiotic resistant pathogens (“Threat Report 2013 I Antimicrobial Resistance I CDC” 2013). At least 2 million people are infected with antibiotic resistant bacteria each year in the US alone, and at least 23,000 people die as a direct result of these infections (“Threat Report 2013 I Antimicrobial Resistance I CDC” 2013). In the European Union, an estimated 400,000 patients present with resistant bacterial strains each year, of which 25,000 patients die (“WHO Europe-Data and Statistics” 2014). Consequently, the World Health Organization has warned that therapeutic coverage will be insufficient within 10 years, putting the world at risk of entering a “post-antibiotic era”, in which antibiotics will no longer be effective against infectious diseases (“WHO I Antimicrobial Resistance” 2013). The CDC considers this phenomenon “one of the world's most pressing health problems in the 21′ century” (“CDC—About Antimicrobial Resistance” 2013; Arias and Murray 2009).
  • Antibiotics under-prescription is not uncommon either. For example up to 15% of adult bacterial pneumonia hospitalized patients in the US receive delayed or no Abx treatment, even though in these instances early treatment can save lives and reduce complications (Houck, P. M. and D. W. Bratzler, et al 2002).
  • Technologies for infectious disease diagnostics have the potential to reduce the associated health and financial burden associated with antibiotics misuse. Ideally, such a technology should: (i) accurately differentiate between a bacterial and viral infections; (ii) be rapid (within minutes); (iii) be able to differentiate between pathogenic and non-pathogenic bacteria that are part of the body's natural flora; (iv) differentiate between mixed co-infections and pure viral infections and (v) be applicable in cases where the pathogen is inaccessible (e.g. sinusitis, pneumonia, otitis-media, bronchitis, etc).
  • Current solutions (such as culture, PCR and immunoassays) do not fulfill all these requirements: (i) Some of the assays yield poor diagnostic accuracy (e.g. low sensitivity or specificity) (Uyeki et al. 2009), and are restricted to a limited set of bacterial or viral strains; (ii) they often require hours to days; (iii) they do not distinguish between pathogenic and non-pathogenic bacteria (Del Mar, C 1992), thus leading to false positives; (iv) they often fail to distinguish between a mixed and a pure viral infections and (v) they require direct sampling of the infection site in which traces of the disease causing agent are searched for, thus prohibiting the diagnosis in cases where the pathogen resides in an inaccessible tissue, which is often the case. Moreover, currently available diagnostic approaches often suffer from reduced clinical utility because they do not distinguish between pathogenic strains of microorganisms and potential colonizers, which can be present as part of the natural microbiota without causing an infection (Kim,
  • Shin, and Kim 2009; Shin, Han, and Kim 2009; Jung, Lee, and Chung 2010; Rhedin et al. 2014). For example, Rhedin and colleagues recently tested the clinical utility of qPCR for common viruses in acute respiratory illness (Rhedin et al. 2014). The authors concluded that qPCR detection of several respiratory viruses including rhinovirus, enterovirus and coronavirus should be interpreted with caution due to high detection rates in asymptomatic children. Other studies reached similar conclusions after analyzing the detection rates of different bacterial strains in asymptomatic patients (Bogaert, De Groot, and Hermans 2004; Spuesens et al. 2013).
  • Consequentially, there is still a diagnostic gap, which in turn often leads physicians to either over-prescribe Abx (the “Just-in-case-approach”), or under-prescribe Abx (the “Wait-and-see-approach”) (Little, P. S. and I. Williamson 1994; Little, P. 2005; Spiro, D. M. and K. Y. Tay, et al 2006), both of which have far reaching health and financial consequences.
  • Accordingly, a need exists for a rapid method that accurately differentiates between bacterial, viral, mixed and non-infectious disease patients that addresses these challenges. An approach that has the potential to address these challenges relies on monitoring the host's immune-response to infection, rather than direct pathogen detection (Cohen et al. 2015). Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogen species (Tang et al. 2007; Limper et al. 2010; Engel et al. 2012; Quenot et al. 2013; van der Meer et al. 2005; Falk and Fahey 2009). Oved et al. 2015 has developed an immune signature, combining both bacterial- and viral-induced circulating host-proteins, which can aid in the correct diagnosis of patients with acute infections.
  • Additional background art includes Ramilo et al., Blood, Mar. 1 2007, Vol 109, No. 5, pages 2066-2077, Zaas et al., Sci Transl Med. 2013 Sep. 18; 5(203) 203ra126. doi:10.1126/scitranslmed.3006280; US Patent Application No. 20080171323, WO2011/132086 and WO2013/117746.
    • SUMMARY OF THE INVENTION
  • According to an aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring the amount of a determinant which is set forth in Tables 1 or 2 in a sample derived from the subject, wherein the amount is indicative of the infection type.
  • According to an aspect of the present invention there is provided a method of distinguishing between an infectious and non-infectious subject comprising measuring the amount of a determinant which is set forth in Table 16E in a sample derived from the subject, wherein the amount is indicative whether the subject is infectious or non-infectious.
  • According to an aspect of the present invention there is provided a method of determining whether a subject has a bacterial or non-bacterial infection comprising measuring the amount of a determinant which is set forth in Table 16D in a sample derived from the subject, wherein the amount is indicative whether the subject has a bacterial or non-bacterial infection.
  • According to an aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring at least two RNAs in a sample derived from the subject, wherein pairs of the at least two RNAs are set forth in Tables 9, 11, 12, 17 or 18 wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to an aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring at least three RNAs in a sample derived from the subject, wherein triplets of the at least three RNAs are set forth in Tables 13, 14, 19 or 20 wherein amount of the at least three RNAs is indicative of the infection type.
  • According to an aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 3 or Table 4, in a sample derived from the subject, wherein no more than 20 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • According to an aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 5 or Table 6A, in a sample derived from the subject, wherein no more than 5 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • According to an aspect of the present invention there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in Table 1 or Table 2, and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6A or Tables 15A-B.
  • According to an aspect of the present invention there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two RNA detection reagents specifically detects a first RNA which is set forth in Table 3 or Table 4, and a second of the at least two RNA detection reagents specifically detects a second RNA which is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • According to an aspect of the present invention there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in any one of Tables 1-6A or Tables 15A-B and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the kit comprises detection reagents that specifically detect no more than 5 determinants.
  • According to an aspect of the present invention there is provided a composition of matter comprising a sample derived from a subject suspected of having an infection and a first agent which binds specifically to at least one determinant of the sample which is set forth in Table 1 or Table 2.
  • According to embodiments of the invention, the amount of the determinant set forth in Table 1 is above a predetermined level, the infection is a viral infection.
  • According to embodiments of the invention, when the amount of the determinant set forth in Table 2 is above a predetermined level, the infection is a bacterial infection.
  • According to embodiments of the invention, the determinant is an RNA.
  • According to embodiments of the invention, the RNA is a protein-coding RNA.
  • According to embodiments of the invention, the RNA is a non protein-coding
  • RNA.
  • According to embodiments of the invention, the determinant is a polypeptide.
  • According to embodiments of the invention, the method further comprises measuring the amount of at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to embodiments of the invention, the at least one additional RNA is set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C, wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to embodiments of the invention, the pairs of the at least two RNAs are set forth in Tables 9, 11, 17 or 18.
  • According to embodiments of the invention, the determinant is selected from the group consisting of OTOF, PI3, CYBRD1, EIF2AK2, CMPK2, CR1, CYP1B1, DDX60, DGAT2, PARP12, PNPT1, PYGL, SULT1B1, TRIB2, USP41, ZCCHC2 and uc003hr1.1.
  • According to embodiments of the invention, the determinant is selected from the group consisting of TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_00072, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 and TCONS_12_00002367-XLOC_12_000720.
  • According to embodiments of the invention, the method further comprises analyzing at least two additional RNAs so as to measure the amount of at least three RNAs, wherein the at least two additional RNAs are set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least three RNAs is indicative of the infection type.
  • According to embodiments of the invention, the at least two additional RNAs are set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • According to embodiments of the invention, the triplets of the at least three RNAs are set forth in Table 13, Table 19 or Table 20.
  • According to embodiments of the invention, the infection is a viral infection, a bacterial infection or a mixed infection.
  • According to embodiments of the invention, the method further comprises analyzing the expression level of CRP polypeptide or TRAIL polypeptide in the sample.
  • According to embodiments of the invention, the determinant is set forth in Table 21.
  • According to embodiments of the invention, no more than 20 RNAs are measured.
  • According to embodiments of the invention, no more than 5 RNAs are measured.
  • According to embodiments of the invention, the at least one RNA is set forth in Table 3.
  • According to embodiments of the invention, when the RNA set forth in Table 3 is above a predetermined level, the infection is a viral infection.
  • According to embodiments of the invention, the method further comprises measuring at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to embodiments of the invention, the at least one additional RNA is set forth in Tables 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • According to embodiments of the invention, the at least one additional RNA set forth in Table 1 is OTOF, PI3, EIF2AK2, CMPK2, PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2 or TCONS_00003184-XLOC_001966.
  • According to embodiments of the invention, the at least one additional RNA set forth in Table 2 is CYBRD1, CR1, DGAT2, PYGL, SULT1B1, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 or TCONS_12_00002367-XLOC_12_000720.
  • According to embodiments of the invention, the at least one RNA is set forth in Table 5.
  • According to embodiments of the invention, when the at least one RNA set forth in Table 5 is above a predetermined level, the infection is a viral infection.
  • According to embodiments of the invention, the method further comprises measuring at least one additional RNA so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6A or Tables 15A-B, wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to embodiments of the invention, the at least one additional RNA is set forth in Table 1, Table 2, Table 3, Table 5 or Tables 16A-C.
  • According to embodiments of the invention, the at least one additional RNA set forth in Table 1 is selected from the group consisting of OTOF, PI3, EIF2AK2, CMPK2,
  • PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2 and TCONS_00003184-XLOC_001966.
  • According to embodiments of the invention, the at least one additional RNA set forth in Table 2 is CYBRD1, CR1, DGAT2, PYGL, SULT1B1, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 or TCONS_12_00002367-XLOC_12_000720.
  • According to embodiments of the invention, the sample is whole blood or a fraction thereof.
  • According to embodiments of the invention, the infection is a viral infection, a bacterial infection or a mixed infection.
  • According to embodiments of the invention, the blood fraction sample comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.
  • According to embodiments of the invention, the blood fraction sample comprises serum or plasma.
  • According to embodiments of the invention, the at least two determinant detection reagents comprise RNA detection reagents.
  • According to embodiments of the invention, the at least two RNA detection reagents are attached to a detectable moiety.
  • According to embodiments of the invention, the first RNA detection reagent comprises a first polynucleotide that hybridizes to the first RNA and the second RNA detection reagent comprises a polynucleotide that hybridizes to the second RNA.
  • According to embodiments of the invention, the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • According to embodiments of the invention, the kit comprises detection reagents that specifically detect no more than 10 RNAs.
  • According to embodiments of the invention, the kit comprises detection reagents that specifically detect no more than 5 RNAs.
  • According to embodiments of the invention, wherein each of the 5 RNAs are set forth in any one of Tables 1-6A or Tables 15A-B.
  • According to embodiments of the invention, the kit comprises detection reagents that specifically detect no more than 5 RNAs.
  • According to embodiments of the invention, the kit comprises detection reagents that specifically detect no more than 3 RNAs.
  • According to embodiments of the invention, the determinant is an RNA.
  • According to embodiments of the invention, the determinant is a polypeptide.
  • According to embodiments of the invention, the composition further comprises an additional agent which binds specifically to an additional determinant which is set forth in any one of Tables 1-6A or Tables 15A-B.
  • According to embodiments of the invention, the infectious subject has sepsis and the non-infectious subject has SIRS without sepsis.
  • Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
    • BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
  • The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
  • Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
  • In the drawings:
  • FIG. 1 is a flow-chart of the clinical study design.
  • FIG. 2 illustrates distinctive gene expression profiles of 6 bacterial and 9 viral patients. Hierarchical clustering of 65 genes using Euclidean distance metric and average linkage. Transformed expression levels are indicated by color scale, with red representing high expression and green indicating low expression.
  • FIG. 3A is a heat map illustrating the classification accuracy in terms of MCC of 9 viral versus 6 bacterial infected patients attained for pairs of the 65 RNA determinants described in Table 8 (according to the serial numbers) using a logistic regression model.
  • FIG. 3B is a heat map illustrating the change in classification accuracy (dMCC) for the 65 RNA determinants described in Table 8 (according to the serial numbers). The change is computed as follows: MCCi,j−max(MCCi, MCCj), where MCCi and MCCj correspond to the MCC obtained for determinant i and j individually and MCCi,j is obtained for the pair. Hot and cold colors indicate pairs of RNA determinants whose combined classification accuracy compared to the individual determinant accuracy is higher and lower.
  • FIG. 4A is a heat map illustrating the classification accuracy in terms of AUC of 9 viral versus 6 bacterial infected patients attained for pairs of the 65 RNA determinants described in Table 8 (according to the serial numbers) using a logistic regression model.
  • FIG. 4B is a heat map illustrating the change in classification accuracy (dAUC) for the 65 RNA determinants described in Table 8 (according to the serial numbers) is computed as follows: AUCi,j−max(AUCi, AUCj), where AUCi and AUCj correspond to the AUC obtained for determinant i and j individually and AUCi,j is obtained for the pair. Hot and cold colors indicate pairs of RNA determinants whose combined classification accuracy compared to the individual determinant accuracy is higher and lower.
  • FIGS. 5A-5K. Differential expression levels of the RNA determinants described in Tables 16A-C in bacterial, viral and non-infectious (including healthy) patients. Red line and circle correspond to group median and average respectively; Wilcoxon rank sum (RS) p-values between bacterial and viral groups and between infectious (bacterial and viral) vs non-infectious (including healthy subjects) are depicted. Gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) are included.
  • FIG. 6 . Volcano plot (scatter-plot) presenting the fold change as well as t-test p-value of the evaluated RNA determinants. Viral- and bacterial-induced RNAs that are described in Tables 16A-C are marked in red and blue “x” (respectively).
  • FIGS. 7A-7I. Expression levels (arbitrary units) of selected RNAs in non-infectious (including healthy) patients as well as in patients with bacterial or various viral infections as indicated. RSV—Respiratory syncytial virus; hMPV—human Metapneumovirus. Red line corresponds to group median.
    •  DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
  • The present invention, in some embodiments thereof, relates to the identification of signatures and determinants associated with bacterial and viral infections. More specifically it was discovered that certain RNA determinants are differentially expressed in a statistically significant manner in subjects with bacterial and viral infections.
  • Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
  • Methods of distinguishing between bacterial and viral infections by analyzing protein determinants have been disclosed in International Patent Application WO2013/117746, to the present inventors. Seeking to expand the number and type of determinants that can aid in accurate diagnosis, the present inventors have now carried out additional clinical experiments and have identified other determinants that can be used for this aim.
  • Correct diagnosis of bacterial patients is of high importance as these patients require antibiotic treatment and in some cases more aggressive management (hospitalization, additional diagnostic tests etc). Misclassification of bacterial patients increases the chance of morbidity and mortality. Therefore, increasing the sensitivity of a biomarker or diagnostic test that distinguishes between bacterial and viral infections may be desired, even though specificity may be reduced.
  • Whilst reducing the present invention to practice, the present inventors studied the gene expression profiles of blood leukocytes obtained from patients with acute infections. The results indicate there is a differential response of the immune system to bacterial and viral infections, which can potentially be used to classify patients with acute infections.
  • Initially, the present inventors identified 65 RNA determinants that were differentially expressed in the bacterial and viral patients tested (Table 8). FIG. 2 presents distinctive gene expression profiles of the identified genes after applying a hierarchical clustering that uses Euclidean distance metric and average linkage. Based on these results the present inventors developed a classifier for distinguishing between bacterial and viral patients using logistic regression.
  • Pairs and triplets that present high accuracy improvement compared to the single RNA determinants were also uncovered (Tables 9, 11, 12, 13 and 14).
  • Whilst subsequently corroborating these results, the present inventors uncovered additional RNA markers that could serve as determinants for distinguishing between bacterial and viral infections (Tables 15A-B). Further analysis identified which of these RNAs are the most differentially expressed between bacterial and viral patients (Tables 16A-C). Particular RNAs were shown to be highly accurate at diagnosing infection type at disease onset (Table 24). Additional RNAs were shown to be accurate at indicating a specific type of viral strain (Table 25).
  • Thus, according to a first aspect of the present invention there is provided a method of determining an infection type in a subject comprising measuring the amount of a determinant which is set forth in Tables 1 and 2 in a sample derived from the subject, wherein the amount is indicative of the infection type.
  • As used herein, the term “determinant” refers to a polypeptide or a polynucleotide (e.g. RNA).
  • The infection type may be a bacterial infection, a viral infection or a mixed infection (a combination of bacterial and viral infection).
  • The bacterial infection may be the result of gram-positive, gram-negative bacteria or atypical bacteria.
  • The term “Gram-positive bacteria” refers to bacteria that are stained dark blue by Gram staining. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall.
  • The term “Gram-negative bacteria” refers to bacteria that do not retain the crystal violet dye in the Gram staining protocol.
  • The term “Atypical bacteria” are bacteria that do not fall into one of the classical “Gram” groups. They are usually, though not always, intracellular bacterial pathogens. They include, without limitations, Mycoplasmas spp., Legionella spp. Rickettsiae spp., and Chlamydiae spp.
  • The infection may be an acute or chronic infection.
  • A chronic infection is an infection that develops slowly and lasts a long time. Viruses that may cause a chronic infection include Hepatitis C and HIV. One difference between acute and chronic infection is that during acute infection the immune system often produces IgM+antibodies against the infectious agent, whereas the chronic phase of the infection is usually characteristic of IgM−/IgG+antibodies. In addition, acute infections cause immune mediated necrotic processes while chronic infections often cause inflammatory mediated fibrotic processes and scaring (e.g. Hepatitis C in the liver). Thus, acute and chronic infections may elicit different underlying immunological mechanisms.
  • In one embodiment, the level of the determinant may be used to rule in an infection type. In another embodiment, the level of the determinant may be used to rule out an infection type.
  • By “ruling in” an infection it is meant that the subject has that type of infection. By “ruling out” an infection it is meant that the subject does not have that type of infection.
  • “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of the determinant within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such determinants.
  • A “sample” in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, saliva, mucus, breath, urine, CSF, sputum, sweat, stool, hair, seminal fluid, biopsy, rhinorrhea, tissue biopsy, cytological sample, platelets, reticulocytes, leukocytes, epithelial cells, or whole blood cells.
  • For measuring protein determinants, preferably the sample is a blood sample—e.g. serum or a sample comprising white blood cells (which is depleted of red blood cells).
  • For measuring RNA determinants, preferably the sample is a blood sample comprising white blood cells such as lymphocytes, monocytes and granulocytes (which is depleted of red blood cells). In one embodiment, the sample is not a serum sample.
  • Methods of depleting red blood cells are known in the art and include for example hemolysis, centrifugation, sedimentation, filtration or combinations thereof.
  • It will be appreciated that when the determinant to be measured is a protein, a protein sample is generated.
  • When the determinant that is measured is an RNA, an RNA sample is generated.
  • The RNA sample may comprise RNA from a heterogeneous population of cells or from a single population of cells. The RNA may comprise total RNA, mRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, viral RNA, cell free RNA, and mixtures thereof. In one embodiment, the RNA sample is devoid of DNA.
  • The sample may be fresh or frozen.
  • Isolation, extraction or derivation of RNA may be carried out by any suitable method. Isolating RNA from a biological sample generally includes treating a biological sample in such a manner that the RNA present in the sample is extracted and made available for analysis. Any isolation method that results in extracted RNA may be used in the practice of the present invention. It will be understood that the particular method used to extract RNA will depend on the nature of the source.
  • Methods of RNA extraction are well-known in the art and further described herein under.
  • Phenol based extraction methods: These single-step RNA isolation methods based on Guanidine isothiocyanate (GITC)/phenol/chloroform extraction require much less time than traditional methods (e.g. CsCl2 ultracentrifugation). Many commercial reagents (e.g. Trizol, RNAzol, RNAWIZ) are based on this principle. The entire procedure can be completed within an hour to produce high yields of total RNA.
  • Silica gel-based purification methods: RNeasy is a purification kit marketed by Qiagen. It uses a silica gel-based membrane in a spin-column to selectively bind RNA larger than 200 bases. The method is quick and does not involve the use of phenol.
  • Oligo-dT based affinity purification of mRNA: Due to the low abundance of mRNA in the total pool of cellular RNA, reducing the amount of rRNA and tRNA in a total RNA preparation greatly increases the relative amount of mRNA. The use of oligo-dT affinity chromatography to selectively enrich poly (A)+RNA has been practiced for over 20 years. The result of the preparation is an enriched mRNA population that has minimal rRNA or other small RNA contamination. mRNA enrichment is essential for construction of cDNA libraries and other applications where intact mRNA is highly desirable. The original method utilized oligo-dT conjugated resin column chromatography and can be time consuming. Recently more convenient formats such as spin-column and magnetic bead based reagent kits have become available.
  • The sample may also be processed prior to carrying out the diagnostic methods of the present invention. Processing of the sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like.
  • After obtaining the RNA sample, cDNA may be generated therefrom. For synthesis of cDNA, template mRNA may be obtained directly from lysed cells or may be purified from a total RNA or mRNA sample. The total RNA sample may be subjected to a force to encourage shearing of the RNA molecules such that the average size of each of the RNA molecules is between 100-300 nucleotides, e.g. about 200 nucleotides. To separate the heterogeneous population of mRNA from the majority of the RNA found in the cell, various technologies may be used which are based on the use of oligo(dT) oligonucleotides attached to a solid support. Examples of such oligo(dT) oligonucleotides include: oligo(dT) cellulose/spin columns, oligo(dT)/magnetic beads, and oligo(dT) oligonucleotide coated plates.
  • Generation of single stranded DNA from RNA requires synthesis of an intermediate RNA-DNA hybrid. For this, a primer is required that hybridizes to the 3′ end of the RNA. Annealing temperature and timing are determined both by the efficiency with which the primer is expected to anneal to a template and the degree of mismatch that is to be tolerated.
  • The annealing temperature is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 50° C. to about 80° C., usually from about 55° C. to about 70° C., and more usually from about 60° C. to about 68° C. Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.
  • According to a specific embodiment, the primer comprises a polydT oligonucleotide sequence.
  • Preferably the polydT sequence comprises at least 5 nucleotides. According to another is between about 5 to 50 nucleotides, more preferably between about 5-25 nucleotides, and even more preferably between about 12 to 14 nucleotides.
  • Following annealing of the primer (e.g. polydT primer) to the RNA sample, an RNA-DNA hybrid is synthesized by reverse transcription using an RNA-dependent DNA polymerase. Suitable RNA-dependent DNA polymerases for use in the methods and compositions of the invention include reverse transcriptases (RTs). Examples of RTs include, but are not limited to, Moloney murine leukemia virus (M-MLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, rous associated virus (RAV) reverse transcriptase, and myeloblastosis associated virus (MAV) reverse transcriptase or other avian sarcoma-leukosis virus (ASLV) reverse transcriptases, and modified RTs derived therefrom. See e.g. U.S. Pat. No. 7,056,716. Many reverse transcriptases, such as those from avian myeloblastosis virus (AMV-RT), and Moloney murine leukemia virus (MMLV-RT) comprise more than one activity (for example, polymerase activity and ribonuclease activity) and can function in the formation of the double stranded cDNA molecules.
  • Additional components required in a reverse transcription reaction include dNTPS (dATP, dCTP, dGTP and dTTP) and optionally a reducing agent such as Dithiothreitol (DTT) and MnCl2.
  • As mentioned, in order to determine the type of infection, at least one determinant (e.g. RNA determinant) of Table 1 or Table 2 is measured.
  • In one embodiment, the RNA determinant is an RNA that codes for a protein.
  • In another embodiment, the RNA determinant does not code for a protein (non-coding RNA).
  • TABLE 1
    Gene symbol REFSEQ NO. Gene Name
    AIM2 NC_000001.11 Absent In Melanoma 2
    NT_004487.20,
    NC_018912.2
    ANKRD2 NC_000010.11 Ankyrin Repeat Domain 2
    NC_018921.2
    NT_030059.14
    BMX NC_000023.11 BMX Non-Receptor
    NT_167197.2, Tyrosine Kinase
    NC_018934.2
    C19orf59 NC_000019.10 Mast Cel4-Expressed
    NC_018930.2 Membrane Protein 1
    NT_011295.12
    CD 177 NC_000019.10 CD 177 Molecule
    NC_018930.2,
    NT_011109.17
    CEACAM1 NC_000019.10 Carcinoembryonic
    NT_011109.17, Antigen-Related Cell
    NC_018930.2 Adhesion Molecule 1
    CLEC4D NC_000012.12 C-Type Lectin Domain
    NT_009714.18, Family 4, Member D
    NC_018923.2
    CMPK2 NC_000002.12 Cytidine/uridine
    NT_005334.17, monophosphate kinase 2
    NC_018913.2
    EIF1AY NC_000024.10 Eukaryotic Translation
    NT_011875.13 Initiation Factor 1A, Y-
    Linked
    EIF2AK2 NC_000002.12 Eukaryotic Translation
    NT_022184.16 Initiation Factor 2-Alpha
    NC_018913.2 Kinase 2
    EPSTI1 NC_000013.ll Epithelial Stromal
    NT_024524.15 Interaction 1 (breast)
    NC_018924.2
    FFAR3 NC_000019.10 Free Fatty Acid Receptor 3
    NT_011109.17
    NC_018930.2
    GALM NC_000002.12 galactose mutarotase
    NT_022184.16 (aldose 1-epimerase)
    NC_018913.2
    IFITM3 NC_000011.10 interferon induced
    NC_018922.2 transmembrane protein 3
    NT_009237.19
    INCA NC_000011.10 caspase recruitment
    NT_033899.9 domain family member 17
    NC_018922.2
    IRF7 NC_000011.10 interferon regulatory
    NT_009237.19 factor 7
    NT_187586.1
    NC_018922.2
    JARID1D NC_000024.10 lysine (K)-specific
    NT_011875.13 demethylase 5D
    JUP NC_000017.il junction plakoglobin
    NT_010783.16
    NC_018928.2
    MT1G NC_000016.10 metallo thionein 1G
    NC_018927.2
    NT_010498.16
    MT2A NC_000016.10 metallothionein 2A
    NC_018927.2
    NT_010498.16
    OTOF NC_000002.12 otoferlin
    NT_022184.16
    NC_018913.2
    PLSCRI NC_000003.12 phospholipid scramblase 1
    NT_005612.17
    NC_018914.2
    PSTPIP2 NC_000018.10 proline-serine-threonine
    NT_010966.15 phosphatase interacting
    NC_018929.2 protein 2
    RGS1 NC_000001.11 regulator of G-protein
    NC_018912.2 signaling 1
    NT_004487.20
    TREML4 NC_000006.12 triggering receptor
    NC_018917.2 expressed on myeloid cells
    NT_007592.16 like 4
    UTY NC_000024.10 ubiquitously transcribed
    NT_011875.13 tetratricopeptide repeat
    containing, Y-linked
    PARP12 NC_000007.14 Poly(ADP-Ribose)
    NC_018918.2 Polymerase Family
    Member 12
    PNPT1 NC_000002.12 Polyribonucleotide
    NC. 0189 13.2 Nucleotidyltransferase 1
    TRIB2 NC_000002.12 Tribbles Pseudokinase 2
    NC_018913.2
    uc003hrl.1 Affymetrix
    transcript ID:
    TC04001372.hg.1
    Start of genomic
    location:
    89178768 of
    chromosome 4
    End of genomic
    location:
    89180508 of
    chromosome 4
    USP41 NC_018933.2 Ubiquitin Specific
    NC_000022.ll Peptidase 41
    ZCCHC2 NC_000018.10 Zinc Finger CCHC-Type
    NC_018929.2 Containing 2
    TCONS_00003184- Affymetrix linc-RNASEHl-12
    XLOC_001966 transcript ID:
    TC02004017.hg.l
    start of genomic
    location:
    6968645
    Chromosome 2
    End of genomic
    location:
    6973662
    Chromosome 2
  • TABLE 2
    Gene
    symbol REFSEQ NO. Gene Name
    CYBRD1 NC_000002.12 Cytochrome B
    NC_018913.2 Reductase 1
    NT_005403.18
    CYP1B1 NC_000002.12 Cytochrome P450,
    NC_018913.2 Family 1, ,
    NT_022184.16 Subfamily B
    Polypeptide 1
    F13A1 NC_000006.12 Coagulation factor
    NT_007592.16 XIII A chain
    NC_018917.2
    FAM101B NC_000017.11 Family With
    NC_018928.2, Sequence
    NT_024972.9 Similarity 101,
    Member B
    RASA4 NC_000007.14 RAS P21 Protein
    NT_007933.16 Activator 4
    NC_018918.2 (clone name
    HLA- NT_167247.2 FLJ21767)
    DQA1 NT_167245.2 major
    NT_167246.2 histocompatibility
    NT_167249.2 complex,
    NC_000006.12 class 11, DQ
    NT_007592.16 alpha l
    NT_113891.3
    NT_167248.2
    NC_018917.2
    LOC100132244 NC_000007.12 uncharacterized
    LOC100132244
    PHOSPHO1 NC_000017.1 phosphatase,
    NT_010783.16 orphan 1
    NC_018928.2
    PI3 NC_000020.il peptidase inhibitor 3
    NC_018931.2
    NT_011362.11
    PPBP NC_000004.12 pro-platelet basic
    NC_018915.2 protein
    NT_016354.20
    SH3BGRL2 NC_000006.12 SH3 domain
    NT_025741.16 binding
    NC_018917.2 glutamate-rich
    protein
    like 2
    TMEM176A NC_000007.14 transmembrane
    NT_007933.16 protein
    NC_018918.2 176A
    CR1 NC_000001.11 Comple merit
    NC_018912.2 C3b/C4b
    Receptor 1 (Knops
    Blood Group)
    DGAT2 NC_000011.10 Diacylglycerol O-
    NC_018922.2 Acyl transferase 2
    PYGL NC_000014.9 Phosphorylase,
    NC_018925.2 Glycogen, Liver
    SULT1B1 NC_000004.12 Sulfotraasferase
    NC_018915.2 Family
    1B Member 1
    TCONS_0 Affymetrix transcript ID: linc-VIPR2-2
    0013664- TC07002730.hg.1
    XLOC_006324 Start of genomic location:
    26392 of chromosome 7
    End of genomic location:
    35472 of chromosome 7
    TCONS_12_00028242- Affymetrix transcript ID: linc-UBE2W
    XLOCJ2_014551 TC08002386.hg.1
    Start of genomic location:
    74817620
    of chromosome 8
    End of genomic location:
    74827134 of chromosome 8
    TCONS12_00001926- Affymetrix transcript ID: linc-OR4F16-1
    XLOC_12_000004 TC01004103.hg.1
    Start of genomic location:
    329783 of chromosome 1
    End of genomic location:
    334271 of chromosome 1
    TCONS_12_000023 Affymetrix transcript ID: linc-OR4F29-1
    86-XLOC_12_000726 TC01005192.hg.1
    Start of genomic location:
    637315 of chromosome 1
    End of genomic location:
    655530 of chromosome 7
    TCONS_02_00002811- Affymetrix transcript ID: linc-PLD5-4
    XLOC_12_001398 TC01006237.hg.1
    Start of genomic location:
    243192814
    of chromosome 1
    End of genomic location:
    243215554
    of chromosome 1
    TCONS_12_00003949- Affymetrix transcript ID: linc-BMS1-9
    XLOC_12_001561 TC10002018.hg.1
    Start of genomic location:
    38742109
    of chromosome 10
    End of genomic location:
    38764837
    of chromosome 10
    TCONS_00019559- Affymetrix transcript ID: linc-CTSD-2
    XLOC_009354 TC11002997.hg.1
    Start of genomic location:
    1792623
    of chromosome 11
    End of genomic location:
    1793111
    of chromosome 11
    TCONS_12_00010440- Affymetrix transcript ID: linc-RBM11-5
    XLOC_12_005352 TC16001727.hg.1
    Start of genomic location:
    90244125
    of chromosome 16
    End of genomic location:
    90289178
    of chromosome 16
    TCONS_12_00016828- Affymetrix transcript ID: linc-HNF1B-4
    XLOC_12_008724 TC20001365.hg.1
    Start of genomic location:
    62921738
    of chromosome 20
    End of genomic location:
    62944485
    of chromosome 20
    TCONS_12_00021682- Affymetrix transcript ID: linc-EXOSC9-l 1
    XLOC_12-010810 TC04002168.hg.l
    Start of genomic location:
    120299287
    of chromosome 4
    End of genomic location:
    120326770
    of chromosome 4
    TCONS_12_00002367- Affymetrix transcript ID: linc-ZNF692-5
    XLOC_12_000720 TCO1006418.hg.1
    Start of genomic location:
    89295
    of chromosome 1
    End of genomic location:
    173864
    of chromosome 1
  • According to particular embodiments, when the level of determinant in Table 1 is above a predetermined level (e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a bacterial infection, as further described herein below), it is indicative that the subject has a viral infection (i.e. a viral infection may be ruled in).
  • Alternatively or additionally, when the level of determinant in Table 1 is above a predetermined level, it is indicative that the subject does not have a bacterial infection (i.e. a bacterial infection is ruled out).
  • According to other embodiments, when the level of determinant in Table 2 is above a predetermined level (e.g. above the level that is present in a sample derived from a non-infectious, preferably healthy subject; or above the level that is present in a sample derived from a subject known to be infected with a viral infection, as further described herein below), it is indicative that the subject has a bacterial infection (i.e. a bacterial infection is ruled in).
  • Alternatively or additionally, when the level of determinant in Table 2 is above a predetermined level, it is indicative that the subject does not have a viral infection (i.e. a viral infection is ruled out).
  • The predetermined level of any of the aspects of the present invention may be a reference value derived from population studies, including without limitation, such subjects having a known infection, subject having the same or similar age range, subjects in the same or similar ethnic group, or relative to the starting sample of a subject undergoing treatment for an infection. Such reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of infection. Reference determinant indices can also be constructed and used using algorithms and other methods of statistical and structural classification.
  • In one embodiment of the present invention, the predetermined level is the amount (i.e. level) of determinants in a control sample derived from one or more subjects who do not have an infection (i.e., healthy, and or non-infectious individuals). In a further embodiment, such subjects are monitored and/or periodically retested for a diagnostically relevant period of time (“longitudinal studies”) following such test to verify continued absence of infection. Such period of time may be one day, two days, two to five days, five days, five to ten days, ten days, or ten or more days from the initial testing date for determination of the reference value. Furthermore, retrospective measurement of determinants in properly banked historical subject samples may be used in establishing these reference values, thus shortening the study time required.
  • A reference value can also comprise the amounts of determinants derived from subjects who show an improvement as a result of treatments and/or therapies for the infection. A reference value can also comprise the amounts of determinants derived from subjects who have confirmed infection by known techniques.
  • An example of a bacterially infected reference value is the mean or median concentrations of that determinant in a statistically significant number of subjects having been diagnosed as having a bacterial infection.
  • An example of a virally infected reference value is the mean or median concentrations of that determinant in a statistically significant number of subjects having been diagnosed as having a viral infection.
  • It will be appreciated that the control sample is the same blood sample type as the sample being analyzed.
  • As well as measuring at least one determinant in Tables 1 or 2, the present inventors contemplate measuring at least one additional (non-identical) determinant (so as to measure at least two determinants), wherein the at least one additional determinant is set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least two determinants is indicative of the infection type.
  • In one embodiment, at least two non-identical RNAs are measured, the first from Tables 1 or 2 and the second from any one of Tables 1-6A, or 16A-C. Preferably the second RNA is set forth in Tables 1, 2, 3 or 5 and even more preferably, the second RNA is set forth in Tables 1 or 2.
  • According to still another embodiment as well as measuring at least one determinant in Tables 1 or 2, the present inventors contemplate measuring at least one additional (non-identical) determinant (so as to measure at least two determinants), wherein the at least one additional determinant is set forth in Table 6B.
  • According to a particular embodiment, at least one of the RNA determinants is a coding RNA e.g. OTOF, PI3, CYBRD1, EIF2AK2 or CMPK2, PARP12, PNPT1, TRIB2, uc003hr1.1, USP41, ZCCHC2.
  • According to a particular embodiment, at least one of the RNA determinants is a non-coding RNA e.g, TCONS_00003184-XLOC_001966.
  • Exemplary pairs contemplated by the present inventors are set forth in Tables 9, 11, 17 and 18 in the Examples section herein below.
  • Other exemplary pairs are provided herein below:
  • CMPK2+CR1, CMPK2+CYP1B1, CMPK2+DDX60, CMPK2+DGAT2, CMPK2+PARP12. CMPK2+PNPT1. CMPK2+PYGL, CMPK2+SULT1B1, CMPK2+TRIB2, CMPK2+uc003hr1.1, CMPK2+USP41, CMPK2+ZCCHC2, CMPK2+n332762, CMPK2+n407780, CMPK2+n332510, CMPK2+n334829, CMPK2+n332456, CMPK2+n333319, CMPK2+TCONS_00003184-XLOC_001966. CMPK2+TCONS_00013664-XLOC_006324. CMPK2+TCONS_12_00028242-XLOC_12_014551, CMPK2+TCONS_12_00001926-XLOC_12_000004, CMPK2+TCONS_12_00002386-XLOC_12_000726, CMPK2+TCONS_12_00002811-XLOC_12_001398. CMPK2+TCONS_12_00003949-XLOC_12_001561. CMPK2+TCONS_00019559-XLOC_009354, CMPK2+TCONS_12_00010440-XLOC_12_005352, CMPK2+TCONS_12_00016828-XLOC_12_008724, CMPK2+TCONS_12_00021682-XLOC_12_010810, CMPK2+TCONS_12_00002367-XLOC_12_000720, CMPK2+FAM89A, CMPK2+MX1. CMPK2+RSAD2, CMPK2+IFI44L, CMPK2+USP18, CMPK2+IFI27, CR1+CYP1B1, CR1+DDX60. CR1+DGAT2, CR1+PARP12, CR1+PNPT1, CR1+PYGL, CR1+SULT1B1, CR1+TRIB2, CR1+uc003hr1.1, CR1+USP41, CR1+ZCCHC2, CR1+n332762, CR1+n407780, CR1+n332510. CR1+n334829, CR1+n332456, CR1+n333319, CR1+TCONS_00003184-XLOC_001966, CR1+TCONS_00013664-XLOC_006324, CR1+TCONS_12_00028242-XLOC_12_014551, CR1+TCONS_12_00001926-XLOC_12_000004, CR1+TCONS_12_00002386-XLOC_12_000726, CR1+TCONS_12_00002811-XLOC_12_001398, CR1+TCONS_12_00003949-XLOC_12_001561, CR 1+TCONS_00019559-XLOC_009354, CR1+TCONS_12_00010440-XLOC_12_005352, CR1+TCONS_12_00016828-XLOC_12_008724, CR1+TCONS_12_00021682-XLOC_12_010810, CR1+TCONS_12_00002367-XLOC_12_000720, CR1+FAM89A, CR1+MX1, CR1+RSAD2, CR1+IFI44L, CR1+USP18, CR1+IFI27, CYP1B1+DDX60, CYP1B1+DGAT2, CYP1B1+PARP12, CYP1B1+PNPT1, CYP1B1+PYGL, CYP1B1+SULT1B1, CYP1B1+TRIB2, CYP1B1+uc003hr1.1, CYP1B1+USP41, CYP1B1+ZCCHC2, CYP1B1+n332762, CYP1B1+n407780, CYP1B1+n332510, CYP1B1+n334829, CYP1B1+n332456, CYP1B1+n333319, CYP1B1+TCONS_00003184-XLOC_001966, CYP1B1+TCONS_00013664-XLOC_006324, CYP1B1+TCONS_12_00028242-XLOC_12_014551, CYP1B1+TCONS_12_00001926-XLOC_12_000004, CYP1B1+TCONS_12_00002386-XLOC_12_000726, CYP1B1+TCONS_12_00002811-XLOC_12_001398, CYP1B1+TCONS_12 00003949-XLOC_12 001561. CYP1B1+TCONS_00019559-XLOC_009354, CYP1B1+TCONS_12_00010440-XLOC_12_005352, CYP1B1+TCONS_12_00016828-XLOC_12_008724, CYP1B1+TCONS_12_00021682-XLOC_12_010810, CYP1B1+TCONS_12_00002367-XLOC_12_000720, CYP1B1+FAM89A, CYP1B1+MX1, CYP1B1+RSAD2, CYP1B1+IFI44L, CYP1B1+USP18, CYP1B1+IFI27, DDX60+DGAT2, DDX60+PARP12, DDX60+PNPT1, DDX60+PYGL, DDX60+SULT1B1, DDX60+TRIB2, DDX60+uc003hr1.1, DDX60+USP41, DDX60+ZCCHC2, DDX60+n332762, DDX60+n407780, DDX60+n332510, DDX60+n334829, DDX60+n332456, DDX60+n333319, DDX60+TCONS_00003184-XLOC_001966, DDX60+TCONS_00013664-XLOC_006324, DDX60+TCONS_12_00028242-XLOC_12_014551, DDX60+TCONS_12_00001926-XLOC_12_000004, DDX60+TCONS_12_00002386-XLOC_12_000726, DDX60+TCONS_12_00002811-XLOC_12_001398, DDX60+TCONS_12_00003949-XLOC_12_001561, DDX60+TCONS_00019559-XLOC_009354, DDX60+TCONS_12_00010440-XLOC_12_005352, DDX60+TCONS_12_00016828-XLOC_12_008724, DDX60+TCONS_12_00021682-XLOC_12_010810, DDX60+TCONS_12_00002367-XLOC_12_000720, DDX60+FAM89A, DDX60+MX1, DDX60+RSAD2, DDX60+IFI44L, DDX60+USP18, DDX60+IFI27, DGAT2+PARP12, DGAT2+PNPT1, DGAT2+PYGL, DGAT2+SULT1B1, DGAT2+TRIB2, DGAT2+uc003hr1.1, DGAT2+USP41, DGAT2+ZCCHC2, DGAT2+n332762, DGAT2+n407780, DGAT2+n332510, DGAT2+n334829, DGAT2+n332456, DGAT2+n333319, DGAT2+TCONS_00003184-XLOC_001966, DGAT2+TCONS_00013664-XLOC_006324, DGAT2+TCONS_12_00028242-XLOC_12_014551, DGAT2+TCONS_12_00001926-XLOC_12_000004, DGAT2+TCONS_12_00002386-XLOC_12_000726, DGAT2+TCONS_12_00002811-XLOC_12_001398, DGAT2+TCONS_12_00003949-XLOC_12_001561, DGAT2+TCONS_00019559-XLOC_009354, DGAT2+TCONS_12_00010440-XLOC_12_005352, DGAT2+TCONS_12 00016828-XLOC_12 008724, DGAT2+TCONS_12 00021682-XLOC_12_010810, DGAT2+TCONS_12_00002367-XLOC_12_000720, DGAT2+FAM89A, DGAT2+MX1, DGAT2+RSAD2, DGAT2+IFI44L, DGAT2+USP18, DGAT2+IFI27, PARP12+PNPT1, PARP12+PYGL, PARP12+SULT1B1, PARP12+TRIB2, PARP12+uc003hr1.1, PARP12+USP41, PARP12+ZCCHC2, PARP12+n332762, PARP12+n407780, PARP12+n332510, PARP12+n334829, PARP12+n332456, PARP12+n333319, PARP12+TCONS_00003184-XLOC_001966, PARP12+TCONS_00013664-XLOC_006324, PARP12+TCONS_12_00028242-XLOC_12_014551, PARP12+TCONS_12_00001926-XLOC_12_000004, PARP12+TCONS_12_00002386-XLOC_12_000726, PARP12+TCONS_12_00002811-XLOC_12_001398, PARP12+TCONS_12_00003949-XLOC_12_001561, PARP12+TCONS_00019559-XLOC_009354, PARP12+TCONS_12_00010440-XLOC_12_005352, PARP12+TCONS_12_00016828-XLOC_12_008724, PARP12+TCONS_12_00021682-XLOC_12_010810, PARP12+TCONS_12_00002367-XLOC_12_000720, PARP12+FAM89A, PARP12+MX1, PARP12+RSAD2, PARP12+IFI44L. PARP12+USP18, PARP12+IFI27, PNPT1+PYGL, PNPT1+SULT1B1, PNPT1+TRIB2, PNPT1+uc003hr1.1, PNPT1+USP41, PNPT1+ZCCHC2, PNPT1+n332762, PNPT1+n407780, PNPT1+n332510, PNPT1+n334829, PNPT1+n332456, PNPT1+n333319, PNPT1+TCONS_00003184-XLOC_001966, PNPT1+TCONS_00013664-XLOC_006324, PNPT1+TCONS_12_00028242-XLOC_12_014551, PNPT1+TCONS_12_00001926-XLOC_12_000004, PNPT1+TCONS_12_00002386-XLOC_12_000726, PNPT1+TCONS_12_00002811-XLOC_12_001398, PNPT1+TCONS_12_00003949-XLOC_12_001561, PNPT1+TCONS_00019559-XLOC_009354, PNPT1+TCONS_12_00010440-XLOC_12_005352, PNPT1+TCONS_12_00016828-XLOC_12_008724, PNPT1+TCONS_12_00021682-XLOC_12_010810, PNPT1+TCONS_12_00002367-XLOC_12_000720, PNPT1+FAM89A, PNPT1+MX1, PNPT1+RSAD2, PNPT1+IFI44L, PNPT1+USP18, PNPT1+IFI27, PYGL+SULT1B1, PYGL+TRIB2, PYGL+uc003hr1.1, PYGL+USP41, PYGL+ZCCHC2, PYGL+n332762, PYGL+n407780, PYGL+n332510, PYGL+n334829, PYGL+n332456, PYGL+n333319, PYGL+TCONS_00003184-XLOC_001966, PYGL+TCONS_00013664-XLOC_006324, PYGL+TCONS_12_00028242-XLOC_12_014551, PYGL+TCONS_12_00001926-XLOC_12_000004, PYGL+TCONS_12_00002386-XLOC_12_000726, PYGL+TCONS_12_00002811-XLOC_12_001398, PYGL+TCONS_12_00003949-XLOC_12_001561, PYGL+TCONS_00019559-XLOC_009354, PYGL+TCONS_12_00010440-XLOC_12_005352, PYGL+TCONS_12_00016828-XLOC_12_008724, PYGL+TCONS_12_00021682-XLOC_12_010810, PYGL+TCONS_12_00002367-XLOC_12_000720, PYGL+FAM89A, PYGL+MX1, PYGL+RSAD2, PYGL+IFI44L, PYGL+USP18, PYGL+IFI27, SULT1B1+TRIB2, SULT1B1+uc003hr1.1, SULT1B1+USP41, SULT1B1+ZCCHC2, SULT1B1+n332762, SULT1B1+n407780, SULT1B1+n332510, SULT1B1+n334829, SULT1B1+n332456, SULT1B1+n333319, SULT1B1+TCONS_00003184-XLOC_001966, SULT1B1+TCONS_00013664-XLOC_006324, SULT1B1+TCONS_12_00028242-XLOC_12_014551, SULT1B1+TCONS_12_00001926-XLOC_12_000004, SULT1B1+TCONS_12_00002386-XLOC_12_000726, SULT1B1+TCONS_12_00002811-XLOC_12_001398, SULT1B1+TCONS_12 00003949-XLOC_12 001561, SULT1B1+TCONS_00019559-XLOC_009354, SULT1B1+TCONS_12_00010440-XLOC_12_005352, SULT1B1+TCONS_12_00016828-XLOC_12_008724, SULT1B1+TCONS_12_00021682-XLOC_12_010810, SULT1B1+TCONS_12_00002367-XLOC_12_000720, SULT1B1+FAM89A, SULT1B1+MX1, SULT1B1+RSAD2, SULT1B1+IFI44L, SULT1B1+USP18, SULT1B1+IFI27, TRIB2+uc003hr1.1, TRIB2+USP41, TRIB2+ZCCHC2, TRIB2+n332762, TRIB2+n407780, TRIB2+n332510, TRIB2+n334829, TRIB2+n332456, TRIB2+n333319, TRIB2+TCONS_00003184-XLOC_001966, TRIB2+TCONS_00013664-XLOC_006324, TRIB2+TCONS_12_00028242-XLOC_12_014551, TRIB2+TCONS_12_00001926-XLOC_12_000004, TRIB2+TCONS_12_00002386-XLOC_12_000726, TRIB2+TCONS_12_00002811-XLOC_12_001398, TRIB2+TCONS_12_00003949-XLOC_12_001561, TRIB2+TCONS_00019559-XLOC_009354, TRIB2+TCONS_12_00010440-XLOC_12_005352, TRIB2+TCONS_12_00016828-XLOC_12_008724, TRIB2+TCONS_12_00021682-XLOC_12_010810, TRIB2+TCONS_12_00002367-XLOC_12_000720, TRIB2+FAM89A, TRIB2+MX1, TRIB2+RSAD2, TRIB2+IFI44L, TRIB2+USP18, TRIB2+IFI27, uc003hr1.1+USP41, uc003hr1.1+ZCCHC2, uc003hr1.1+n332762, uc003hr1.1+n407780, uc003hr1.1+n332510, uc003hr1.1+n334829, uc003hr1.1+n332456, uc003hr1.1+n333319, uc003hr1.1+TCONS_00003184-XLOC_001966, uc003hr1.1+TCONS_00013664-XLOC_006324, uc003hr1.1+TCONS_12_00028242-XLOC_12_014551, uc003hr1.1+TCONS_12_00001926-XLOC_12_000004, uc003hr1.1+TCONS_12_00002386-XLOC_12_000726, uc003hr1.1+TCONS_12_00002811-XLOC_12_001398, uc003hr1.1+TCONS_12_00003949-XLOC_12_001561, uc003hr1.1+TCONS_00019559-XLOC_009354, uc003hr1.1+TCONS_12_00010440-XLOC_12_005352, uc003hr1.1+TCONS_12_00016828-XLOC_12_008724, uc003hr1.1+TCONS_12 00021682-XLOC_12 010810, uc003hr1.1+TCONS_12_00002367-XLOC_12_000720, uc003hr1.1+FAM89A, uc003hr1.1+MX1, uc003hr1.1+RSAD2, uc003hr1.1+IFI44L, uc003hr1.1+USP18, uc003hr1.1+IFI27, USP41+ZCCHC2, USP41+n332762, USP41+n407780, USP41+n332510, USP41+n334829, USP41+n332456, USP41+n333319, USP41+TCONS_00003184-XLOC_001966, USP41+TCONS_00013664-XLOC_006324, USP41+TCONS_12_00028242-XLOC_12_014551, USP41+TCONS_12_00001926-XLOC_12_000004, USP41+TCONS_12_00002386-XLOC_12_000726, USP41+TCONS_12_00002811-XLOC_12_001398, USP41+TCONS_12_00003949-XLOC_2_001561, USP41+TCONS_00019559-XLOC_009354, USP41+TCONS_12_00010440-XLOC_12_005352, USP41+TCONS_12_00016828-XLOC_12_008724, USP41+TCONS_12_00021682-XLOC_12_010810, USP41+TCONS_12_00002367-XLOC_12_000720, USP41+FAM89A, USP41+MX1, USP41+RSAD2, USP41+IFI44L, USP41+USP18, USP41+IFI27, ZCCHC2+n332762, ZCCHC2+n407780, ZCCHC2+n332510, ZCCHC2+n334829, ZCCHC2+n332456, ZCCHC2+n333319, ZCCHC2+TCONS_00003184-XLOC_001966, ZCCHC2+TCONS_00013664-XLOC_006324, ZCCHC2+TCONS_12_00028242-XLOC_12_014551, ZCCHC2+TCONS_12_00001926-XLOC_12_000004, ZCCHC2+TCONS_12_00002386-XLOC_12_000726, ZCCHC2+TCONS_12_00002811-XLOC_12_001398, ZCCHC2+TCONS_12_00003949-XLOC_12_001561, ZCCHC2+TCONS_00019559-XLOC_009354, ZCCHC2+TCONS_12_00010440-XLOC_12_005352, ZCCHC2+TCONS_12_00016828-XLOC_12_008724, ZCCHC2+TCONS_12_00021682-XLOC_12_010810, ZCCHC2+TCONS_12_00002367-XLOC_12_000720, ZCCHC2+FAM89A, ZCCHC2+MX1, ZCCHC2+RSAD2, ZCCHC2+IFI44L, ZCCHC2+USP18, ZCCHC2+IFI27, n332762+n407780, n332762+n332510, n332762+n334829, n332762+n332456, n332762+n333319, n332762+TCONS_00003184-XLOC_001966, n332762+TCONS_00013664-XLOC_006324, n332762+TCONS_12_00028242-XLOC_12_014551, n332762+TCONS_12_00001926-XLOC_12_000004, n332762+TCONS_12_00002386-XLOC_12_000726, n332762+TCONS_12_00002811-XLOC_12_001398, n332762+TCONS_12_00003949-XLOC_12_001561, n332762+TCONS_00019559-XLOC_009354, n332762+TCONS_12_00010440-XLOC_12_005352, n332762+TCONS_12_00016828-XLOC_12_008724, n332762+TCONS_12_00021682-XLOC_12_010810, n332762+TCONS_12_00002367-XLOC_12_000720, n332762+FAM89A, n332762+MX1, n332762+RSAD2, n332762+IFI44L, n332762+USP18, n332762+IFI27, n407780+n332510, n407780+n334829, n407780+n332456, n407780+n333319, n407780+TCONS_00003184-XLOC_001966, n407780+TCONS_00013664-XLOC_006324, n407780+TCONS_12_00028242-XLOC_12_014551, n407780+TCONS_12 00001926-XLOC_12 000004, n407780+TCONS_12 00002386-XLOC_12_000726, n407780+TCONS_12_00002811-XLOC_12_001398, n407780+TCONS_12_00003949-XLOC_12_001561, n407780+TCONS_00019559-XLOC_009354, n407780+TCONS_12_00010440-XLOC_12_005352, n407780+TCONS_12_00016828-XLOC_12_008724, n407780+TCONS_12_00021682-XLOC_12_010810, n407780+TCONS_12_00002367-XLOC_12_000720, n407780+FAM89A, n407780+MX1, n407780+RSAD2, n407780+IFI44L, n407780+USP18, n407780+IFI27, n332510+n334829, n332510+n332456, n332510+n333319, n332510+TCONS_00003184-XLOC_001966, n332510+TCONS_00013664-XLOC_006324, n332510+TCONS_12_00028242-XLOC_12_014551, n332510+TCONS_12_00001926-XLOC_12_000004, n332510+TCONS_12_00002386-XLOC_12_000726, n332510+TCONS_12_00002811-XLOC_12_001398, n332510+TCONS_12_00003949-XLOC_12_001561, n332510+TCONS_00019559-XLOC_009354, n332510+TCONS_12_00010440-XLOC_12_005352, n332510+TCONS_12_00016828-XLOC_12_008724, n332510+TCONS_12_00021682-XLOC_12_010810, n332510+TCONS_12_00002367-XLOC_12_000720, n332510+FAM89A, n332510+MX1, n332510+RSAD2, n332510+IFI44L, n332510+USP18, n332510+IFI27, n334829+n332456, n334829+n333319, n334829+TCONS_00003184-XLOC_001966, n334829+TCONS_00013664-XLOC_006324, n334829+TCONS_12_00028242-XLOC_12_014551, n334829+TCONS_12_00001926-XLOC_12_000004, n334829+TCONS_12_00002386-XLOC_12_000726, n334829+TCONS_12_00002811-XLOC_12_001398, n334829+TCONS_12_00003949-XLOC_12_001561, n334829+TCONS_00019559-XLOC_009354, n334829+TCONS_12 00010440-XLOC_12_005352, n334829+TCONS_12_00016828-XLOC_12_008724, n334829+TCONS_12_00021682-XLOC_12_010810, n334829+TCONS_12_00002367-XLOC_12_000720, n334829+FAM89A, n334829+MX1, n334829+RSAD2, n334829+IFI44L, n334829+USP18, n334829+IFI27, n332456+n333319, n332456+TCONS_00003184-XLOC_001966, n332456+TCONS_00013664-XLOC_006324, n332456+TCONS_12_00028242-XLOC_12_014551, n332456+TCONS_12_00001926-XLOC_12_000004, n332456+TCONS_12_00002386-XLOC_12_000726, n332456+TCONS_12_00002811-XLOC_12_001398, n332456+TCONS_12 00003949-XLOC_12 001561, n332456+TCONS_00019559-XLOC_009354, n332456+TCONS_12_00010440-XLOC_12_005352, n332456+TCONS_12_00016828-XLOC_12_008724, n332456+TCONS_12_00021682-XLOC_12_010810, n332456+TCONS_12_00002367-XLOC_12_000720, n332456+FAM89A, n332456+MX1, n332456+RSAD2, n332456+IFI44L, n332456+USP18, n332456+IFI27, n333319+TCONS_00003184-XLOC_001966, n333319+TCONS_00013664-XLOC_006324, n333319+TCONS_12_00028242-XLOC_12_014551, n333319+TCONS_12_00001926-XLOC_12_000004, n333319+TCONS_12_00002386-XLOC_12_000726, n333319+TCONS_12_00002811-XLOC_12_001398, n333319+TCONS_12_00003949-XLOC_12_001561, n333319+TCONS_00019559-XLOC_009354, n333319+TCONS_12_00010440-XLOC_12_005352, n333319+TCONS_12_00016828-XLOC_12_008724, n333319+TCONS_12_00021682-XLOC_12_010810, n333319+TCONS_12_00002367-XLOC_12_000720, n333319+FAM89A, n333319+MX1, n333319+RSAD2, n333319+IFI44L, n333319+USP18, n333319+IFI27, TCONS_00003184-XLOC_001966+TCONS_00013664-XLOC_006324, TCONS_00003184-XLOC_001966+TCONS_12_00028242-XLOC_12_014551, TCONS_00003184-XLOC_001966+TCONS_12_00001926-XLOC_12_000004, TCONS_00003184-XLOC_001966+TCONS_12_00002386-XLOC_12_000726, TCONS_00003184-XLOC_001966+TCONS_12_00002811-XLOC_12_001398, TCONS_00003184-XLOC_001966+TCONS_12_00003949-XLOC_12_001561, TCONS_00003184-XLOC_001966+TCONS_00019559-XLOC_009354, TCONS_00003184-XLOC_001966+TCONS_12_00010440-XLOC_12_005352, TCONS_00003184-XLOC_001966+TCONS_12_00016828-XLOC_12_008724, TCONS_00003184-XLOC_001966+TCONS_12_00021682-XLOC_12_010810, TCONS_00003184-XLOC_001966+TCONS_12_00002367-XLOC_12_000720, TCONS_00003184-XLOC_001966+FAM89A, TCONS_00003184-XLOC_001966+MX1, TCONS_00003184-XLOC_001966+RSAD2, TCONS_00003184-XLOC_001966+IFI44L, TCONS_00003184-XLOC_001966+USP18, TCONS_00003184-XLOC_001966+IFI27, TCONS_00013664-XLOC_006324+TCONS_12_00028242-XLOC_12_014551, TCONS_00013664-XLOC_006324+TCONS_12_00001926-XLOC_12_000004, TCONS_00013664-XLOC_006324+TCONS_12 00002386-XLOC_12 000726, TCONS_00013664-XLOC_006324+TCONS_12_00002811-XLOC_12_001398, TCONS_00013664-XLOC_006324+TCONS_12_00003949-XLOC_12_001561, TCONS_00013664-XLOC_006324+TCONS_00019559-XLOC_009354, TCONS_00013664-XLOC_006324+TCONS_12_00010440-XLOC_12_005352, TCONS_00013664-XLOC_006324+TCONS_12_00016828-XLOC_12_008724, TCONS_00013664-XLOC_006324+TCONS_12_00021682-XLOC_12_010810, TCONS_00013664-XLOC_006324+TCONS_12_00002367-XLOC_12_000720, TCONS_00013664-XLOC_006324+FAM89A, TCONS_00013664-XLOC_006324+MX1, TCONS_00013664-XLOC_006324+RSAD2, TCONS_00013664-XLOC_006324+IFI44L, TCONS_00013664-XLOC_006324+USP18, TCONS_00013664-XLOC_006324+IFI27, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00001926-XLOC_12_00(004, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00002386-XLOC_12_000726, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00002811-XLOC_12_001398, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00003949-XLOC_12_001561, TCONS_12_00028242-XLOC_12_014551+TCONS_00019559-XLOC_009354, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00010440-XLOC_12_005352, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00016828-XLOC_12_008724, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00028242-XLOC_12_014551+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00028242-XLOC_12_014551+FAM89A, TCONS_12_00028242-XLOC_12_014551+MX1, TCONS_12_00028242-XLOC_12_014551+RSAD2, TCONS_12_00028242-XLOC_12_014551+IFI44L, TCONS_12_00028242-XLOC_12_014551+USP18, TCONS_12_00028242-XLOC_12_014551+IFI27, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00002386-XLOC_12_000726, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00002811-XLOC_12_001398, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00003949-XLOC_12_001561, TCONS_12_00001926-XLOC_12_000004+TCONS_00019559-XLOC_009354, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00010440-XLOC_12_005352, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00016828-XLOC_12_008724, TCONS_12_00001926-XLOC_12_000004+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00001926-XLOC_12 000004+TCONS_12 00002367-XLOC_12 000720, TCONS_12 00001926-XLOC_12_000004+FAM89A, TCONS_12_00001926-XLOC_12_000004+MX1, TCONS_12_00001926-XLOC_12_000004+RSAD2, TCONS_12_00001926-XLOC_12_000004+IFI44L, TCONS_12_00001926-XLOC_12_000004+USP18, TCONS_12_00001926-XLOC_12_000004+IFI27, TCONS_12_00002386-XLOC_12_000726+TCONS_12_00002811-XLOC_12_001398, TCONS_12_00002386-XLOC_12_000726+TCONS_12_00003949-XLOC_12_001561, TCONS_12_00002386-XLOC_12_000726+TCONS_00019559-XLOC_009354, TCONS_12_00002386-XLOC_12_000726+TCONS_12_00010440-XLOC_12_005352, TCONS_12_00002386-XLOC_12_000726+TCONS_12_00016828-XLOC_12_008724, TCONS_12_00002386-XLOC_12_000726+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00002386-XLOC_12_000726+TCONS_12 00002367-XLOC_121000720, TCONS_12_00002386-XLOC_12_000726+FAM89A, TCONS_12_00002386-XLOC_12_000726+MX1, TCONS_12_00002386-XLOC_12_000726+RSAD2, TCONS_12_00002386-XLOC_12_000726+IFI44L, TCONS_12_00002386-XLOC_12_000726+USP18, TCONS_12_00002386-XLOC_12_000726+IFI27, TCONS_12_00002811-XLOC_12_001398+TCONS_12_00003949-XLOC_12_001561, TCONS_12_00002811-XLOC_12_001398+TCONS_00019559-XLOC_009354, TCONS_12_00002811-XLOC_12_001398+TCONS_12_00010440-XLOC_12_005352, TCONS_12_00002811-XLOC_12_001398+TCONS_12_00016828-XLOC_12_008724, TCONS_12_00002811-XLOC_12_001398+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00002811-XLOC_12_001398+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00002811-XLOC_12_001398+FAM89A, TCONS_12_00002811-XLOC_12_001398+MX1, TCONS_12_00002811-XLOC_12_001398+RSAD2, TCONS_12_00002811-XLOC_12_001398+IFI44L, TCONS_12_00002811-XLOC_12_001398+USP18, TCONS_12_00002811-XLOC_12_001398+IFI27, TCONS_12_00003949-XLOC_12_001561+TCONS_00019559-XLOC_009354, TCONS_12_00003949-XLOC_12_001561+TCONS_12_00010440-XLOC_12_005352, TCONS_12_00003949-XLOC_12 001561+TCONS_12 00016828-XLOC_12 008724, TCONS_12 00003949-XLOC_12_001561+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00003949-XLOC_12_001561+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00003949-XLOC_12_001561+FAM89A, TCONS_12_00003949-XLOC_12_001561+MX1, TCONS_12_00003949-XLOC_12_001561+RSAD2, TCONS_12_00003949-XLOC_12_001561+IFI44L, TCONS_12_00003949-XLOC_12_001561+USP18, TCONS_12_00003949-XLOC_12_001561+IFI27, TCONS_00019559-XLOC_009354+TCONS_12_00010440-XLOC_12_005352, TCONS_00019559-XLOC_009354+TCONS_12_00016828-XLOC_12_008724, TCONS_00019559-XLOC_009354+TCONS_12_00021682-XLOC_12_010810, TCONS_00019559-XLOC_009354+TCONS_12_00002367-XLOC_12_000720, TCONS_00019559-XLOC_009354+FAM89A, TCONS_00019559-XLOC_009354+MX1, TCONS_00019559-XLOC_009354+RSAD2, TCONS_00019559-XLOC_009354+IFI44L, TCONS_00019559-XLOC_009354+USP18, TCONS_00019559-XLOC_009354+IFI27, TCONS_12_00010440-XLOC_12_005352+TCONS_12 00016828-XLOC_12 008724, TCONS_12_00010440-XLOC_12_005352+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00010440-XLOC_12_005352+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00010440-XLOC_12_005352+FAM89A, TCONS_12_00010440-XLOC_12_005352+MX1, TCONS_12_00010440-XLOC_12_005352+RSAD2, TCONS_12_00010440-XLOC_12_005352+IFI44L, TCONS_12_00010440-XLOC_12_005352+USP18, TCONS_12_00010440-XLOC_12_005352+IFI27, TCONS_12_00016828-XLOC_12_008724+TCONS_12_00021682-XLOC_12_010810, TCONS_12_00016828-XLOC_12_008724+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00016828-XLOC_12_008724+FAM89A, TCONS_12_00016828-XLOC_12_008724+MX1, TCONS_12_00016828-XLOC_12_008724+RSAD2, TCONS_12_00016828-XLOC_12_008724+IFI44L, TCONS_12_00016828-XLOC_12_008724+USP18, TCONS_12_00016828-XLOC_12_008724+IFI27, TCONS_12_00021682-XLOC_12_010810+TCONS_12_00002367-XLOC_12_000720, TCONS_12_00021682-XLOC_12_010810+FAM89A, TCONS_12_00021682-XLOC_12_010810+MX1, TCONS_12_00021682-XLOC_12_010810+RSAD2, TCONS_12_00021682-XLOC_12_010810+IFI44L, TCONS_12_00021682-XLOC_12_010810+USP18, TCONS_12_00021682-XLOC_12_010810+IFI27, TCONS_12_00002367-XLOC_12_000720+FAM89A, TCONS_12_00002367-XLOC_12_000720+MX1, TCONS_12_00002367-XLOC_12_000720+RSAD2, TCONS_12_00002367-XLOC_12_000720+IFI44L, TCONS_12_00002367-XLOC_12_000720+USP18, TCONS_12_00002367-XLOC_12_000720+IFI27, FAM89A+MX1, FAM89A+RSAD2, FAM89A+IFI44L. FAM89A+USP18. FAM89A+IFI27, MX1+RSAD2. MX1+IFI44L, MX1+USP18, MX1+IFI27, RSAD2+IFI44L, RSAD2+USP18, RSAD2+IFI27, IFI44L+USP18, IFI44L+IFI27, USP18+IFI27
  • In another embodiment, at least three determinants are measured, wherein the at least two additional determinant are set forth in any one of Tables 1-6A or 15A-B, wherein the amount of the at least three determinants is indicative of the infection type.
  • In one embodiment, at least three non-identical RNAs are measured, the first from Tables 1 or 2 and the second and third from any one of Tables 1-6A or 16A-C. Preferably the second and third RNA is set forth in Tables 1, 2, 3, 5 or 16A-C and even more preferably, the second RNA is set forth in Tables 1 or 2.
  • Exemplary triplets contemplated by the present inventors are set forth in Tables 13, 19 or 20 of the Examples section herein below.
  • Other exemplary triplets which may be measured according to this aspect of the present invention are provided herein below:
  • CMPK2+CR1+CYP1B1, CMPK2+CR1+DDX60, CMPK2+CR1+DGAT2, CMPK2+CR1+PARP12, CMPK2+CR1+PNPT1, CMPK2+CR1+PYGL, CMPK2+CR1+SULT1B1, CMPK2+CR1+TRIB2, CMPK2+CR1+uc003hr1.1, CMPK2+CR1+USP41, CMPK2+CR1+ZCCHC2, CMPK2+CYP1B1+DDX60, CMPK2+CYP1B1+DGAT2, CMPK2+CYP1B1+PARP12, CMPK2+CYP1B1+PNPT1, CMPK2+CYP1B1+PYGL, CMPK2+CYP1B1+SULT1B1, CMPK2+CYP1B1+TRIB2, CMPK2+CYP1B1+uc003hr1.1, CMPK2+CYP1B1+USP41, CMPK2+CYP1B1+ZCCHC2, CMPK2+DDX60+DGAT2, CMPK2+DDX60+PARP12, CMPK2+DDX60+PNPT1, CMPK2+DDX60+PYGL, CMPK2+DDX60+SULT1B1, CMPK2+DDX60+TRIB2, CMPK2+DDX60+uc003hr1.1, CMPK2+DDX60+USP41, CMPK2+DDX60+ZCCHC2, CMPK2+DGAT2+PARP12, CMPK2+DGAT2+PNPT1, CMPK2+DGAT2+PYGL, CMPK2+DGAT2+SULT1B1, CMPK2+DGAT2+TRIB2, CMPK2+DGAT2+uc003hr1.1, CMPK2+DGAT2+USP41, CMPK2+DGAT2+ZCCHC2, CMPK2+PARP12+PNPT1, CMPK2+PARP12+PYGL, CMPK2+PARP12+SULT1B1, CMPK2+PARP12+TRIB2, CMPK2+PARP12+uc003hr1.1, CMPK2+PARP12+USP41, CMPK2+PARP12+ZCCHC2, CMPK2+PNPT1+PYGL, CMPK2+PNPT1+SULT1B1, CMPK2+PNPT1+TRIB2, CMPK2+PNPT1+uc003hr1.1, CMPK2+PNPT1+USP41, CMPK2+PNPT1+ZCCHC2, CMPK2+PYGL+SULT1B1, CMPK2+PYGL+TRIB2, CMPK2+PYGL+uc003hr1.1, CMPK2+PYGL+USP41, CMPK2+PYGL+ZCCHC2, CMPK2+SULT1B1+TRIB2, CMPK2+SULT1B1+uc003hr1.1, CMPK2+SULT1B1+USP41, CMPK2+SULT1B1+ZCCHC2, CMPK2+TRIB2+uc003hr1.1, CMPK2+TRIB2+USP41, CMPK2+TRIB2+ZCCHC2, CMPK2+uc003hr1.1+USP41, CMPK2+uc003hr1.1+ZCCHC2, CMPK2+USP41+ZCCHC2, CR1+CYP1B1+DDX60, CR1+CYP1B1+DGAT2, CR1+CYP1B1+PARP12, CR1+CYP1B1+PNPT1, CR1+CYP1B1+PYGL, CR1+CYP1B1+SULT1B1, CR1+CYP1B1+TRIB2, CR1+CYP1B1+uc003hr1.1, CR1+CYP1B1+USP41, CR1+CYP1B1+ZCCHC2, CR1+DDX60+DGAT2, CR1+DDX60+PARP12, CR1+DDX60+PNPT1, CR1+DDX60+PYGL, CR1+DDX60+SULT1B1, CR1+DDX60+TRIB2, CR1+DDX60+uc003hr1.1, CR1+DDX60+USP41, CR1+DDX60+ZCCHC2, CR1+DGAT2+PARP12, CR1+DGAT2+PNPT1, CR1+DGAT2+PYGL, CR1+DGAT2+SULT1B1, CR1+DGAT2+TRIB2, CR1+DGAT2+uc003hr1.1, CR1+DGAT2+USP41, CR1+DGAT2+ZCCHC2, CR1+PARP12+PNPT1, CR1+PARP12+PYGL, CR1+PARP12+SULT1B1, CR1+PARP12+TRIB2, CR1+PARP12+uc003hr1.1, CR1+PARP12+USP41, CR1+PARP12+ZCCHC2, CR1+PNPT1+PYGL, CR1+PNPT1+SULT1B1, CR1+PNPT1+TRIB2, CR1+PNPT1+uc003hr1.1, CR1+PNPT1+USP41, CR1+PNPT1+ZCCHC2, CR1+PYGL+SULT1B1, CR1+PYGL+TRIB2, CR1+PYGL+uc003hr1.1, CR1+PYGL+USP41, CR1+PYGL+ZCCHC2, CR1+SULT1B1+TRIB2, CR1+SULT1B1+uc003hr1.1, CR1+SULT1B1+USP41, CR1+SULT1B1+ZCCHC2, CR1+TRIB2+uc003hr1.1, CR1+TRIB2+USP41, CR1+TRIB2+ZCCHC2, CR1+uc003hr1.1+USP41, CR1+uc003hr1.1+ZCCHC2, CR1+USP41+ZCCHC2, CYP1B1+DDX60+DGAT2, CYP1B1+DDX60+PARP12, CYP1B1+DDX60+PNPT1, CYP1B1+DDX60+PYGL, CYP1B1+DDX60+SULT1B1, CYP1B1+DDX60+TRIB2, CYP1B1+DDX60+uc003hr1.1, CYP1B1+DDX60+USP41, CYP1B1+DDX60+ZCCHC2, CYP1B1+DGAT2+PARP12, CYP1B1+DGAT2+PNPT1, CYP1B1+DGAT2+PYGL, CYP1B1+DGAT2+SULT1B1, CYP1B1+DGAT2+TRIB2, CYP1B1+DGAT2+uc003hr1.1, CYP1B1+DGAT2+USP41, CYP1B1+DGAT2+ZCCHC2, CYP1B1+PARP12+PNPT1, CYP1B1+PARP12+PYGL, CYP1B1+PARP12+SULT1B1, CYP1B1+PARP12+TRIB2, CYP1B1+PARP12+uc003hr1.1, CYP1B1+PARP12+USP41, CYP1B1+PARP12+ZCCHC2, CYP1B1+PNPT1+PYGL, CYP1B1+PNPT1+SULT1B1, CYP1B1+PNPT1+TRIB2, CYP1B1+PNPT1+uc003hr1.1, CYP1B1+PNPT1+USP41, CYP1B1+PNPT1+ZCCHC2, CYP1B1+PYGL+SULT1B1, CYP1B1+PYGL+TRIB2, CYP1B1+PYGL+uc003hr1.1, CYP1B1+PYGL+USP41, CYP1B1+PYGL+ZCCHC2, CYP1B1+SULT1B1+TRIB2, CYP1B1+SULT1B1+uc003hr1.1, CYP1B1+SULT1B1+USP41, CYP1B1+SULT1B1+ZCCHC2, CYP1B1+TRIB2+uc003hr1.1, CYP1B1+TRIB2+USP41, CYP1B1+TRIB2+ZCCHC2, CYP1B1+uc003hr1.1+USP41, CYP1B1+uc003hr1.1+ZCCHC2, CYP1B1+USP41+ZCCHC2, DDX60+DGAT2+PARP12, DDX60+DGAT2+PNPT1, DDX60+DGAT2+PYGL, DDX60+DGAT2+SULT1B1, DDX60+DGAT2+TRIB2, DDX60+DGAT2+uc003hr1.1, DDX60+DGAT2+USP41, DDX60+DGAT2+ZCCHC2, DDX60+PARP12+PNPT1, DDX60+PARP12+PYGL, DDX60+PARP12+SULT1B1, DDX60+PARP12+TRIB2, DDX60+PARP12+uc003hr1.1, DDX60+PARP12+USP41, DDX60+PARP12+ZCCHC2, DDX60+PNPT1+PYGL, DDX60+PNPT1+SULT1B1, DDX60+PNPT1+TRIB2, DDX60+PNPT1+uc003hr1.1, DDX60+PNPT1+USP41, DDX60+PNPT1+ZCCHC2, DDX60+PYGL+SULT1B1, DDX60+PYGL+TRIB2, DDX60+PYGL+uc003hr1.1, DDX60+PYGL+USP41, DDX60+PYGL+ZCCHC2, DDX60+SULT1B1+TRIB2, DDX60+SULT1B1+uc003hr1.1, DDX60+SULT1B1+USP41, DDX60+SULT1B1+ZCCHC2, DDX60+TRIB2+uc003hr1.1, DDX60+TRIB2+USP41, DDX60+TRIB2+ZCCHC2, DDX60+uc003hr1.1+USP41, DDX60+uc003hr1.1+ZCCHC2, DDX60+USP41+ZCCHC2, DGAT2+PARP12+PNPT1, DGAT2+PARP12+PYGL, DGAT2+PARP12+SULT1B1, DGAT2+PARP12+TRIB2, DGAT2+PARP12+uc003hr1.1, DGAT2+PARP12+USP41, DGAT2+PARP12+ZCCHC2, DGAT2+PNPT1+PYGL, DGAT2+PNPT1+SULT1B1, DGAT2+PNPT1+TRIB2, DGAT2+PNPT1+uc003hr1.1, DGAT2+PNPT1+USP41, DGAT2+PNPT1+ZCCHC2, DGAT2+PYGL+SULT1B1, DGAT2+PYGL+TRIB2, DGAT2+PYGL+uc003hr1.1, DGAT2+PYGL+USP41, DGAT2+PYGL+ZCCHC2, DGAT2+SULT1B1+TRIB2, DGAT2+SULT1B1+uc003 hr1.1, DGAT2+SULT1B1+USP41, DGAT2+SULT1B1+ZCCHC2, DGAT2+TRIB2+uc003hr1.1, DGAT2+TRIB2+USP41, DGAT2+TRIB2+ZCCHC2, DGAT2+uc003hr1.1+USP41, DGAT2+uc003hr1.1+ZCCHC2, DGAT2+USP41+ZCCHC2, PARP12+PNPT1+PYGL, PARP12+PNPT1+SULT1B1, PARP12+PNPT1+TRIB2, PARP12+PNPT1+uc003 hr1.1, PARP12+PNPT1+USP41, PARP12+PNPT1+ZCCHC2, PARP12+PYGL+SULT1B1, PARP12+PYGL+TRIB2, PARP12+PYGL+uc003 hr1.1, PARP12+PYGL+USP41, PARP12+PYGL+ZCCHC2, PARP12+SULT1B1+TRIB2, PARP12+SULT1B1+uc003hr1.1, PARP12+SULT1B1+USP41, PARP12+SULT1B1+ZCCHC2, PARP12+TRIB2+uc003hr1.1, PARP12+TRIB2+USP41, PARP12+TRIB2+ZCCHC2, PARP12+uc003 hr1.1+USP41, PARP12+uc003 hr1.1+ZCCHC2, PARP12+USP41+ZCCHC2, PNPT1+PYGL+SULT1B1, PNPT1+PYGL+TRIB2, PNPT1+PYGL+uc003hr1.1, PNPT1+PYGL+USP41, PNPT1+PYGL+ZCCHC2, PNPT1+SULT1B1+TRIB2, PNPT1+SULT1B1+uc003hr1.1, PNPT1+SULT1B1+USP41, PNPT1+SULT1B1+ZCCHC2, PNPT1+TRIB2+uc003hr1.1, PNPT1+TRIB2+USP41, PNPT1+TRIB2+ZCCHC2, PNPT1+uc003hr1.1+USP41, PNPT1+uc003hr1.1+ZCCHC2, PNPT1+USP41+ZCCHC2, PYGL+SULT1B1+TRIB2, PYGL+SULT1B1+uc003hr1.1, PYGL+SULT1B1+USP41, PYGL+SULT1B1+ZCCHC2, PYGL+TRIB2+uc003hr1.1, PYGL+TRIB2+USP41, PYGL+TRIB2+ZCCHC2, PYGL+uc003hr1.1+USP41, PYGL+uc003hr1.1+ZCCHC2, PYGL+USP41+ZCCHC2, SULT1B1+TRIB2+uc003hr1.1, SULT1B1+TRIB2+USP41, SULT1B1+TRIB2+ZCCHC2, SULT1B1+uc003hr1.1+USP41, SULT1B1+uc003hr1.1+ZCCHC2, SULT1B1+USP41+ZCCHC2, TRIB2+uc003hr1.1+USP41, TRIB2+uc003hr1.1+ZCCHC2, TRIB2+USP41+ZCCHC2, uc003hr1.1+USP41+ZCCHC2
  • According to this aspect of the present invention, in order to distinguish between the different infection types, no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • TABLE 3
    Gene symbol Gene name
    CCL2 NC_000017.11 chemokine (C—C motif) ligand 2
    NC_018928.2
    NT_010783.16
    HERC5 NC_000004.12 HECT and RLD domain
    NT_016354.20 containing E3 ubiquitin protein
    NC_018915.2 ligase 5
    IFI44L NC_000001.11 interferon induced protein 44 like
    NT_032977.10
    NC_018912.2
    IFI6 NC_000001.11 interferon, alpha-inducible
    NC_018912.2 protein 6
    NT_032977.10
    IFIT1 NC_000010.11 interferon induced protein with
    NC_018921.2 tetratricopeptide repeats 1
    NT_030059.14
    ISG15 NC_000001.11 ISG15 ubiquitin-like modifier
    NC_018912.2
    NT_032977.10
    LAMP3 NC_000003.12 lysosomal associated membrane
    NT_005612.17 protein 3
    NC_018914.2
    LOC26010 NC_000002.12 spermatogenesis associated,
    (SPATS2L) NT_005403.18 serine rich 2 like
    NC_018913.2
    LY6E NC_000008.11 lymphocyte antigen 6 complex,
    NC_018919.2 locus E
    NT_008046.17
    NT_187573.1
    MX1 NC_000021.9 MX dynamin-like GTPase 1
    NT_011512.12
    NC_018932.2
    OAS3 NC_000012.12 2′-5′-oligoadenylate synthetase 3
    NT_029419.13
    NC_018923.2
    RTP4 NC_000003.12 receptor (chemosensory)
    NC_018914.2 transporter protein 4
    NT_005612.17
    SERPING1 NC_000011.10 serpin peptidase inhibitor, clade
    NC_018922.2 G (C1 inhibitor), member 1
    NT_167190.2
    SIGLEC1 NC_000020.11 sialic acid binding Ig like lectin 1
    NT_011387.9
    NC_018931.2
    TNFAIP6 NC_000002.12 TNF alpha induced protein 6
    NT_005403.18
    NC_018913.2
    USP18 NC_000022.11 ubiquitin specific peptidase 18
    NT_187355.1
    NC_018933.2
    XAF1 NC_000017.11 XIAP associated factor 1
    NT_010718.17
    NC_018928.2
    CXCL10 NC_000004.12 chemokine (C—X—C motif) ligand
    NC_018915.2 10
    NT_016354.20
    OAS1 NC_000012.12 2′-5′-Oligoadenylate Synthetase
    NT_029419.13 1, 40/46 kDa
    NC_018923.2
    DDX60 NC_000004.12 DEXD/H-box helicase 60
    NT_016354.20
    NC_018915.2
    HERC6 NC_000004.12 HECT and RLD domain
    NT_016354.20 containing E3 ubiquitin protein
    NC_018915.2 ligase family member 6
    PPM1K NC_000004.12 Protein Phosphatase,
    NC_018915.2 Mg2+/Mn2+ Dependent 1K
  • TABLE 4
    Gene symbol Gene Name
    APOBEC3C NC_000022.11 apolipoprotein B mRNA editing
    NC_018933.2 enzyme catalytic polypeptide
    NT_011520.13 like 3C
    BST2 NC_000019.10 bone marrow stromal cell
    NC_018930.2 antigen 2
    NT_011295.12
    C1orf29 NC_000001.11 interferon induced protein 44
    NT_032977.10 like
    NC_018912.2
    CD44 NC_000011.10 CD44 molecule (Indian blood
    NT_009237.19 group)
    NC_018922.2
    dJ507I15.1 NC_000023.11 ribosomal protein L36a
    NC_018934.2 pseudogene
    NT_011786.17
    DNAPTP6 NC_000002.12 spermatogenesis associated,
    NT_005403.18 serine rich 2 like
    NC_018913.2
    EEF1G NC_000011.10 eukaryotic translation
    NC_018922.2 elongation factor 1 gamma
    NT_167190.2
    EIF3S5 NC_000011.10 eukaryotic translation initiation
    NC_018922.2 factor 3 subunit F
    NT_009237.19
    EIF3S7 NC_000022.11 eukaryotic translation initiation
    NT_011520.13 factor 3 subunit D
    NC_018933.2
    EIF4B NC_000012.12 eukaryotic translation initiation
    NT_029419.13 factor 4B
    NC_018923.2
    G1P2 NC_000001.11 ISG15 ubiquitin-like modifier
    NC_018912.2
    NT_032977.10
    GPATCH11 NC_000002.12 GPATCH11;
    NC_018913.2 G-patch domain containing 11
    NT_022184.16
    HADHA NC_000002.12 hydroxyacyl-CoA
    NC_018913.2 dehydrogenase/3-ketoacyl-CoA
    NT_022184.16 thiolase/enoyl-CoA hydratase
    (trifunctional protein), alpha
    subunit
    IFI35 NC_000017.11 interferon induced protein 35
    NT_010783.16
    NC_018928.2
    MLEC NC_000012.12 MLEC; malectin
    NT_029419.13
    NC_018923.2
    PCBP2 NC_000012.12 poly(rC) binding protein 2
    NC_018923.2
    NT_029419.13
    PFDN5 NC_000012.12 prefoldin subunit 5
    NC_018923.2
    NT_029419.13
    PHACTR2 NC_000006.12 phosphatase and actin regulator
    NC_018917.2 2
    NT_025741.16
    QARS NC_000003.12 glutaminyl-tRNA synthetase
    NT_022517.19
    NC_018914.2
    RPL31 NC_000002.12 ribosomal protein L31
    NC_018913.2
    NT_005403.18
    RPL4 NC_000015.10 ribosomal protein L4
    NC_018926.2
    NT_010194.18
    SON NC_000021.9 SON DNA binding protein
    NC_018932.2
    NT_011512.12
    TRIM14 NC_000009.12 tripartite motif containing 14
    NT_008470.20
    NC_018920.2
    ZBP1 NC_000020.11 Z-DNA binding protein 1
    NT_011362.11
    NC_018931.2
    ADAR NC_000001.11 adenosine deaminase, RNA-
    NT_004487.20 specific
    NC_018912.2
    ATF3 NC_000001.11 activating transcription factor 3
    NT_004487.20
    NC_018912.2
    KIAA0226L NC_000013.11 KIAA0226L;
    NC_018924.2 KIAA0226-like
    NT_024524.15
    CTSL NC_000009.12 CTSL;
    NT_008470.20 cathepsin L
    NC_018920.2
    CUZD1 NC_000010.11 CUB and zona pellucida-like
    NC_018921.2 domains 1
    NT_030059.14
    DDX58 NC_000009.12 DEXD/H-box helicase 58
    NC_018920.2
    NT_008413.19
    DHX58 NC_000017.11 DEXH-box helicase 58
    NT_010783.16
    NC_018928.2
    ENOSF1 NC_000018.10 enolase superfamily member 1
    NT_010859.15
    NC_018929.2
    ETV7 NC_000006.12 ets variant 7
    NT_007592.16
    NC_018917.2
    FCGR1A NC_000001.11 Fc fragment of IgG receptor Ia
    NT_004487.20
    NC_018912.2
    FCGR1B NC_000001.11 Fc fragment of IgG receptor Ib
    NC_018912.2
    NT_032977.10
    GAPDH NC_000012.12 glyceraldehyde-3-phosphate
    NC_018923.2 dehydrogenase
    NT_009759.17
    GBP1 NC_000001.11 guanylate binding protein 1
    NT_032977.10
    NC_018912.2
    GM2A NC_000005.10 GM2 ganglioside activator
    NC_018916.2
    NT_029289.12
    HESX1 NC_000003.12 HESX homeobox 1
    NT_022517.19
    NC_018914.2
    HLA-DOB NC_000006.12 major histocompatibility
    NC_018917.2 complex, class II, DO beta
    NT_007592.16
    NT_113891.3
    NT_167244.2
    NT_167245.2
    NT_167247.2
    NT_167249.2
    IFIT5 NC_000010.11 interferon induced protein with
    NC_018921.2 tetratricopeptide repeats 5
    NT_030059.14
    IL16 NC_000015.10 interleukin 16
    NT_010194.18
    NC_018926.2
    IDO1 NC_000008.11 IDO1;
    NC_018919.2 indoleamine 2,3-dioxygenase 1
    NT_167187.2
    LAP3 NC_000004.12 leucine aminopeptidase 3
    NC_018915.2
    NT_006316.17
    LILRB2 NC_000019.10 leukocyte immunoglobulin like
    NT_011109.17 receptor B2
    NT_187693.1
    NC_018930.2
    LOC727996 NC_000022.10 hypothetical LOC727996
    MS4A4A NC_000011.10 membrane spanning 4-domains
    NC_018922.2 A4A
    NT_167190.2
    NDUFA10 NC_000002.12 NADH:ubiquinone
    NT_005403.18 oxidoreductase subunit A10
    NC_018913.2
    NLRP3 NC_000001.11 NLR family, pyrin domain
    NT_167186.2 containing 3
    NC_018912.2
    NOD2 NC_000016.10 nucleotide binding
    NT_010498.16 oligomerization domain
    NC_018927.2 containing 2
    PML NC_000015.10 promyelocytic leukemia
    NC_018926.2
    NT_010194.18
    PRSS21 NC_000016.10 protease, serine 21
    NC_018927.2
    NT_010393.17
    SEPT4 NC_000017.11 septin 4
    NT_010783.16
    NC_018928.2
    SOCS1 NC_000016.10 suppressor of cytokine signaling
    NC_018927.2 1
    NT_010393.17
    SOCS2 NC_000012.12 suppressor of cytokine signaling
    NT_029419.13 2
    NC_018923.2
    STAT1 NC_000002.12 signal transducer and activator
    NT_005403.18 of transcription 1
    NC_018913.2
    XAF1 NC_000017.11 XIAP associated factor 1
    NT_010718.17
    NC_018928.2
  • TABLE 5
    Gene symbol Gene name
    RSAD2 NC_000002.12 radical S-adenosyl methionine
    NT_005334.17 domain containing 2
    NC_018913.2
    OAS2 NC_000012.12 2′-5′-oligoadenylate synthetase 2
    NT_029419.13
    NC_018923.2
    OASL NC_000012.12 2′-5′-oligoadenylate synthetase-
    NC_018923.2 like
    NT_029419.13
    IFI27 NC_018925.2 interferon, alpha-inducible
    NT_187601.1 protein 27
    NC_000014.9
    NT_026437.13
    IFI44 NC_000001.11 interferon induced protein 44
    NT_032977.10
    NC_018912.2
    IFIT2 NC_000010.11 interferon induced protein with
    NC_018921.2 tetratricopeptide repeats 2
    NT_030059.14
    IFIT3 NC_000010.11 interferon induced protein with
    NC_018921.2 tetratricopeptide repeats 3
    NT_030059.14
  • TABLE 6A
    Gene symbol Gene name
    BTN3A3 NC_000006.12 butyrophilin subfamily 3 member
    NC_018917.2 A3
    NT_007592.16
    RNF213 NC_000017.11 RNF213;
    NT_010783.16 ring finger protein 213
    NC_018928.2
    PARP9 NC_000003.12 poly(ADP-ribose) polymerase
    NT_005612.17 family member 9
    NC_018914.2
  • TABLE 6B
    Gene
    Name/mRNA
    Probe ID accession number
    TC01004260.hg n363820
    TC02002080.hg ENST00000443397
    TC02004398.hg n336681
    TC16001577.hg n375375
    TC16001578.hg n406211
    TC22000951.hg n384079
    TC22001004.hg n387236
    TC22001243.hg n332472
    TC22001248.hg n346241
    TC17000386.hg CCL8
    TC09001536.hg CDK5RAP2
    TC04002928.hg FAM200B
    TC09000608.hg GSN
    TC02004983.hg IGKV3D15
    TC02000720.hg IL1RN
    TC17002906.hg KRT19
    TC07000704.hg LRRN3
    TC22001450.hg MIR650
    TC16002035.hg MT1A
    TC16000470.hg MT1DP
    TC16000468.hg MT1E
    TC16000475.hg MT1IP
    TC16002074.hg MT1M
    TC06000961.hg NCOA7
    TC01000789.hg NEXN
    TC22000376.hg PRR5
    TC01001522.hg RABGAP1L
    TC09001730.hg SDCCAG3
    TC03000198.hg TTC21A
  • According to another aspect of the present invention, there is provided a method of determining an infection type in a subject comprising measuring at least two RNAs in a sample derived from the subject, wherein pairs of the at least two RNAs are set forth in Tables 9, 11, 12, 18 or 19, wherein the amount of the at least two RNAs is indicative of the infection type.
  • According to this aspect of the present invention, in order to distinguish between the different infection types, no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • According to yet another aspect of the present invention, there is provided a method of determining an infection type in a subject comprising measuring at least three RNAs in a sample derived from the subject, wherein triplets of the at least three RNAs are set forth in Tables 13, 14, 19 or 20 wherein amount of the at least three RNAs is indicative of the infection type.
  • According to this aspect of the present invention, in order to distinguish between the different infection types, no more than 30 determinants are measured, no more than 25 determinants are measured, no more than 20 determinants are measured, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, or even no more than 3 determinants are measured.
  • According to yet another aspect of the present invention, there is provided a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 3 or Table 4, in a sample derived from the subject, wherein no more than 20 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • In one embodiment, no more than 15 determinants are measured, no more than 10 determinants are measured, no more than 5 determinants are measured, no more than 4 determinants are measured, no more than 3 determinants are measured or even no more than 2 determinants are measured.
  • According to this aspect of the present invention, preferably the at least one RNA is set forth in Table 3. When the level of RNA set forth in Table 3 is above a predetermined level, it is indicative of a viral infection.
  • When additional determinants are measured according to this aspect, preferably the additional determinant is set forth in any one of Tables 1-6A or Tables 15A-B.
  • Other determinants that may be measured according to aspects of the present invention include pathogen (bacterial or viral) specific RNA determinants. For example, RNA determinants that can distinguish between influenza and parainfluenza are described in Table 25 of the Examples section, herein below. This may be carried out in order to aid in identification of a specific pathogen. The measurements may be effected simultaneously with the above described measurements or consecutively. The measurement may be performed on the biological sample used to determine the patient immune response (as described herein above) or on a different biological patient-derived sample (e.g., blood sample; serum sample; saliva; nasopharyngeal sample; etc.). In one embodiment, the host immune RNA determinants are measured in a patient derived serum sample and analysis of the pathogen specific RNA determinants is performed on a nasopharyngeal sample.
  • According to a particular embodiment, the additional RNA is set forth in Table 1 (e.g. OTOF, PI3, EIF2AK2 and CMPK2), Table 2 (e.g. CYBRD1), Table 3, Table 5 or Tables 16A-C (protein-coding RNAs such as CR1, CYP1B1, DDX60, DGAT2,
  • PARP12, PNPT1, PYGL, SULT1B1, TRIB2, USP41, ZCCHC2 or uc003hr1.1; non-protein coding RNAs such as TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_00072, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 and TCONS_12_00002367-XLOC_12_000720).
  • According to still another aspect of the present invention, there is provided a method of determining an infection type in a subject comprising measuring the amount of at least one RNA as set forth in Table 5 or Table 6A, in a sample derived from the subject, wherein no more than 5 RNAs are measured, wherein the amount of at least one RNA is indicative of the infection type.
  • According to a particular embodiment of this aspect of the present invention, the at least one RNA is set forth in Table 5.
  • When the RNA of Table 5 is above a predetermined level, the infection may be classified as a viral infection.
  • According to this aspect additional RNAs may be measured so as to measure at least two RNAs, wherein the at least one additional RNA is set forth in any one of Tables 1-6, wherein the amount of the at least two RNAs is indicative of the infection type.
  • Preferably, the at least one additional RNA is set forth in Table 1 (e.g., PI3, EIF2AK2 and CMPK2), Table 2 (e.g. CYBRD1), Table 3,Table 5 or Tables 16A-C(e.g. coding RNAs such as CR1, CYP1B1, DDX60, DGAT2, PARP12, PNPT1, PYGL, SULT1B1, TRIB2, USP41, ZCCHC2, uc003hr1.1; or non-coding RNAs such as TCONS_00003184-XLOC_001966, TCONS_00013664-XLOC_006324, TCONS_12_00028242-XLOC_12_014551, TCONS_12_00001926-XLOC_12_000004, TCONS_12_00002386-XLOC_12_000726, TCONS_12_00002811-XLOC_12_001398, TCONS_12_00003949-XLOC_12_001561, TCONS_00019559-XLOC_009354, TCONS_12_00010440-XLOC_12_005352, TCONS_12_00016828-XLOC_12_008724, TCONS_12_00021682-XLOC_12_010810 and TCONS_12_00002367-XLOC_12_000720.
  • Methods of analyzing the amount of RNA are known in the art and are summarized infra:
  • Northern Blot analysis: This method involves the detection of a particular RNA in a mixture of RNAs. An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere. The membrane is then exposed to labeled DNA probes. Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
  • RT-PCR analysis: This method uses PCR amplification of relatively rare RNAs molecules. First, RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine. Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.
  • RNA in situ hybridization stain: In this method DNA or RNA probes are attached to the RNA molecules present in the cells. Generally, the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe. The hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe. Those of skills in the art are capable of adjusting the hybridization conditions (i.e., temperature, concentration of salts and formamide and the like) to specific probes and types of cells. Following hybridization, any unbound probe is washed off and the bound probe is detected using known methods. For example, if a radio-labeled probe is used, then the slide is subjected to a photographic emulsion which reveals signals generated using radio-labeled probes; if the probe was labeled with an enzyme then the enzyme-specific substrate is added for the formation of a colorimetric reaction; if the probe is labeled using a fluorescent label, then the bound probe is revealed using a fluorescent microscope; if the probe is labeled using a tag (e.g., digoxigenin, biotin, and the like) then the bound probe can be detected following interaction with a tag-specific antibody which can be detected using known methods.
  • In situ RT-PCR stain: This method is described in Nuovo G J, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, Calif.).
  • DNA Microarrays/DNA Chips:
  • The expression of thousands of genes may be analyzed simultaneously using DNA microarrays, allowing analysis of the complete transcriptional program of an organism during specific developmental processes or physiological responses. DNA microarrays consist of thousands of individual gene sequences attached to closely packed areas on the surface of a support such as a glass microscope slide. Various methods have been developed for preparing DNA microarrays. In one method, an approximately 1 kilobase segment of the coding region of each gene for analysis is individually PCR amplified. A robotic apparatus is employed to apply each amplified DNA sample to closely spaced zones on the surface of a glass microscope slide, which is subsequently processed by thermal and chemical treatment to bind the DNA sequences to the surface of the support and denature them. Typically, such arrays are about 2×2 cm and contain about individual nucleic acids 6000 spots. In a variant of the technique, multiple DNA oligonucleotides, usually 20 nucleotides in length, are synthesized from an initial nucleotide that is covalently bound to the surface of a support, such that tens of thousands of identical oligonucleotides are synthesized in a small square zone on the surface of the support. Multiple oligonucleotide sequences from a single gene are synthesized in neighboring regions of the slide for analysis of expression of that gene. Hence, thousands of genes can be represented on one glass slide. Such arrays of synthetic oligonucleotides may be referred to in the art as “DNA chips”, as opposed to “DNA microarrays”, as described above [Lodish et al. (eds.). Chapter 7.8: DNA Microarrays: Analyzing Genome-Wide Expression. In: Molecular Cell Biology, 4th ed., W. H. Freeman, New York. (2000)].
  • Oligonucleotide microarray—In this method oligonucleotide probes capable of specifically hybridizing with the polynucleotides of some embodiments of the invention are attached to a solid surface (e.g., a glass wafer). Each oligonucleotide probe is of approximately 20-25 nucleic acids in length. To detect the expression pattern of the polynucleotides of some embodiments of the invention in a specific cell sample (e.g., blood cells), RNA is extracted from the cell sample using methods known in the art (using e.g., a TRIZOL solution, Gibco BRL, USA). Hybridization can take place using either labeled oligonucleotide probes (e.g., 5′-biotinylated probes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA). Briefly, double stranded cDNA is prepared from the RNA using reverse transcriptase (RT) (e.g., Superscript II RT), DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, Md., USA). To prepare labeled cRNA, the double stranded cDNA is subjected to an in vitro transcription reaction in the presence of biotinylated nucleotides using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, Affymetrix Santa Clara Calif.). For efficient hybridization the labeled cRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate for 35 minutes at 94° C. Following hybridization, the microarray is washed and the hybridization signal is scanned using a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • For example, in the Affymetrix microarray (Affymetrix®, Santa Clara, Calif.) each gene on the array is represented by a series of different oligonucleotide probes, of which, each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. While the perfect match probe has a sequence exactly complimentary to the particular gene, thus enabling the measurement of the level of expression of the particular gene, the mismatch probe differs from the perfect match probe by a single base substitution at the center base position. The hybridization signal is scanned using the Agilent scanner, and the Microarray Suite software subtracts the non-specific signal resulting from the mismatch probe from the signal resulting from the perfect match probe.
  • RNA sequencing: Methods for RNA sequence determination are generally known to the person skilled in the art. Preferred sequencing methods are next generation sequencing methods or parallel high throughput sequencing methods. An example of an envisaged sequence method is pyrosequencing, in particular 454 pyrosequencing, e.g. based on the Roche 454 Genome Sequencer. This method amplifies DNA inside water droplets in an oil solution with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. Yet another envisaged example is Illumina or Solexa sequencing, e.g. by using the Illumina Genome
  • Analyzer technology, which is based on reversible dye-terminators. DNA molecules are typically attached to primers on a slide and amplified so that local clonal colonies are formed. Subsequently one type of nucleotide at a time may be added, and non-incorporated nucleotides are washed away. Subsequently, images of the fluorescently labeled nucleotides may be taken and the dye is chemically removed from the DNA, allowing a next cycle. Yet another example is the use of Applied Biosystems' SOLiD technology, which employs sequencing by ligation. This method is based on the use of a pool of all possible oligonucleotides of a fixed length, which are labeled according to the sequenced position. Such oligonucleotides are annealed and ligated. Subsequently, the preferential ligation by DNA ligase for matching sequences typically results in a signal informative of the nucleotide at that position. Since the DNA is typically amplified by emulsion PCR, the resulting bead, each containing only copies of the same DNA molecule, can be deposited on a glass slide resulting in sequences of quantities and lengths comparable to Illumina sequencing. A further method is based on Helicos' Heliscope technology, wherein fragments are captured by polyT oligomers tethered to an array. At each sequencing cycle, polymerase and single fluorescently labeled nucleotides are added and the array is imaged. The fluorescent tag is subsequently removed and the cycle is repeated. Further examples of sequencing techniques encompassed within the methods of the present invention are sequencing by hybridization, sequencing by use of nanopores, microscopy-based sequencing techniques, microfluidic Sanger sequencing, or microchip-based sequencing methods. The present invention also envisages further developments of these techniques, e.g. further improvements of the accuracy of the sequence determination, or the time needed for the determination of the genomic sequence of an organism etc.
  • According to one embodiment, the sequencing method comprises deep sequencing.
  • As used herein, the term “deep sequencing” refers to a sequencing method wherein the target sequence is read multiple times in the single test. A single deep sequencing run is composed of a multitude of sequencing reactions run on the same target sequence and each, generating independent sequence readout.
  • It will be appreciated that in order to analyze the amount of an RNA, oligonucleotides may be used that are capable of hybridizing thereto or to cDNA generated therefrom. According to one embodiment a single oligonucleotide is used to determine the presence of a particular determinant, at least two oligonucleotides are used to determine the presence of a particular determinant, at least five oligonucleotides are used to determine the presence of a particular determinant, at least four oligonucleotides are used to determine the presence of a particular determinant, at least five or more oligonucleotides are used to determine the presence of a particular determinant.
  • When more than one oligonucleotide is used, the sequence of the oligonucleotides may be selected such that they hybridize to the same exon of the RNA determinant or different exons of the RNA determinant. In one embodiment, at least one of the oligonucleotides hybridizes to the 3′ exon of the RNA determinant. In another embodiment, at least one of the oligonucleotides hybridizes to the 5′ exon of the RNA determinant.
  • In one embodiment, the method of this aspect of the present invention is carried out using an isolated oligonucleotide which hybridizes to the RNA or cDNA of any of the determinants listed in Tables 1-6 by complementary base-pairing in a sequence specific manner, and discriminates the determinant sequence from other nucleic acid sequence in the sample. Oligonucleotides (e.g. DNA or RNA oligonucleotides) typically comprises a region of complementary nucleotide sequence that hybridizes under stringent conditions to at least about 8, 10, 13, 16, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule. Depending on the particular assay, the consecutive nucleotides include the determinant nucleic acid sequence.
  • The term “isolated”, as used herein in reference to an oligonucleotide, means an oligonucleotide, which by virtue of its origin or manipulation, is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained. By “isolated”, it is alternatively or additionally meant that the oligonucleotide of interest is produced or synthesized by the hand of man.
  • In order to identify an oligonucleotide specific for any of the determinant sequences, the gene/transcript of interest is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligonucleotides of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, lack predicted secondary structure that may interfere with hybridization, and/or possess other desired characteristics or that lack other undesired characteristics.
  • Following identification of the oligonucleotide it may be tested for specificity towards the determinant under wet or dry conditions. Thus, for example, in the case where the oligonucleotide is a primer, the primer may be tested for its ability to amplify a sequence of the determinant using PCR to generate a detectable product and for its non ability to amplify other determinants in the sample. The products of the PCR reaction may be analyzed on a gel and verified according to presence and/or size.
  • Additionally, or alternatively, the sequence of the oligonucleotide may be analyzed by computer analysis to see if it is homologous (or is capable of hybridizing to) other known sequences. A BLAST 2.2.10 (Basic Local Alignment Search Tool) analysis may be performed on the chosen oligonucleotide (worldwidewebdotncbidotnlmdotnihdotgov/blast/). The BLAST program finds regions of local similarity between sequences. It compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches thereby providing valuable information about the possible identity and integrity of the ‘query’ sequences.
  • According to one embodiment, the oligonucleotide is a probe. As used herein, the term “probe” refers to an oligonucleotide which hybridizes to the determinant specific nucleic acid sequence to provide a detectable signal under experimental conditions and which does not hybridize to additional determinant sequences to provide a detectable signal under identical experimental conditions.
  • The probes of this embodiment of this aspect of the present invention may be, for example, affixed to a solid support (e.g., arrays or beads).
  • Solid supports are solid-state substrates or supports onto which the nucleic acid molecules of the present invention may be associated. The nucleic acids may be associated directly or indirectly. Solid-state substrates for use in solid supports can include any solid material with which components can be associated, directly or indirectly. This includes materials such as acrylamide, agarose, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can have any useful form including thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or a combination. Solid-state substrates and solid supports can be porous or non-porous. A chip is a rectangular or square small piece of material. Preferred forms for solid-state substrates are thin films, beads, or chips. A useful form for a solid-state substrate is a microtiter dish. In some embodiments, a multiwell glass slide can be employed.
  • In one embodiment, the solid support is an array which comprises a plurality of nucleic acids of the present invention immobilized at identified or predefined locations on the solid support. Each predefined location on the solid support generally has one type of component (that is, all the components at that location are the same). Alternatively, multiple types of components can be immobilized in the same predefined location on a solid support. Each location will have multiple copies of the given components. The spatial separation of different components on the solid support allows separate detection and identification.
  • According to particular embodiments, the array does not comprise nucleic acids that specifically bind to more than 50 determinants, more than 40 determinants, 30 determinants, 20 determinants, 15 determinants, 10 determinants, 5 determinants or even 3 determinants.
  • Methods for immobilization of oligonucleotides to solid-state substrates are well established. Oligonucleotides, including address probes and detection probes, can be coupled to substrates using established coupling methods. For example, suitable attachment methods are described by Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and Khrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method for immobilization of 3′-amine oligonucleotides on casein-coated slides is described by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383 (1995). A useful method of attaching oligonucleotides to solid-state substrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465 (1994).
  • According to another embodiment, the oligonucleotide is a primer of a primer pair. As used herein, the term “primer” refers to an oligonucleotide which acts as a point of initiation of a template-directed synthesis using methods such as PCR (polymerase chain reaction) or LCR (ligase chain reaction) under appropriate conditions (e.g., in the presence of four different nucleotide triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse-transcriptase, DNA ligase, etc, in an appropriate buffer solution containing any necessary co-factors and at suitable temperature(s)). Such a template directed synthesis is also called “primer extension”. For example, a primer pair may be designed to amplify a region of DNA using PCR. Such a pair will include a “forward primer” and a “reverse primer” that hybridize to complementary strands of a DNA molecule and that delimit a region to be synthesized/amplified. A primer of this aspect of the present invention is capable of amplifying, together with its pair (e.g. by PCR) a determinant specific nucleic acid sequence to provide a detectable signal under experimental conditions and which does not amplify other determinant nucleic acid sequence to provide a detectable signal under identical experimental conditions.
  • According to additional embodiments, the oligonucleotide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. While the maximal length of a probe can be as long as the target sequence to be detected, depending on the type of assay in which it is employed, it is typically less than about 50, 60, 65, or 70 nucleotides in length. In the case of a primer, it is typically less than about 30 nucleotides in length. In a specific preferred embodiment of the invention, a primer or a probe is within the length of about 18 and about 28 nucleotides. It will be appreciated that when attached to a solid support, the probe may be of about 30-70, 75, 80, 90, 100, or more nucleotides in length.
  • The oligonucleotide of this aspect of the present invention need not reflect the exact sequence of the determinant nucleic acid sequence (i.e. need not be fully complementary), but must be sufficiently complementary to hybridize with the determinant nucleic acid sequence under the particular experimental conditions. Accordingly, the sequence of the oligonucleotide typically has at least 70% homology, preferably at least 80%, 90%, 95%, 97%, 99% or 100% homology, for example over a region of at least 13 or more contiguous nucleotides with the target determinant nucleic acid sequence. The conditions are selected such that hybridization of the oligonucleotide to the determinant nucleic acid sequence is favored and hybridization to other determinant nucleic acid sequences is minimized.
  • By way of example, hybridization of short nucleic acids (below 200 bp in length, e.g. 13-50 bp in length) can be effected by the following hybridization protocols depending on the desired stringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm (stringent hybridization conditions) (ii) hybridization solution of 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final wash solution of 6×SSC, and final wash at 22° C. (stringent to moderate hybridization conditions); and (iii) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature at 2.5-3° C. below the Tm and final wash solution of 6×SSC at 22° C. (moderate hybridization solution).
  • Oligonucleotides of the invention may be prepared by any of a variety of methods (see, for example, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, M. A. Innis (Ed.), Academic Press: New York, N.Y.; P. Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, M. A. Innis (Ed.), Academic Press: New York, N.Y.; and “Short Protocols in Molecular Biology”, 2002, F. M. Ausubel (Ed.), 5.sup.th Ed., John Wiley & Sons: Secaucus, N.J.). For example, oligonucleotides may be prepared using any of a variety of chemical techniques well-known in the art, including, for example, chemical synthesis and polymerization based on a template as described, for example, in S. A. Narang et al., Meth. Enzymol. 1979, 68: 90-98; E. L. Brown et al., Meth. Enzymol. 1979, 68: 109-151; E. S. Belousov et al., Nucleic Acids Res. 1997, 25: 3440-3444; D. Guschin et al., Anal. Biochem. 1997, 250: 203-211; M. J. Blommers et al., Biochemistry, 1994, 33: 7886-7896; and K. Frenkel et al., Free Radic. Biol. Med. 1995, 19: 373-380; and U.S. Pat. No. 4,458,066.
  • For example, oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach. In such a method, each nucleotide is individually added to the 5′-end of the growing oligonucleotide chain, which is attached at the 3′-end to a solid support. The added nucleotides are in the form of trivalent 3′-phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5′-position. After base-induced phosphoramidite coupling, mild oxidation to give a pentavalent phosphotriester intermediate and DMT removal provides a new site for oligonucleotide elongation. The oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide. These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively, oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc. (Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.
  • Purification of the oligonucleotides of the invention, where necessary or desirable, may be carried out by any of a variety of methods well-known in the art. Purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC as described, for example, by J. D. Pearson and F. E. Regnier (J. Chrom., 1983, 255: 137-149) or by reverse phase HPLC (G. D. McFarland and P. N. Borer, Nucleic Acids Res., 1979, 7: 1067-1080).
  • The sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation (A. M. Maxam and W. Gilbert, Methods of Enzymology, 1980, 65: 499-560), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (U. Pieles et al., Nucleic Acids Res., 1993, 21: 3191-3196), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (H. Wu and H. Aboleneen, Anal. Biochem., 2001, 290: 347-352), and the like.
  • As already mentioned above, modified oligonucleotides may be prepared using any of several means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc). Oligonucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc), intercalators (e.g., acridine, psoralen, etc), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc), and alkylators. The oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the oligonucleotide sequences of the present invention may also be modified with a label.
  • In certain embodiments, the detection probes or amplification primers or both probes and primers are labeled with a detectable agent or moiety before being used in amplification/detection assays. In certain embodiments, the detection probes are labeled with a detectable agent. Preferably, a detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.
  • The association between the oligonucleotide and detectable agent can be covalent or non-covalent. Labeled detection probes can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g., through a linker). Linkers or spacer arms of various lengths are known in the art and are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules (see, for example, E. S. Mansfield et al., Mol. Cell. Probes, 1995, 9: 145-156).
  • Methods for labeling nucleic acid molecules are well-known in the art. For a review of labeling protocols, label detection techniques, and recent developments in the field, see, for example, L. J. Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153. Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (L. M. Smith et al., Nucl. Acids Res., 1985, 13: 2399-2412) or of enzymes (B. A. Connoly and O. Rider, Nucl. Acids. Res., 1985, 13: 4485-4502); chemical modifications of nucleic acid molecules making them detectable immunochemically or by other affinity reactions (T. R. Broker et al., Nucl. Acids Res. 1978, 5: 363-384; E. A. Bayer et al., Methods of Biochem. Analysis, 1980, 26: 1-45; R. Langer et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 6633-6637; R. W. Richardson et al., Nucl. Acids Res. 1983, 11: 6167-6184; D. J. Brigati et al., Virol. 1983, 126: 32-50; P. Tchen et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 3466-3470; J. E. Landegent et al., Exp. Cell Res. 1984, 15: 61-72; and A. H. Hopman et al., Exp. Cell Res. 1987, 169: 357-368); and enzyme-mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase (for a review on enzymatic labeling, see, for example, J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232). More recently developed nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of mono-reactive cisplatin derivatives with the N7 position of guanine moieties in DNA (R. J. Heetebrij et al., Cytogenet. Cell. Genet. 1999, 87: 47-52), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (C. Levenson et al., Methods Enzymol. 1990, 184: 577-583; and C. Pfannschmidt et al., Nucleic Acids Res. 1996, 24: 1702-1709), photoreactive azido derivatives (C. Neves et al., Bioconjugate Chem. 2000, 11: 51-55), and DNA alkylating agents (M. G. Sebestyen et al., Nat. Biotechnol. 1998, 16: 568-576).
  • Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to, various ligands, radionuclides (such as, for example, 32P, 35S, 3H 14C, 125I, 131I, and the like); fluorescent dyes (for specific exemplary fluorescent dyes, see below); chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper and platinum) or nanoclusters; enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (such as, for example, dyes, colloidal gold, and the like); magnetic labels (such as, for example, Dynabeads™); and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
  • In certain embodiments, the inventive detection probes are fluorescently labeled. Numerous known fluorescent labeling moieties of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of this invention. Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6 carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800), and the like. For more examples of suitable fluorescent dyes and methods for linking or incorporating fluorescent dyes to nucleic acid molecules see, for example, “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg. Fluorescent dyes as well as labeling kits are commercially available from, for example, Amersham Biosciences, Inc. (Piscataway, N.J.), Molecular Probes Inc. (Eugene, Oreg.), and New England Biolabs Inc. (Berverly, Mass.).
  • As mentioned, identification of the determinant may be carried out using an amplification reaction.
  • As used herein, the term “amplification” refers to a process that increases the representation of a population of specific nucleic acid sequences in a sample by producing multiple (i.e., at least 2) copies of the desired sequences. Methods for nucleic acid amplification are known in the art and include, but are not limited to, polymerase chain reaction (PCR) and ligase chain reaction (LCR). In a typical PCR amplification reaction, a nucleic acid sequence of interest is often amplified at least fifty thousand fold in amount over its amount in the starting sample. A “copy” or “amplicon” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.
  • A typical amplification reaction is carried out by contacting a forward and reverse primer (a primer pair) to the sample DNA together with any additional amplification reaction reagents under conditions which allow amplification of the target sequence.
  • The terms “forward primer” and “forward amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the target (template strand). The terms “reverse primer” and “reverse amplification primer” are used herein interchangeably, and refer to a primer that hybridizes (or anneals) to the complementary target strand. The forward primer hybridizes with the target sequence 5′ with respect to the reverse primer.
  • The term “amplification conditions”, as used herein, refers to conditions that promote annealing and/or extension of primer sequences. Such conditions are well-known in the art and depend on the amplification method selected. Thus, for example, in a PCR reaction, amplification conditions generally comprise thermal cycling, i.e., cycling of the reaction mixture between two or more temperatures. In isothermal amplification reactions, amplification occurs without thermal cycling although an initial temperature increase may be required to initiate the reaction. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and temperature cycling, buffer, salt, ionic strength, and pH, and the like.
  • As used herein, the term “amplification reaction reagents”, refers to reagents used in nucleic acid amplification reactions and may include, but are not limited to, buffers, reagents, enzymes having reverse transcriptase and/or polymerase activity or exonuclease activity, enzyme cofactors such as magnesium or manganese, salts, nicotinamide adenine dinuclease (NAD) and deoxynucleoside triphosphates (dNTPs), such as deoxyadenosine triphospate, deoxyguano sine triphosphate, deoxycytidine triphosphate and thymidine triphosphate. Amplification reaction reagents may readily be selected by one skilled in the art depending on the amplification method used.
  • According to this aspect of the present invention, the amplifying may be effected using techniques such as polymerase chain reaction (PCR), which includes, but is not limited to Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence-specific PCR (ISSR), Inverse PCR, Ligation-mediated PCR, Methylation-specific PCR (MSP), Miniprimer PCR, Multiplex Ligation-dependent Probe Amplification, Multiplex-PCR, Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR), Reverse Transcription PCR (RT-PCR), Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal ‘step’ to favour solid support priming), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR (Step-down PCR), PAN-AC and Universal Fast Walking.
  • The PCR (or polymerase chain reaction) technique is well-known in the art and has been disclosed, for example, in K. B. Mullis and F. A. Faloona, Methods Enzymol., 1987, 155: 350-355 and U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,800,159 (each of which is incorporated herein by reference in its entirety). In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A plurality of reaction cycles, each cycle comprising: a denaturation step, an annealing step, and a polymerization step, results in the exponential accumulation of a specific DNA fragment (“PCR Protocols: A Guide to Methods and Applications”, M. A. Innis (Ed.), 1990, Academic Press: New York; “PCR Strategies”, M. A. Innis (Ed.), 1995, Academic Press: New York; “Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach”, McPherson et al. (Eds.), 1991, IRL Press: Oxford; R. K. Saiki et al., Nature, 1986, 324: 163-166). The termini of the amplified fragments are defined as the 5′ ends of the primers. Examples of DNA polymerases capable of producing amplification products in PCR reactions include, but are not limited to: E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcus litoralis (“Vent” polymerase, New England Biolabs). RNA target sequences may be amplified by reverse transcribing the mRNA into cDNA, and then performing PCR (RT-PCR), as described above. Alternatively, a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770.
  • The duration and temperature of each step of a PCR cycle, as well as the number of cycles, are generally adjusted according to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated. The ability to optimize the reaction cycle conditions is well within the knowledge of one of ordinary skill in the art. Although the number of reaction cycles may vary depending on the detection analysis being performed, it usually is at least 15, more usually at least 20, and may be as high as 60 or higher. However, in many situations, the number of reaction cycles typically ranges from about 20 to about 40.
  • The denaturation step of a PCR cycle generally comprises heating the reaction mixture to an elevated temperature and maintaining the mixture at the elevated temperature for a period of time sufficient for any double-stranded or hybridized nucleic acid present in the reaction mixture to dissociate. For denaturation, the temperature of the reaction mixture is usually raised to, and maintained at, a temperature ranging from about 85° C. to about 100° C., usually from about 90° C. to about 98° C., and more usually from about 93° C. to about 96° C. for a period of time ranging from about 3 to about 120 seconds, usually from about 5 to about 30 seconds.
  • Following denaturation, the reaction mixture is subjected to conditions sufficient for primer annealing to template DNA present in the mixture. The temperature to which the reaction mixture is lowered to achieve these conditions is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 50° C. to about ° C., usually from about 55° C. to about 70° C., and more usually from about 60° C. to about 68° C. Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 5 minutes.
  • Following annealing of primer to template DNA or during annealing of primer to template DNA, the reaction mixture is subjected to conditions sufficient to provide for polymerization of nucleotides to the primer's end in a such manner that the primer is extended in a 5′ to 3′ direction using the DNA to which it is hybridized as a template, (i.e., conditions sufficient for enzymatic production of primer extension product). To achieve primer extension conditions, the temperature of the reaction mixture is typically raised to a temperature ranging from about 65° C. to about 75° C., usually from about 67° C. to about 73° C., and maintained at that temperature for a period of time ranging from about 15 seconds to about 20 minutes, usually from about 30 seconds to about 5 minutes.
  • The above cycles of denaturation, annealing, and polymerization may be performed using an automated device typically known as a thermal cycler or thermocycler. Thermal cyclers that may be employed are described in U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610 (each of which is incorporated herein by reference in its entirety). Thermal cyclers are commercially available, for example, from Perkin Elmer-Applied Biosystems (Norwalk, Conn.), BioRad (Hercules, Calif.), Roche Applied Science (Indianapolis, Ind.), and Stratagene (La Jolla, Calif.).
  • Amplification products obtained using primers of the present invention may be detected using agarose gel electrophoresis and visualization by ethidium bromide staining and exposure to ultraviolet (UV) light or by sequence analysis of the amplification product.
  • According to one embodiment, the amplification and quantification of the amplification product may be effected in real-time (qRT-PCR). Typically, QRT-PCR methods use double stranded DNA detecting molecules to measure the amount of amplified product in real time.
  • As used herein the phrase “double stranded DNA detecting molecule” refers to a double stranded DNA interacting molecule that produces a quantifiable signal (e.g., fluorescent signal). For example such a double stranded DNA detecting molecule can be a fluorescent dye that (1) interacts with a fragment of DNA or an amplicon and (2) emits at a different wavelength in the presence of an amplicon in duplex formation than in the presence of the amplicon in separation. A double stranded DNA detecting molecule can be a double stranded DNA intercalating detecting molecule or a primer-based double stranded DNA detecting molecule.
  • A double stranded DNA intercalating detecting molecule is not covalently linked to a primer, an amplicon or a nucleic acid template. The detecting molecule increases its emission in the presence of double stranded DNA and decreases its emission when duplex DNA unwinds. Examples include, but are not limited to, ethidium bromide, YO-PRO-1, Hoechst 33258, SYBR Gold, and SYBR Green I. Ethidium bromide is a fluorescent chemical that intercalates between base pairs in a double stranded DNA fragment and is commonly used to detect DNA following gel electrophoresis. When excited by ultraviolet light between 254 nm and 366 nm, it emits fluorescent light at 590 nm. The DNA-ethidium bromide complex produces about 50 times more fluorescence than ethidium bromide in the presence of single stranded DNA. SYBR Green I is excited at 497 nm and emits at 520 nm. The fluorescence intensity of SYBR Green I increases over 100 fold upon binding to double stranded DNA against single stranded DNA. An alternative to SYBR Green I is SYBR Gold introduced by Molecular Probes Inc. Similar to SYBR Green I, the fluorescence emission of SYBR Gold enhances in the presence of DNA in duplex and decreases when double stranded DNA unwinds. However, SYBR Gold's excitation peak is at 495 nm and the emission peak is at 537 nm. SYBR Gold reportedly appears more stable than SYBR Green I. Hoechst 33258 is a known bisbenzimide double stranded DNA detecting molecule that binds to the AT rich regions of DNA in duplex. Hoechst 33258 excites at 350 nm and emits at 450 nm. YO-PRO-1, exciting at 450 nm and emitting at 550 nm, has been reported to be a double stranded DNA specific detecting molecule. In a particular embodiment of the present invention, the double stranded DNA detecting molecule is SYBR Green I.
  • A primer-based double stranded DNA detecting molecule is covalently linked to a primer and either increases or decreases fluorescence emission when amplicons form a duplex structure. Increased fluorescence emission is observed when a primer-based double stranded DNA detecting molecule is attached close to the 3′ end of a primer and the primer terminal base is either dG or dC. The detecting molecule is quenched in the proximity of terminal dC-dG and dG-dC base pairs and dequenched as a result of duplex formation of the amplicon when the detecting molecule is located internally at least 6 nucleotides away from the ends of the primer. The dequenching results in a substantial increase in fluorescence emission. Examples of these type of detecting molecules include but are not limited to fluorescein (exciting at 488 nm and emitting at 530 nm), FAM (exciting at 494 nm and emitting at 518 nm), JOE (exciting at 527 and emitting at 548), HEX (exciting at 535 nm and emitting at 556 nm), TET (exciting at 521 nm and emitting at 536 nm), Alexa Fluor 594 (exciting at 590 nm and emitting at 615 nm), ROX (exciting at 575 nm and emitting at 602 nm), and TAMRA (exciting at 555 nm and emitting at 580 nm). In contrast, some primer-based double stranded DNA detecting molecules decrease their emission in the presence of double stranded DNA against single stranded DNA. Examples include, but are not limited to, rhodamine, and BODIPY-FI (exciting at 504 nm and emitting at 513 nm). These detecting molecules are usually covalently conjugated to a primer at the 5′ terminal dC or dG and emit less fluorescence when amplicons are in duplex. It is believed that the decrease of fluorescence upon the formation of duplex is due to the quenching of guanosine in the complementary strand in close proximity to the detecting molecule or the quenching of the terminal dC-dG base pairs.
  • According to one embodiment, the primer-based double stranded DNA detecting molecule is a 5′ nuclease probe. Such probes incorporate a fluorescent reporter molecule at either the 5′ or 3′ end of an oligonucleotide and a quencher at the opposite end. The first step of the amplification process involves heating to denature the double stranded DNA target molecule into a single stranded DNA. During the second step, a forward primer anneals to the target strand of the DNA and is extended by Taq polymerase. A reverse primer and a 5′ nuclease probe then anneal to this newly replicated strand.
  • In this embodiment, at least one of the primer pairs or 5′ nuclease probe should hybridize with a unique determinant sequence. The polymerase extends and cleaves the probe from the target strand. Upon cleavage, the reporter is no longer quenched by its proximity to the quencher and fluorescence is released. Each replication will result in the cleavage of a probe. As a result, the fluorescent signal will increase proportionally to the amount of amplification product.
  • As well as distinguishing between viral and bacterial infections, the determinants described in this application may be used to distinguish between an infectious (bacterial or viral) and non-infectious subject. In one embodiment the determinant is one which appears in Table 16E. For a determinant in Table 16E which is characterized as increasing during an infection, when that determinant is above a predetermined threshold it is indicative that the subject has an infection. For a determinant in Table 16E which is characterized as increasing not due to an infection, then when the determinant is above a predetermined threshold it is indicative that the subject does not have an infection.
  • In one embodiment, the non-infectious subject of this aspect of the present invention has SIRS and the infectious subject has sepsis.
  • SIRS is a serious condition related to systemic inflammation, organ dysfunction, and organ failure. It is defined as 2 or more of the following variables: fever of more than 38° C. (100.4° F.) or less than 36° C. (96.8° F.); heart rate of more than 90 beats per minute; respiratory rate of more than 20 breaths per minute or arterial carbon dioxide tension (PaCO2) of less than 32 mm Hg; abnormal white blood cell count (>12,000/μL or <4,000/μL or >10% immature [band] forms). SIRS is nonspecific and can be caused by ischemia, inflammation, trauma, infection, or several insults combined. Thus, SIRS is not always related to infection.
  • Sepsis is a life-threatening condition that is caused by inflammatory response to an infection. It is currently diagnosed as the presence of SIRS criteria in the presence of a known infection. The early diagnosis of sepsis is essential for clinical intervention before the disease rapidly progresses beyond initial stages to the more severe stages, such as severe sepsis or septic shock, which are associated with high mortality. However, non-infectious SIRS associated with acute tissue injury and innate immune activation can induce clinical syndromes analogous to sepsis, including multiple trauma, pancreatitis, burns, and autoimmune diseases. Current diagnostics are limited in their ability to distinguish between SIRS and sepsis. Therefore, there is a need for new biomarkers or combinations of biomarkers that can provide added value in the accurate and timely diagnosis of sepsis.
  • Thus, the method of this aspect of the present invention may be used to distinguish between a subject who has SIRS (and no infection) and a subject who has sepsis. If the subject has sepsis then at least one of the RNAs from Table 16E is above a predetermined level (e.g. above the amount in a subject who does not have an infection, such as a healthy subject).
  • Contemplated pairs or triplets of RNA determinants that may be used to distinguish between non-infectious SIRS and infectious sepsis include any pair or triplet of determinants that appears in Table 16E.
  • Exemplary pairs of RNA determinants that may be used to distinguish between non-infectious SIRS and infectious sepsis include but are not limited to:
  • FCGR1C+SGK1, FCGR1A+SGK1, FCGR1B+SGK1, AIM2+SGK1, TIFA+SGK1, LMNB1+SGK1, SGK1+MYBL1, SGK1+ZNF438, SGK1+KLRB1, TLR5+SGK1, SGK1+CD274, SGK1+ANKRD22, SGK1+CARD17, SGK1+LILRA5, SGK1+CLEC4D, SGK1+KLRG1, SGK1+MAP2K6, SGK1+PSTPIP2, KLRB1+CASS4, DDX60L+SGK1, SGK1+TNFSF13B, SGK1+C19orf59, ZNF438+CASS4, OR56B1+CD177P1, OAS1+KLRB1, OAS1+MAP2K6, AIM2+CASS4, CHI3L1+CARD17, TLR5+CASS4 and PLSCR1+SGK1.
  • Furthermore, the determinants described in this application may be used to distinguish between a bacterial infection and a non-bacterial infection. In one embodiment the determinant is one which appears in Table 16D. For a determinant in Table 16D which is characterized as increasing during a bacterial infection, when that determinant is above a predetermined threshold it is indicative that the subject has a bacterial infection. For a determinant in Table 16D which is characterized as increasing due to a non-bacterial infection, then when the determinant is above a predetermined threshold it is indicative that the subject does not have a bacterial infection.
  • mRNA is the biological precursor of proteins and changes in its expression levels are expected to precede those of its protein counterparts. Consequently, protein and RNA biomarkers may differ in their temporal dynamics patterns and can complement each other to detect infection prior to symptom onset or following convalescence. Therefore, it will be appreciated that as well as determining the level of the RNA determinants described herein, the present inventors also contemplate combining these measurements with measurements of polypeptide determinants that are known to be indicative of infection type. Examples of polypeptide determinants that are contemplated by the present invention include those that are described in WO 2013/117746, WO 2011/132086, PCT Application IL 2015/051024 and PCT Application IL 2015/051201, the contents of each are incorporated herein by reference. Other polypeptide determinants contemplated by the present inventors are the polypeptide counterparts of the RNA determinants described herein.
  • Examples of polypeptides contemplated by the present inventors are those set forth in Table 15 herein below. Other examples include, but are not limited to: TRAIL, CRP, IP-10, MX1, RSAD2, PCT, OTOF, PI3, CYBRD1, EIF2AK2 and CMPK2.
  • TABLE 15
    RefSeq DNA
    Protein symbol Full Gene Name sequence RefSeq proteins
    CRP C-reactive protein, pentraxin- NC_000001.11 NP_000558.2
    related NT_004487.20
    NC_018912.2
    TRAIL Tumor necrosis factor NC_000003.12 NP_001177871.1
    superfamily member 10 NC_018914.2 NP_001177872.1
    NT_005612.17 NP_003801.1
    IP-10 Chemokine (C—X—C motif) NC_000004.12 NP_001556.2
    ligand 10 NC_018915.2
    NT_016354.20
    IL1R/IL1R1/IL1RA Interleukin 1 receptor, type I NC_000002.12 NP_000868.1
    NT_005403.18 NP_001275635.1
    NC_018913.2
    Procalcitonin (PCT) Calcitonin-related NC_000011.10 NP_001029124.1
    polypeptide alpha NC_018922.2 NP_001029125.1
    NT_009237.19 NP_001732.1
    SAA/SAA1 Serum amyloid A1 NC_000011.10 NP_000322.2
    NC_018922.2 NP_001171477.1
    NT_009237.19 NP_954630.1
    TREM1 Triggering receptor expressed NC_000006.12 NP_001229518.1
    on myeloid cells 1 NT_007592.16 NP_001229519.1
    NC_018917.2 NP_061113.1
    TREM2 Triggering receptor expressed NC_000006.12 NP_001258750.1
    on myeloid cells 2 NT_007592.16 NP_061838.1
    NC_018917.2
    RSAD2 Radical S-adenosyl NC_000002.12 NP_542388.2
    methionine domain NT_005334.17
    containing 2 NC_018913.2
    NGAL Lipocalin 2 NC_000009.12 NP_005555.2
    NC_018920.2
    NT_008470.20
    MMP8 Matrix metallopeptidase 8 NC_000011.10 NP_001291370.1
    NT_033899.9 NP_001291371.1
    NC_018922.2 NP_002415.1
    MX1 MX Dynamin-Like GTPase 1 NC_000021.9 NP_001138397.1
    NT_011512.12 NP_001171517.1
    NC_018932.2 NP_001269849.1
    NP_002453.2
  • Examples of polypeptide combinations contemplated by the present inventors include, but are not limited to: TRAIL+CRP+IP-10; MX1+CRP; MX1+RSAD2; CRP+RSAD2; MX1+CRP+RSAD2; MX1+CRP+PCT; MX1+CRP+TRAIL; TRAIL+CRP+RSAD2; and OTOF, PI3, CYBRD1, EIF2AK2 and CMPK2.
  • Particular combinations of RNA determinants and polypeptide determinants include but are not limited to:
  • TRAIL polypeptide+RSAD2 RNA;
  • TRAIL polypeptide+MX1 RNA;
  • TRAIL polypeptide+IFI44 RNA;
  • TRAIL polypeptide+IFI44L RNA;
  • TRAIL polypeptide+IFI27 RNA;
  • CRP polypeptide+RSAD2 RNA;
  • CRP polypeptide+MX1 RNA;
  • CRP polypeptide+IFI44 RNA;
  • CRP polypeptide+IFI44L RNA;
  • CRP polypeptide+IFI27 RNA;
  • TRAIL polypeptide+CRP polypeptide+RSAD2 RNA;
  • TRAIL polypeptide+CRP polypeptide+MX1 RNA;
  • TRAIL polypeptide+CRP polypeptide+IFI44 RNA;
  • TRAIL polypeptide+CRP polypeptide+IFI44L RNA;
  • TRAIL polypeptide+CRP polypeptide+IFI27 RNA;
  • Therefore, in another aspect of the present invention there is provided a method of distinguishing between bacterial and viral infected patients, the method comprising analyzing one of more RNA determinant selected from Tables 1-6 or 15A-B and analyzing a polypeptide determinant indicative of infection type for example CRP or TRAIL. Exemplary contemplated combinations of polypeptide determinants and RNA determinants are provided in Table 21 of the Examples section herein below.
  • The measurements of the RNA and protein determinants may be combined to provide a single predictive score. In another embodiment, the level of the polypeptide determinant is used for initial screening of subjects with suspected infection and the level of the RNA determinant is used as a follow-up test only in cases where the polypeptide test is inconclusive (e.g. higher/lower than a predetermined threshold). Alternatively, the level of the RNA determinant is used for initial screening of subjects with suspected infection and the level of the polypeptide determinant is used as a follow-up test only in cases where the polypeptide test is inconclusive (e.g. higher/lower than a predetermined threshold). In yet a different embodiment of the invention, RNA signatures comprised of the RNA determinants set forth in Tables 1-6 are combined with their protein counterparts in order to improve diagnostic accuracy.
  • Methods of measuring the levels of polypeptides are well known in the art and include, e.g., immunoassays based on antibodies to proteins, aptamers or molecular imprints.
  • The polypeptide determinants can be detected in any suitable manner, but are typically detected by contacting a sample from the subject with an antibody, which binds the determinant and then detecting the presence or absence of a reaction product. The antibody may be monoclonal, polyclonal, chimeric, or a fragment of the foregoing, as discussed in detail above, and the step of detecting the reaction product may be carried out with any suitable immunoassay. The sample from the subject is typically a biological sample as described above, and may be the same sample of biological sample used to conduct the method described above.
  • In one embodiment, the antibody which specifically binds the determinant is attached (either directly or indirectly) to a signal producing label, including but not limited to a radioactive label, an enzymatic label, a hapten, a reporter dye or a fluorescent label.
  • Immunoassays carried out in accordance with some embodiments of the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody (e.g., anti-determinant antibody), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution. Immunochemical labels, which may be employed, include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.
  • In a heterogeneous assay approach, the reagents are usually the sample, the antibody, and means for producing a detectable signal. Samples as described above may be used. The antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, or enzyme labels. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are oligonucleotides, immunoblotting, immunofluorescence methods, immunoprecipitation, chemiluminescence methods, electrochemiluminescence (ECL) or enzyme-linked immunoassays.
  • Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al., titled “Methods for Modulating Ligand-Receptor Interactions and their Application,” U.S. Pat. No. 4,659,678 to Forrest et al., titled “Immunoassay of Antigens,” U.S. Pat. No. 4,376,110 to David et al., titled “Immunometric Assays Using Monoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled “Macromolecular Environment Control in Specific Receptor Assays,” U.S. Pat. No. 4,233,402 to Maggio et al., titled “Reagents and Method Employing Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled “Heterogenous Specific Binding Assay Employing a Coenzyme as Label.” The determinant can also be detected with antibodies using flow cytometry. Those skilled in the art will be familiar with flow cytometric techniques which may be useful in carrying out the methods disclosed herein(Shapiro 2005). These include, without limitation, Cytokine Bead Array (Becton Dickinson) and Luminex technology.
  • Antibodies can be conjugated to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding. Antibodies as described herein may likewise be conjugated to detectable labels or groups such as radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques.
  • Antibodies can also be useful for detecting post-translational modifications of determinant proteins, polypeptides, mutations, and polymorphisms, such as tyrosine phosphorylation, threonine phosphorylation, serine phosphorylation, glycosylation (e.g., O-GlcNAc). Such antibodies specifically detect the phosphorylated amino acids in a protein or proteins of interest, and can be used in immunoblotting, immunofluorescence, and ELISA assays described herein. These antibodies are well-known to those skilled in the art, and commercially available. Post-translational modifications can also be determined using metastable ions in reflector matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Wirth U. and Muller D. 2002).
  • For determinant-proteins, polypeptides, mutations, and polymorphisms known to have enzymatic activity, the activities can be determined in vitro using enzyme assays known in the art. Such assays include, without limitation, kinase assays, phosphatase assays, reductase assays, among many others. Modulation of the kinetics of enzyme activities can be determined by measuring the rate constant KM using known algorithms, such as the Hill plot, Michaelis-Menten equation, linear regression plots such as Lineweaver-Burk analysis, and Scatchard plot.
  • Suitable sources for antibodies for the detection of determinants include commercially available sources such as, for example, Abazyme, Abnova, AssayPro, Affinity Biologicals, AntibodyShop, Aviva bioscience, Biogenesis, Biosense Laboratories, Calbiochem, Cell Sciences, Chemicon International, Chemokine, Clontech, Cytolab, DAKO, Diagnostic BioSystems, eBioscience, Endocrine Technologies, Enzo Biochem, Eurogentec, Fusion Antibodies, Genesis Biotech, GloboZymes, Haematologic Technologies, Immunodetect, Immunodiagnostik, Immunometrics, Immunostar, Immunovision, Biogenex, Invitrogen, Jackson ImmunoResearch Laboratory, KMI Diagnostics, Koma Biotech, LabFrontier Life Science Institute, Lee Laboratories, Lifescreen, Maine Biotechnology Services, Mediclone, MicroPharm Ltd., ModiQuest, Molecular Innovations, Molecular Probes, Neoclone, Neuromics, New England Biolabs, Novocastra, Novus Biologicals, Oncogene Research Products, Orbigen, Oxford Biotechnology, Panvera, PerkinElmer Life Sciences, Pharmingen, Phoenix Pharmaceuticals, Pierce Chemical Company, Polymun Scientific, Polysiences, Inc., Promega Corporation, Proteogenix, Protos Immunoresearch, QED Biosciences, Inc., R&D Systems, Repligen, Research Diagnostics, Roboscreen, Santa Cruz Biotechnology, Seikagaku America, Serological Corporation, Serotec, SigmaAldrich, StemCell
  • Technologies, Synaptic Systems GmbH, Technopharm, Terra Nova Biotechnology, TiterMax, Trillium Diagnostics, Upstate Biotechnology, US Biological, Vector Laboratories, Wako Pure Chemical Industries, and Zeptometrix. However, the skilled artisan can routinely make antibodies, against any of the polypeptide determinants described herein.
  • A “subject” in the context of the present invention may be a mammal (e.g. human, dog, cat, horse, cow, sheep, pig, goat). According to another embodiment, the subject is a bird (e.g. chicken, turkey, duck or goose). According to a particular embodiment, the subject is a human. The subject can be male or female. The subject may be an adult (e.g. older than 18, 21, or 22 years or a child (e.g. younger than 18, 21 or 22 years). In another embodiment, the subject is an adolescent (between 12 and 21 years), an infant (29 days to less than 2 years of age) or a neonate (birth through the first 28 days of life). The subject can be one who has been previously diagnosed or identified as having an infection, and optionally has already undergone, or is undergoing, a therapeutic intervention for the infection. Alternatively, a subject can also be one who has not been previously diagnosed as having an infection. For example, a subject can be one who exhibits one or more risk factors for having an infection.
  • It will be appreciated that the marker which is used to diagnose the infection may be selected according to the particular subject.
  • Particularly accurate RNA determinants for ruling in infection type in children (e.g. below 21 years, or below 18 years), include but are not limited to SIGLEC1, IFI27, n334829, LY6E, USP41, USP18, uc003hr1.1, n332456, n332510, GAS7, TCONS_00003184-XLOC_001966, OAS1, n333319, OASL, IFI44L, SULT1B1.
  • Particularly accurate RNA determinants for ruling in infection type in adults include but are not limited to IFI44, DGAT2, PYGL, n407780, ZCCHC2, PARP12, TRIB2.
  • Particularly accurate RNA determinants for ruling in infection type in male subjects include, but are not limited to DDX60, ZCCHC2, LY6E, IFI44, n332510, IFIT1, OAS1, TCONS_00003184-XLOC_001966, PNPT1.
  • Particularly accurate RNA determinants for ruling in infection type in female subjects include but are not limited to n407780, CYP1B1, IMPA2, KREMEN1, n332456, CR1, GAS7, PARP12, n333319, PPM1K, n334829, IFI27, uc003hr1.1, SULT1B1, LTA4H.
  • In a particular embodiment, the subject presents with symptoms of pneumonia and the test is carried out to determine if the source of the pneumonia is from a bacterial or viral infection. Particular recommended determinants for this use are at least one of those listed in Table 23 of the Examples section herein below.
  • In another embodiment, the subjects present with symptoms of influenza or parainfluenza. Particular RNA determinants for ruling in influenza include SIGLEC1, n332456, DDX60, HER6 and TCONS_00003184-XLOC_001966. Particular RNA determinants for ruling in parainfluenza include CYP1B1, GAS7 and TCONS_00003184-XLOC_001966. When CYP1B1, GAS7 are below a predetermined level (e.g. below the level found in an identical sample type of a healthy or non-infected subject), a parainfluenza infection is ruled in. When TCONS_00003184-XLOC_001966 is above a predetermined level (e.g. above the level found in an identical sample type of a healthy or non-infected subject, a parainfluenza infection is ruled in. When SIGLEC1, n332456, DDX60, HER6 or TCONS_00003184-XLOC_001966 is above a predetermined level, an influenza infection is ruled in.
  • In still another embodiment, the RNA determinant is used for ruling in CMV, EBV or Bocavirus. Thus, for example when the level of SLUT1B1 is below a predetermined level (e.g. below the level found in an identical sample type of a healthy or non-infected subject), a CMV, EBV or Bocavirus infection is ruled in.
  • Particular RNA determinants have also been shown to be highly accurate in distinguishing between viral infections and bacterial infections of a gram negative bacteria. Such RNA determinants are set forth in Table 22 of the Examples section herein below.
  • Kits
  • Some aspects of the invention also include a determinant-detection reagent such as an oligonucleotide or antibody packaged together in the form of a kit. The kit may contain in separate containers oligonucleotides or antibodies (either already bound to a solid matrix or packaged separately with reagents for binding them to the matrix), control formulations (positive and/or negative), and/or a detectable label such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among others. The detectable label may be attached to a secondary antibody which binds to the Fc portion of the antibody which recognizes the determinant. The detectable label may also be attached to the oligonucleotides. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may be included in the kit.
  • The kits of this aspect of the present invention may comprise additional components that aid in the detection of the determinants such as enzymes, salts, buffers etc. necessary to carry out the detection reactions.
  • Thus, according to another aspect of the present invention, there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in Table 1 or Table 2, and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6 or Tables 15A-B.
  • According to this aspect of the present invention, the at least two determinant detection reagents comprise RNA detection reagents (e.g. oligonucleotides, as described herein above) or protein detection reagents (antibodies, as described herein above).
  • The detection reagents may be attached to detectable moieties as described herein above.
  • Preferably, the kit contains a number of detection reagents such that no more than 20 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit contains a number of detection reagents such that no more than 10 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit contains a number of detection reagents such that no more than 5 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit contains a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit contains a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • In one embodiment, the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-6 or Tables 15A-B.
  • In another embodiment, the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-6 or Tables 16A.
  • In another embodiment, the detection reagents in the kit are only capable of detecting determinants which appear in Tables 1-2.
  • According to further embodiments, the kits of the present invention comprise detection reagents such that pairs of determinants set forth in Tables 9, 11, 12, 17 or 18 may be analyzed.
  • According to further embodiments, the kits of the present invention comprise detection reagents such that triplets of determinants set forth in Tables 13,14, 19 or 20 may be analyzed.
  • According to yet another aspect of the present invention there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two RNA detection reagents specifically detects a first RNA which is set forth in Table 3 or Table 4, and a second of the at least two RNA detection reagents specifically detects a second RNA which is set forth in any one of
  • Tables 1-6 or Tables 15A-B, wherein the kit comprises detection reagents that specifically detect no more than 20 RNAs.
  • Preferably, the kit of this aspect of the present invention contains a number of detection reagents such that no more than 10 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit of this aspect of the present invention contains a number of detection reagents such that no more than 5 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit of this aspect of the present invention contains a number of detection reagents such that no more than 3 determinants (e.g. RNAs) can be detected.
  • Preferably, the kit of this aspect of the present invention contains a number of detection reagents such that no more than 2 determinants (e.g. RNAs) can be detected.
  • According to still another aspect there is provided a kit for diagnosing an infection type comprising at least two determinant detection reagents, wherein the first of the at least two determinant detection reagents specifically detects a first determinant which is set forth in any one of Tables 1-6 or 16A and a second of the at least two determinant detection reagents specifically detects a second determinant which is set forth in any one of Tables 1-6 or 16A, wherein the kit comprises detection reagents that specifically detect no more than 5 determinants.
  • Some aspects of the present invention can also be used to screen patient or subject populations in any number of settings. For example, a health maintenance organization, public health entity or school health program can screen a group of subjects to identify those requiring interventions, as described above, or for the collection of epidemiological data. Insurance companies (e.g., health, life or disability) may screen applicants in the process of determining coverage or pricing, or existing clients for possible intervention. Data collected in such population screens, particularly when tied to any clinical progression to conditions like infection, will be of value in the operations of, for example, health maintenance organizations, public health programs and insurance companies. Such data arrays or collections can be stored in machine-readable media and used in any number of health-related data management systems to provide improved healthcare services, cost effective healthcare, improved insurance operation, etc. See, for example, U.S. Patent Application No. 2002/0038227; U.S. Patent Application No. US 2004/0122296; U.S. Patent Application No. US 2004/0122297; and U.S. Pat. No. 5,018,067. Such systems can access the data directly from internal data storage or remotely from one or more data storage sites as further detailed herein.
  • A machine-readable storage medium can comprise a data storage material encoded with machine readable data or data arrays which, when using a machine programmed with instructions for using the data, is capable of use for a variety of purposes. Measurements of effective amounts of the biomarkers of the invention and/or the resulting evaluation of risk from those biomarkers can be implemented in computer programs executing on programmable computers, comprising, inter alia, a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code can be applied to input data to perform the functions described above and generate output information. The output information can be applied to one or more output devices, according to methods known in the art. The computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. The language can be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette or others as defined elsewhere in this disclosure) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The health-related data management system used in some aspects of the invention may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform various functions described herein.
  • The determinants of the present invention, in some embodiments thereof, can be used to generate a “reference determinant profile” of those subjects who do not have an infection. The determinants disclosed herein can also be used to generate a “subject determinant profile” taken from subjects who have an infection. The subject determinant profiles can be compared to a reference determinant profile to diagnose or identify subjects with an infection. The subject determinant profile of different infection types can be compared to diagnose or identify the type of infection. The reference and subject determinant profiles of the present invention, in some embodiments thereof, can be contained in a machine-readable medium, such as but not limited to, analog tapes like those readable by a VCR, CD-ROM, DVD-ROM, USB flash media, among others. Such machine-readable media can also contain additional test results, such as, without limitation, measurements of clinical parameters and traditional laboratory risk factors. Alternatively or additionally, the machine-readable media can also comprise subject information such as medical history and any relevant family history. The machine-readable media can also contain information relating to other disease-risk algorithms and computed indices such as those described herein.
  • The effectiveness of a treatment regimen can be monitored by detecting a determinant in an effective amount (which may be one or more) of samples obtained from a subject over time and comparing the amount of determinants detected. For example, a first sample can be obtained prior to the subject receiving treatment and one or more subsequent samples are taken after or during treatment of the subject.
  • For example, the methods of the invention can be used to discriminate between bacterial, viral and mixed infections (i.e. bacterial and viral co-infections.) This will allow patients to be stratified and treated accordingly.
  • In a specific embodiment of the invention a treatment recommendation (i.e., selecting a treatment regimen) for a subject is provided by identifying the type infection (i.e., bacterial, viral, mixed infection or no infection) in the subject according to the method of any of the disclosed methods and recommending that the subject receive an antibiotic treatment if the subject is identified as having bacterial infection or a mixed infection; or an anti-viral treatment is if the subject is identified as having a viral infection.
  • In another embodiment, the methods of the invention can be used to prompt additional targeted diagnosis such as pathogen specific PCRs, chest-X-ray, cultures etc. For example, a diagnosis that indicates a viral infection according to embodiments of this invention, may prompt the usage of additional viral specific multiplex-PCRs, whereas a diagnosis that indicates a bacterial infection according to embodiments of this invention may prompt the usage of a bacterial specific multiplex-PCR. Thus, one can reduce the costs of unwarranted expensive diagnostics.
  • In a specific embodiment, a diagnostic test recommendation for a subject is provided by identifying the infection type (i.e., bacterial, viral, mixed infection or no infection) in the subject according to any of the disclosed methods and recommending a test to determine the source of the bacterial infection if the subject is identified as having a bacterial infection or a mixed infection; or a test to determine the source of the viral infection if the subject is identified as having a viral infection.
  • Performance and Accuracy Measures of the Invention.
  • The performance and thus absolute and relative clinical usefulness of the invention may be assessed in multiple ways as noted above. Amongst the various assessments of performance, some aspects of the invention are intended to provide accuracy in clinical diagnosis and prognosis. The accuracy of a diagnostic or prognostic test, assay, or method concerns the ability of the test, assay, or method to distinguish between subjects having an infection is based on whether the subjects have, a “significant alteration” (e.g., clinically significant and diagnostically significant) in the levels of a determinant. By “effective amount” it is meant that the measurement of an appropriate number of determinants (which may be one or more) to produce a “significant alteration” (e.g. level of expression or activity of a determinant) that is different than the predetermined cut-off point (or threshold value) for that determinant (s) and therefore indicates that the subject has an infection for which the determinant (s) is an indication. The difference in the level of determinant is preferably statistically significant. As noted below, and without any limitation of the invention, achieving statistical significance, and thus the preferred analytical, diagnostic, and clinical accuracy, may require that combinations of several determinants be used together in panels and combined with mathematical algorithms in order to achieve a statistically significant determinant index.
  • In the categorical diagnosis of a disease state, changing the cut point or threshold value of a test (or assay) usually changes the sensitivity and specificity, but in a qualitatively inverse relationship. Therefore, in assessing the accuracy and usefulness of a proposed medical test, assay, or method for assessing a subject's condition, one should always take both sensitivity and specificity into account and be mindful of what the cut point is at which the sensitivity and specificity are being reported because sensitivity and specificity may vary significantly over the range of cut points. One way to achieve this is by using the Matthews correlation coefficient (MCC) metric, which depends upon both sensitivity and specificity. Use of statistics such as area under the ROC curve (AUC), encompassing all potential cut point values, is preferred for most categorical risk measures when using some aspects of the invention, while for continuous risk measures, statistics of goodness-of-fit and calibration to observed results or other gold standards, are preferred.
  • By predetermined level of predictability it is meant that the method provides an acceptable level of clinical or diagnostic accuracy. Using such statistics, an “acceptable degree of diagnostic accuracy”, is herein defined as a test or assay (such as the test used in some aspects of the invention for determining the clinically significant presence of determinants, which thereby indicates the presence an infection type) in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.
  • By a “very high degree of diagnostic accuracy”, it is meant a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.75, 0.80, desirably at least 0.85, more desirably at least 0.875, preferably at least 0.90, more preferably at least 0.925, and most preferably at least 0.95.
  • Alternatively, the methods predict the presence or absence of an infection or response to therapy with at least 75% total accuracy, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater total accuracy.
  • Alternatively, the methods predict the presence of a bacterial infection or response to therapy with at least 75% sensitivity, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater sensitivity.
  • Alternatively, the methods predict the presence of a viral infection or response to therapy with at least 75% specificity, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater specificity. Alternatively, the methods predict the presence or absence of an infection or response to therapy with an MCC larger than 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.
  • In general, alternative methods of determining diagnostic accuracy are commonly used for continuous measures, when a disease category has not yet been clearly defined by the relevant medical societies and practice of medicine, where thresholds for therapeutic use are not yet established, or where there is no existing gold standard for diagnosis of the pre-disease. For continuous measures of risk, measures of diagnostic accuracy for a calculated index are typically based on curve fit and calibration between the predicted continuous value and the actual observed values (or a historical index calculated value) and utilize measures such as R squared, Hosmer-Lemeshow P-value statistics and confidence intervals. It is not unusual for predicted values using such algorithms to be reported including a confidence interval (usually 90% or 95% CI) based on a historical observed cohort's predictions, as in the test for risk of future breast cancer recurrence commercialized by Genomic Health, Inc. (Redwood City, Calif.).
  • In general, by defining the degree of diagnostic accuracy, i.e., cut points on a ROC curve, defining an acceptable AUC value, and determining the acceptable ranges in relative concentration of what constitutes an effective amount of the determinants of the invention allows for one of skill in the art to use the determinants to identify, diagnose, or prognose subjects with a pre-determined level of predictability and performance.
  • Furthermore, other unlisted biomarkers will be very highly correlated with the determinants (for the purpose of this application, any two variables will be considered to be “very highly correlated” when they have a Coefficient of Determination (R2) of 0.5 or greater). Some aspects of the present invention encompass such functional and statistical equivalents to the aforementioned determinants. Furthermore, the statistical utility of such additional determinants is substantially dependent on the cross-correlation between multiple biomarkers and any new biomarkers will often be required to operate within a panel in order to elaborate the meaning of the underlying biology.
  • One or more of the listed determinants can be detected in the practice of the present invention, in some embodiments thereof. For example, two (2), three (3), four (4), five (5), ten (10), fifteen (15), twenty (20), forty (40), or more determinants can be detected.
  • In some aspects, all determinants listed herein can be detected. Preferred ranges from which the number of determinants can be detected include ranges bounded by any minimum selected from between one and, particularly two, three, four, five, six, seven, eight, nine ten, twenty, or forty. Particularly preferred ranges include two to five (2-5), two to ten (2-10), two to twenty (2-20), or two to forty (2-40).
  • Construction of Determinant Panels
  • Groupings of determinants can be included in “panels”, also called “determinant-signatures”, “determinant signatures”, or “multi-determinant signatures.” A “panel” within the context of the present invention means a group of biomarkers (whether they are determinants, clinical parameters, or traditional laboratory risk factors) that includes one or more determinants. A panel can also comprise additional biomarkers, e.g., clinical parameters, traditional laboratory risk factors, known to be present or associated with infection, in combination with a selected group of the determinants listed herein.
  • As noted above, many of the individual determinants, clinical parameters, and traditional laboratory risk factors listed, when used alone and not as a member of a multi-biomarker panel of determinants, have little or no clinical use in reliably distinguishing individual normal subjects, subjects at risk for having an infection (e.g., bacterial, viral or co-infection), and thus cannot reliably be used alone in classifying any subject between those three states. Even where there are statistically significant differences in their mean measurements in each of these populations, as commonly occurs in studies which are sufficiently powered, such biomarkers may remain limited in their applicability to an individual subject, and contribute little to diagnostic or prognostic predictions for that subject. A common measure of statistical significance is the p-value, which indicates the probability that an observation has arisen by chance alone; preferably, such p-values are 0.05 or less, representing a 5% or less chance that the observation of interest arose by chance. Such p-values depend significantly on the power of the study performed.
  • Despite this individual determinant performance, and the general performance of formulas combining only the traditional clinical parameters and few traditional laboratory risk factors, the present inventors have noted that certain specific combinations of two or more determinants can also be used as multi-biomarker panels comprising combinations of determinants that are known to be involved in one or more physiological or biological pathways, and that such information can be combined and made clinically useful through the use of various formulae, including statistical classification algorithms and others, combining and in many cases extending the performance characteristics of the combination beyond that of the individual determinants. These specific combinations show an acceptable level of diagnostic accuracy, and, when sufficient information from multiple determinants is combined in a trained formula, they often reliably achieve a high level of diagnostic accuracy transportable from one population to another.
  • The general concept of how two less specific or lower performing determinants are combined into novel and more useful combinations for the intended indications, is a key aspect of some embodiments of the invention. Multiple biomarkers can yield significant improvement in performance compared to the individual components when proper mathematical and clinical algorithms are used; this is often evident in both sensitivity and specificity, and results in a greater AUC or MCC. Significant improvement in performance could mean an increase of 1%, 2%, 3%, 4%, 5%, 8%, 10% or higher than 10% in different measures of accuracy such as total accuracy, AUC, MCC, sensitivity, specificity, PPV or NPV. Secondly, there is often novel unperceived information in the existing biomarkers, as such was necessary in order to achieve through the new formula an improved level of sensitivity or specificity. This hidden information may hold true even for biomarkers which are generally regarded to have suboptimal clinical performance on their own. In fact, the suboptimal performance in terms of high false positive rates on a single biomarker measured alone may very well be an indicator that some important additional information is contained within the biomarker results—information which would not be elucidated absent the combination with a second biomarker and a mathematical formula.
  • On the other hand, it is often useful to restrict the number of measured diagnostic determinants (e.g., RNA or protein biomarkers), as this allows significant cost reduction and reduces required sample volume and assay complexity. Accordingly, even when two signatures have similar diagnostic performance (e.g., similar AUC or sensitivity), one which incorporates less RNAs could have significant utility and ability to reduce to practice. For example, a signature that includes 5 genes compared to 10 genes and performs similarly has many advantages in real world clinical setting and thus is desirable. Therefore, there is value and invention in being able to reduce the number of genes incorporated in a signature while retaining similar levels of accuracy. In this context similar levels of accuracy could mean plus or minus 1%, 2%, 3%, 4%, 5%, 8%, or 10% in different measures of accuracy such as total accuracy, AUC, MCC, sensitivity, specificity, PPV or NPV; a significant reduction in the number of genes of a signature includes reducing the number of genes by 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 genes.
  • Several statistical and modeling algorithms known in the art can be used to both assist in determinant selection choices and optimize the algorithms combining these choices. Statistical tools such as factor and cross-biomarker correlation/covariance analyses allow more rationale approaches to panel construction. Mathematical clustering and classification tree showing the Euclidean standardized distance between the determinants can be advantageously used. Pathway informed seeding of such statistical classification techniques also may be employed, as may rational approaches based on the selection of individual determinants based on their participation across in particular pathways or physiological functions.
  • Ultimately, formula such as statistical classification algorithms can be directly used to both select determinants and to generate and train the optimal formula necessary to combine the results from multiple determinants into a single index. Often, techniques such as forward (from zero potential explanatory parameters) and backwards selection (from all available potential explanatory parameters) are used, and information criteria, such as AIC or BIC, are used to quantify the tradeoff between the performance and diagnostic accuracy of the panel and the number of determinants used. The position of the individual determinant on a forward or backwards selected panel can be closely related to its provision of incremental information content for the algorithm, so the order of contribution is highly dependent on the other constituent determinants in the panel.
  • Construction of Clinical Algorithms
  • Any formula may be used to combine determinant results into indices useful in the practice of the invention. As indicated above, and without limitation, such indices may indicate, among the various other indications, the probability, likelihood, absolute or relative risk, time to or rate of conversion from one to another disease states, or make predictions of future biomarker measurements of infection. This may be for a specific time period or horizon, or for remaining lifetime risk, or simply be provided as an index relative to another reference subject population.
  • Although various preferred formula are described here, several other model and formula types beyond those mentioned herein and in the definitions above are well known to one skilled in the art. The actual model type or formula used may itself be selected from the field of potential models based on the performance and diagnostic accuracy characteristics of its results in a training population. The specifics of the formula itself may commonly be derived from determinant results in the relevant training population. Amongst other uses, such formula may be intended to map the feature space derived from one or more determinant inputs to a set of subject classes (e.g. useful in predicting class membership of subjects as normal, having an infection), to derive an estimation of a probability function of risk using a Bayesian approach, or to estimate the class-conditional probabilities, then use Bayes' rule to produce the class probability function as in the previous case.
  • Preferred formulas include the broad class of statistical classification algorithms, and in particular the use of discriminant analysis. The goal of discriminant analysis is to predict class membership from a previously identified set of features. In the case of linear discriminant analysis (LDA), the linear combination of features is identified that maximizes the separation among groups by some criteria. Features can be identified for LDA using an eigengene based approach with different thresholds (ELDA) or a stepping algorithm based on a multivariate analysis of variance (MANOVA). Forward, backward, and stepwise algorithms can be performed that minimize the probability of no separation based on the Hotelling-Lawley statistic.
  • Eigengene-based Linear Discriminant Analysis (ELDA) is a feature selection technique developed by Shen et al. (2006). The formula selects features (e.g. biomarkers) in a multivariate framework using a modified eigen analysis to identify features associated with the most important eigenvectors. “Important” is defined as those eigenvectors that explain the most variance in the differences among samples that are trying to be classified relative to some threshold.
  • A support vector machine (SVM) is a classification formula that attempts to find a hyperplane that separates two classes. This hyperplane contains support vectors, data points that are exactly the margin distance away from the hyperplane. In the likely event that no separating hyperplane exists in the current dimensions of the data, the dimensionality is expanded greatly by projecting the data into larger dimensions by taking non-linear functions of the original variables (Venables and Ripley, 2002). Although not required, filtering of features for SVM often improves prediction. Features (e.g., biomarkers) can be identified for a support vector machine using a non-parametric Kruskal-Wallis (KW) test to select the best univariate features. A random forest (RF, Breiman, 2001) or recursive partitioning (RPART, Breiman et al., 1984) can also be used separately or in combination to identify biomarker combinations that are most important. Both KW and RF require that a number of features be selected from the total. RPART creates a single classification tree using a subset of available biomarkers.
  • Other formula may be used in order to pre-process the results of individual determinant measurements into more valuable forms of information, prior to their presentation to the predictive formula. Most notably, normalization of biomarker results, using either common mathematical transformations such as logarithmic or logistic functions, as normal or other distribution positions, in reference to a population's mean values, etc. are all well known to those skilled in the art. Of particular interest are a set of normalizations based on clinical-determinants such as time from symptoms, gender, race, or sex, where specific formula are used solely on subjects within a class or continuously combining a clinical-determinants as an input. In other cases, analyte-based biomarkers can be combined into calculated variables which are subsequently presented to a formula.
  • In addition to the individual parameter values of one subject potentially being normalized, an overall predictive formula for all subjects, or any known class of subjects, may itself be recalibrated or otherwise adjusted based on adjustment for a population's expected prevalence and mean biomarker parameter values, according to the technique outlined in D'Agostino et al., (2001) JAMA 286:180-187, or other similar normalization and recalibration techniques. Such epidemiological adjustment statistics may be captured, confirmed, improved and updated continuously through a registry of past data presented to the model, which may be machine readable or otherwise, or occasionally through the retrospective query of stored samples or reference to historical studies of such parameters and statistics. Additional examples that may be the subject of formula recalibration or other adjustments include statistics used in studies by Pepe, M. S. et al., 2004 on the limitations of odds ratios; Cook, N. R., 2007 relating to ROC curves. Finally, the numeric result of a classifier formula itself may be transformed post-processing by its reference to an actual clinical population and study results and observed endpoints, in order to calibrate to absolute risk and provide confidence intervals for varying numeric results of the classifier or risk formula.
  • Some determinants may exhibit trends that depends on the patient age (e.g. the population baseline may rise or fall as a function of age). One can use a ‘Age dependent normalization or stratification’ scheme to adjust for age related differences. Performing age dependent normalization, stratification or distinct mathematical formulas can be used to improve the accuracy of determinants for differentiating between different types of infections. For example, one skilled in the art can generate a function that fits the population mean levels of each determinant as function of age and use it to normalize the determinant of individual subjects levels across different ages. Another example is to stratify subjects according to their age and determine age specific thresholds or index values for each age group independently.
  • It will be appreciated that if the determinant which is used to diagnose infection type is an RNA which is located on a sex chromosomes, the patient gender may influence the diagnostic accuracy of an RNA based diagnostic signature. Thus, it is proposed that when the RNA determinants EIF1AY and UTY (which are located on the Y chromosome) or the RNA determinant BMX (which is located on the X chromosome) are used, the sex of the subject is taken into account. For example, in one embodiment, the RNA determinants EIF1AY and UTY are used to diagnose male subjects.
  • In the context of the present invention the following statistical terms may be used:
  • “TP” is true positive, means positive test result that accurately reflects the tested-for activity. For example in the context of the present invention a TP, is for example but not limited to, truly classifying a bacterial infection as such.
  • “TN” is true negative, means negative test result that accurately reflects the tested-for activity. For example in the context of the present invention a TN, is for example but not limited to, truly classifying a viral infection as such.
  • “FN” is false negative, means a result that appears negative but fails to reveal a situation. For example in the context of the present invention a FN, is for example but not limited to, falsely classifying a bacterial infection as a viral infection.
  • “FP” is false positive, means test result that is erroneously classified in a positive category. For example in the context of the present invention a FP, is for example but not limited to, falsely classifying a viral infection as a bacterial infection.
  • “Sensitivity” is calculated by TP/(TP+FN) or the true positive fraction of disease subjects.
  • “Specificity” is calculated by TN/(TN+FP) or the true negative fraction of non-disease or normal subjects.
  • “Total accuracy” is calculated by (TN+TP)/(TN+FP+TP+FN).
  • “Positive predictive value” or “PPV” is calculated by TP/(TP+FP) or the true positive fraction of all positive test results. It is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested.
  • “Negative predictive value” or “NPV” is calculated by TN/(TN+FN) or the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. See, e.g., O'Marcaigh A S, Jacobson R M, “Estimating The Predictive Value Of A Diagnostic Test, How To Prevent Misleading Or Confusing Results,” Clin. Ped. 1993, 32(8): 485-491, which discusses specificity, sensitivity, and positive and negative predictive values of a test, e.g., a clinical diagnostic test.
  • “MCC” (Mathews Correlation coefficient) is calculated as follows: MCC=(TP*TN−FP*FN)/{(TP+FN)*(TP+FP)*(TN+FP)*(TN+FN)}{circumflex over ( )}0.5 where TP, FP, TN, FN are true-positives, false-positives, true-negatives, and false-negatives, respectively. Note that MCC values range between −1 to +1, indicating completely wrong and perfect classification, respectively. An MCC of 0 indicates random classification. MCC has been shown to be a useful for combining sensitivity and specificity into a single metric (Baldi, Brunak et al. 2000). It is also useful for measuring and optimizing classification accuracy in cases of unbalanced class sizes (Baldi, Brunak et al. 2000).
  • Often, for binary disease state classification approaches using a continuous diagnostic test measurement, the sensitivity and specificity is summarized by a Receiver Operating Characteristics (ROC) curve according to Pepe et al., “Limitations of the Odds Ratio in Gauging the Performance of a Diagnostic, Prognostic, or Screening Marker,” Am. J. Epidemiol 2004, 159 (9): 882-890, and summarized by the Area Under the Curve (AUC) or c-statistic, an indicator that allows representation of the sensitivity and specificity of a test, assay, or method over the entire range of test (or assay) cut points with just a single value. See also, e.g., Shultz, “Clinical Interpretation Of Laboratory Procedures,” chapter 14 in Teitz, Fundamentals of Clinical Chemistry, Burtis and Ashwood (eds.), 4th edition 1996, W.B. Saunders Company, pages 192-199; and Zweig et al., “ROC Curve Analysis: An Example Showing The Relationships Among Serum Lipid And Apolipoprotein Concentrations In Identifying Subjects With Coronory Artery Disease,” Clin. Chem., 1992, 38(8): 1425-1428. An alternative approach using likelihood functions, odds ratios, information theory, predictive values, calibration (including goodness-of-fit), and reclassification measurements is summarized according to Cook, “Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction,” Circulation 2007, 115: 928-935.
  • “Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), Mathews correlation coefficient (MCC), or as a likelihood, odds ratio, Receiver Operating Characteristic (ROC) curve, Area Under the Curve (AUC) among other measures.
  • A “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an “index” or “index value”. Non-limiting examples of “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical-determinants, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations. Of particular use in combining determinants are linear and non-linear equations and statistical classification analyses to determine the relationship between levels of determinants detected in a subject sample and the subject's probability of having an infection or a certain type of infection. In panel and combination construction, of particular interest are structural and syntactic statistical classification algorithms, and methods of index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, and Hidden Markov Models, among others. Other techniques may be used in survival and time to event hazard analysis, including Cox, Weibull, Kaplan-Meier and Greenwood models well known to those of skill in the art. Many of these techniques are useful either combined with a determinant selection technique, such as forward selection, backwards selection, or stepwise selection, complete enumeration of all potential panels of a given size, genetic algorithms, or they may themselves include biomarker selection methodologies in their own technique. These may be coupled with information criteria, such as Akaike's Information Criterion (AIC) or Bayes Information Criterion (BIC), in order to quantify the tradeoff between additional biomarkers and model improvement, and to aid in minimizing overfit. The resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV). At various steps, false discovery rates may be estimated by value permutation according to techniques known in the art. A “health economic utility function” is a formula that is derived from a combination of the expected probability of a range of clinical outcomes in an idealized applicable patient population, both before and after the introduction of a diagnostic or therapeutic intervention into the standard of care. It encompasses estimates of the accuracy, effectiveness and performance characteristics of such intervention, and a cost and/or value measurement (a utility) associated with each outcome, which may be derived from actual health system costs of care (services, supplies, devices and drugs, etc.) and/or as an estimated acceptable value per quality adjusted life year (QALY) resulting in each outcome. The sum, across all predicted outcomes, of the product of the predicted population size for an outcome multiplied by the respective outcome's expected utility is the total health economic utility of a given standard of care. The difference between (i) the total health economic utility calculated for the standard of care with the intervention versus (ii) the total health economic utility for the standard of care without the intervention results in an overall measure of the health economic cost or value of the intervention. This may itself be divided amongst the entire patient group being analyzed (or solely amongst the intervention group) to arrive at a cost per unit intervention, and to guide such decisions as market positioning, pricing, and assumptions of health system acceptance. Such health economic utility functions are commonly used to compare the cost-effectiveness of the intervention, but may also be transformed to estimate the acceptable value per QALY the health care system is willing to pay, or the acceptable cost-effective clinical performance characteristics required of a new intervention.
  • For diagnostic (or prognostic) interventions of the invention, as each outcome (which in a disease classifying diagnostic test may be a TP, FP, TN, or FN) bears a different cost, a health economic utility function may preferentially favor sensitivity over specificity, or PPV over NPV based on the clinical situation and individual outcome costs and value, and thus provides another measure of health economic performance and value which may be different from more direct clinical or analytical performance measures. These different measurements and relative trade-offs generally will converge only in the case of a perfect test, with zero error rate (a.k.a., zero predicted subject outcome misclassifications or FP and FN), which all performance measures will favor over imperfection, but to differing degrees.
  • “Analytical accuracy” refers to the reproducibility and predictability of the measurement process itself, and may be summarized in such measurements as coefficients of variation (CV), Pearson correlation, and tests of concordance and calibration of the same samples or controls with different times, users, equipment and/or reagents. These and other considerations in evaluating new biomarkers are also summarized in Vasan, 2006.
  • “Performance” is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g., stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate “performance metrics,” such as AUC and MCC, time to result, shelf life, etc. as relevant.
  • By “statistically significant”, it is meant that the alteration is greater than what might be expected to happen by chance alone (which could be a “false positive”). Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is often considered highly significant at a p-value of 0.05 or less.
  • In the context of the present invention the following abbreviations may be used: Antibiotics (Abx), Adverse Event (AE), Arbitrary Units (A.U.), Complete Blood Count (CBC), Case Report Form (CRF), Chest X-Ray (CXR), Electronic Case Report Form (eCRF), Food and Drug Administration (FDA), Good Clinical Practice (GCP), Gastrointestinal (GI),Gastroenteritis (GE), International Conference on Harmonization (ICH), Infectious Disease (ID), In vitro diagnostics (IVD), Lower Respiratory Tract Infection (LRTI), Myocardial infarction (MI), Polymerase chain reaction (PCR), Per-oss (P.O), Per-rectum (P.R), Standard of Care (SoC), Standard Operating Procedure (SOP), Urinary Tract Infection (UTI), Upper Respiratory Tract Infection (URTI).
  • As used herein the term “about” refers to ±10%.
  • The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • The term “consisting of” means “including and limited to”.
  • The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • Throughout this application, various embodiments of this invention may 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, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
  • Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
  • All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
  • Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
    • EXAMPLES
  • Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
  • Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
    • Example 1 Gene Expression Profiles Discriminate Between Patients with Bacterial and Viral Infections
  • Materials and Methods
  • Patient recruitment Patients were prospectively recruited as part of the ‘Curiosity’ clinical study (NCT01917461; (Oved et al. 2015)). Informed consent was obtained from each participant or legal guardian, as applicable. Inclusion criteria for the infectious disease cohort included: clinical suspicion of an acute infectious disease, peak fever >37.5° C. since symptoms onset, and duration of symptoms ≤12 days. Inclusion criteria for the control group included: clinical impression of a non-infectious disease (e.g. trauma, stroke and myocardial infarction), or healthy subjects. Exclusion criteria included: evidence of any episode of acute infectious disease in the two weeks preceding enrollment; diagnosed congenital immune deficiency; current treatment with immunosuppressive or immunomodulatory therapy; active malignancy, proven or suspected human immunodeficiency virus (HIV)-1, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection. Importantly, in order to enable broad generalization, antibiotic treatment at enrollment did not cause exclusion from the study. An overview of study workflow is depicted in FIG. 1 .
  • Enrollment process and data collection: For each patient, the following baseline variables were recorded: demographics, physical examination, medical history (e.g. main complaints, underlying diseases, chronically-administered medications, comorbidities, time of symptom onset, and peak temperature), complete blood count (CBC) obtained at enrollment, and chemistry panel (e.g. creatinine, urea, electrolytes, and liver enzymes). A nasal swab was obtained from each patient for further microbiological investigation, and a blood sample was obtained for protein screening and validation. Additional samples were obtained as deemed appropriate by the physician (e.g. urine and stool samples in cases of suspected urinary tract infection [UTI], and gastroenteritis [GI] respectively). Radiological tests were obtained at the discretion of the physician (e.g. chest X-ray for suspected lower respiratory tract infection [LRTI]). Thirty days after enrollment, disease course and response to treatment were recorded. All information was recorded in a custom electronic case report form (eCRF).
  • Microbiological investigation: Patients underwent two multiplex-PCR diagnostic assays from nasal swab samples: (i) Seeplex® RV15, for detection of parainfluenza virus 1, 2, 3, and 4, coronavirus 229E/NL63, adenovirus A/B/C/D/E, bocavirus 1/2/3/4, influenza virus A and B, metapneumovirus, coronavirus 0C43, rhinovirus A/B/C, respiratory syncytial virus A and B, and Enterovirus, and (ii) Seeplex® PB6 for detection of Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Legionella pneumophila, Bordetella pertussis, and Mycoplasma pneumoniae. Multiplex-PCR assays were performed by a certified service laboratory. Patients were also tested for additional pathogens according to their suspected clinical syndrome, including: blood culture, urine culture and stool culture for Shigella spp., Campylobacter spp. and Salmonella spp.; serological testing (IgM and/or IgG) for cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma Pneumonia, and Coxiella burnetii (Q-Fever).
  • Establishing the reference standard: Currently, no single reference standard exists for determining bacterial and viral infections in a wide range of clinical syndromes. Therefore, a rigorous reference standard was created following recommendations of the Standards for Reporting of Diagnostic Accuracy (STARD) (Bossuyt et al. 2003). First, a thorough clinical and microbiological investigation was performed for each patient as described above. Then, all the data collected throughout the disease course was reviewed by a panel of three physicians (the attending pediatrician, an infectious disease expert and a senior attending pediatrician). Each panel member assigned one of the following diagnostic labels to each patient: (i) bacterial; (ii) viral; (iii) no apparent infectious disease or healthy (controls); and (iv) indeterminate. Final diagnosis was determined by consensus agreement of all three panel members. Importantly, the panel members were blinded to the labeling of their peers and to the results of the signature.
  • Samples, procedures and RNA purification: Nasal swabs and stool samples were stored at 4° C. for up to 72 hours and subsequently transported to a certified service laboratory for multiplex PCR-based assay. Venous blood samples were collected in EDTA contained CBC tube and stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma and cell pellet. Red cells were lysed using Red Cell Lysis Buffer (RCLB) at room temperature (RT) and total RNA was purified using RNeasy plus Mini Kit (QIAGEN, Cat. 74134) according to manufacturer recommended protocols.
  • Microarray experiments A total of 10 μl of 200 ng/3 μl (66.67 ng/μl) RNA were transferred to microarray chip hybridization. Amplified cRNA was prepared from 200 ng total RNA using the WT cDNA Synthesis and WT cDNA Amplification Kits (900672, AFFYMETRIX™). Biotinylated single-stranded cDNA was generated from the amplified cRNA and then fragmented and labeled with the WT Terminal Labeling Kit (AFFYMETRIX™), following manufacturer protocol. Samples were hybridized to Human Gene 1.0 ST Arrays (AFFYMETRIX™) in which the probes are distributed across the full length of the gene, providing a more complete and accurate picture of overall gene expression. This array interrogates 28,869 well-annotated genes with 764,885 distinct probes. In addition, it contains a subset of probes from Exon 1.0 ST Arrays that focuses on well-annotated content. Arrays were scanned using the AFFYMETRIX™ GENECHIP™ Scanner 3000 7G. Partek Genomic Suite software was then used to extract raw data, perform mean probe summarization, RMA and quintile normalization and GC content correction (Downey 2006).
  • Statistical analysis Primary analysis was based on area under the receiver operating curve (AUC), Matthews correlation coefficient (MCC), sensitivity, specificity, and total accuracy. These measures are defined as follows:
  • Sensitivity = TP TP + FN ⁢ Specificity = TN TN + FP ⁢ total ⁢ accuracy = TP + TN TP + FN + TN + FP ⁢ MCC = TP × TN - FP × FN ( TP + FP ) ⁢ ( TP + FN ) ⁢ ( TN + FP ) ⁢ ( TN + FN )
  • P, N, TP, FP, TN, FN are positives, negatives, true-positives, false-positives, true-negatives, and false-negatives, respectively. Unless mentioned otherwise, positives and negatives refer to patients with bacterial and viral infections, respectively.
  • Results
  • Patients characteristics The studied group of pediatric patients included 7 females (47%) and 8 males (53%) aged 7 months to 16 years. The patients presented with a variety of clinical syndromes affecting different physiological systems (e.g., respiratory, urinal, central nervous system, systemic). Detailed characterization of studied patients is depicted in Table 7.
  • TABLE 7
    Detailed description of studied patients. UTI-Urinal tract infection; URTI-
    Upper respiratory tract infection; WBC-white blood count.
    Time
    Maximal from Hospitalization
    Patient Age Clinical temperature symptoms Duration WBC
    number Gender (y) Etiology Syndrome (° C.) (d) (d) (×1000)
    390 Male 6 Bacterial Pneumonia 38 3 4 8.3
    392 Female 2 Bacterial Acute 38.7 2 4 19.5
    otitis
    media
    406 Female 16 Bacterial Pharyngitis 39 6 3 7.5
    417 Female 5 Bacterial UTI 40 4 3 12.2
    418 Male 2 Bacterial Pneumonia 38.1 4 2 14.9
    420 Male 3 Bacterial Pneumonia 39.5 2 1 27.5
    384 Male 3 Viral Bronchitis 39.3 1 0 11.3
    397 Female 2 Viral Fever 38.4 4 1 15.3
    Without
    Source
    404 Female 1 Viral Fever 38.6 1 0 8.2
    Without
    Source
    405 Female 0.7 Viral Bronchiolitis 39.6 2 2 13.9
    408 Male 1.1 Viral Fever 39.7 2 1 6
    Without
    Source
    416 Male 10.5 Viral Meningitis 38.4 5 2 3.8
    421 Male 3 Viral URTI 39.9 2 0 9.4
    422 Male 2 Viral URTI 39 5 0 16.7
    423 Female 1.8 Viral Fever 39.5 3 2 9.1
    Without
    Source
  • Single and combinations of RNA determinants can distinguish between bacterial and viral patients The gene expression profiles of blood leukocytes obtained from the described acute infection patients using the Human Gene 1.0 ST Array (Affymetrix) were studied. The results suggest a differential response of the immune system to bacterial and viral infections, which can potentially be used classify acute infection patients. 65 RNA determinants were identified that were differentially expressed in the bacterial and viral patients tested (Table 8; log2-fold change was calculated compared to bacterial patients baseline). FIG. 2 presents distinctive gene expression profiles of the identified genes after applying a hierarchical clustering that uses Euclidean distance metric and average linkage. Based on these results, a classifier was developed for distinguishing between bacterial and viral patients using logistic regression. The present inventors further calculated for these RNA determinants the measures of accuracy in distinguishing between bacterial and viral patients including AUC, MCC, total accuracy, sensitivity, specificity and Wilcoxon ranksum P-value (Table 8).
  • TABLE 8
    Differentially expressed RNA determinants and their measures of accuracy in
    differentiating between bacterial or mixed versus viral infected subjects. Changes in
    expression levels were calculated as log2 (fold change bacterial)-log2 (fold change
    viral).
    Log2
    (fold
    change
    bacterial)-
    Bacterial log2
    or (fold ranksum
    Serial viral change Total P-
    number DETERMINANT induced viral) AUC MCC accuracy Sensitivity Specificity value
    1 AIM2 Viral −1.56 0.722 0.167 0.667 0.667 0.667 1.81E−
    01
    2 ANKRD2 Viral −2.13 0.648 0.444 0.8 0.667 0.889 3.88E−
    01
    3 BMX Viral −1.53 0.667 0.444 0.733 0.667 0.778 3.28E−
    01
    4 C19orf59 Viral −1.51 0.611 0.167 0.533 0.5 0.556 5.29E−
    01
    5 CCL2 Viral −0.95 0.87 0.764 0.867 1 0.778 1.76E−
    02
    6 CD177 Viral −1.64 0.63 0.167 0.667 0.5 0.778 4.56E−
    01
    7 CEACAM1 Viral −1.88 0.796 0.444 0.8 0.833 0.778 6.63E−
    02
    8 CLEC4D Viral −1.48 0.685 0.444 0.733 0.667 0.778 2.72E−
    01
    9 CMPK2 Viral −2.44 0.87 0.444 0.8 0.667 0.889 1.76E−
    02
    10 CXCL10 Viral −2.08 0.704 0.491 0.733 0.833 0.667 2.24E−
    01
    11 CYBRD1 Bacterial 1.35 0.852 0.444 0.8 0.833 0.778 2.56E−
    02
    12 CYP1B1 Bacterial 1.38 0.63 0.218 0.733 0.333 1 4.56E−
    01
    13 DDX60 Viral −1.79 0.889 0.327 0.8 0.667 0.889 1.20E−
    02
    14 EIF1AY Viral −2.14 0.611 0.492 0.8 0.667 0.889 5.29E−
    01
    15 EIF2AK2 Viral −1.50 0.833 0.289 0.8 0.833 0.778 3.60E−
    02
    16 EPSTI1 Viral −2.32 0.833 0.577 0.867 0.667 1 3.60E−
    02
    17 F13A1 Bacterial 1.03 0.87 0.6 0.933 0.833 1 1.76E−
    02
    18 FAM101B Bacterial 1.21 0.852 0.327 0.8 0.667 0.889 2.56E−
    02
    19 FFAR3 Viral −1.57 0.741 0.491 0.8 1 0.667 1.81E−
    01
    20 RASA4 Bacterial 0.44 0.722 0.389 0.733 0.667 0.778 2.72E−
    01
    21 GALM Viral −1.46 0.944 0.6 0.933 0.833 1 2.80E−
    03
    22 HERC5 Viral −2.45 0.833 0.327 0.733 0.833 0.667 3.60E−
    02
    23 HLA-DQA1 Bacterial 0.40 0.611 0.389 0.6 0.833 0.444 6.07E−
    01
    24 IFI27 Viral −2.96 1 0.873 0.933 1 0.889 4.00E−
    04
    25 IFI44 Viral −2.42 0.852 0.327 0.733 0.833 0.667 2.56E−
    02
    26 IFI44L Viral −3.86 0.889 0.444 0.733 0.833 0.667 1.20E−
    02
    27 IFI6 Viral −1.80 0.815 0.327 0.733 0.833 0.667 4.96E−
    02
    28 IFIT1 Viral −2.28 0.889 0.6 0.8 0.833 0.778 1.20E−
    02
    29 IFIT2 Viral −1.83 0.852 0.327 0.733 0.833 0.667 2.56E−
    02
    30 IFIT3 Viral −2.26 0.796 0.444 0.8 0.667 0.889 6.63E−
    02
    31 IFITM3 Viral −1.53 0.759 0.289 0.733 0.833 0.667 1.13E−
    01
    32 INCA Viral −1.92 0.741 0.444 0.8 0.833 0.778 1.45E−
    01
    33 IRF7 Viral −1.69 0.741 0.289 0.733 0.667 0.778 1.45E−
    01
    34 ISG15 Viral −1.22 0.907 0.6 0.867 0.833 0.889 7.59E−
    03
    35 JARID1D Viral −1.56 0.611 0.577 0.8 0.833 0.778 5.29E−
    01
    36 JUP Viral −1.47 0.796 0.218 0.733 0.667 0.778 6.63E−
    02
    37 LAMP3 Viral −2.36 0.907 0.6 0.867 0.833 0.889 7.59E−
    03
    38 LOC100132244 Bacterial 1.08 0.685 0.218 0.667 0.667 0.667 2.72E−
    01
    39 LOC26010 Viral −2.23 0.87 0.444 0.867 1 0.778 1.76E−
    (SPATS2L) 02
    40 LY6E Viral −1.86 0.889 0.6 0.8 0.833 0.778 1.20E−
    02
    41 MT1G Viral −1.71 0.87 0.6 0.8 0.833 0.778 1.76E−
    02
    42 MT2A Viral −1.56 0.889 0.722 0.867 0.833 0.889 4.80E−
    03
    43 MX1 Viral −2.57 0.87 0.6 0.8 0.833 0.778 1.76E−
    02
    44 OAS1 Viral −2.49 0.852 0.444 0.8 0.667 0.889 2.56E−
    02
    45 OAS2 Viral −1.89 0.907 0.444 0.867 0.667 1 7.59E−
    03
    46 OAS3 Viral −2.97 0.87 0.444 0.8 0.667 0.889 1.76E−
    02
    47 OASL Viral −2.22 0.889 0.491 0.733 0.833 0.667 1.20E−
    02
    48 OTOF Viral −0.83 0.907 0.6 0.867 1 0.778 7.59E−
    03
    49 PHOSPHO1 Bacterial 1.01 0.722 0.327 0.733 0.833 0.667 1.81E−
    01
    50 PI3 Bacterial 1.91 0.87 0.444 0.867 1 0.778 1.76E−
    02
    51 PLSCR1 Viral −1.68 0.704 0.289 0.733 0.5 0.889 2.24E−
    01
    52 PPBP Bacterial 1.01 0.796 0.444 0.8 0.833 0.778 6.63E−
    02
    53 PSTPIP2 Viral −1.61 0.611 0.327 0.733 0.667 0.778 5.29E−
    01
    54 RGS1 Viral −0.53 0.944 0.491 0.933 0.833 1 2.80E−
    03
    55 RSAD2 Viral −3.93 0.889 0.444 0.8 0.667 0.889 1.20E−
    02
    56 RTP4 Viral −1.72 0.889 0.6 0.867 0.833 0.889 1.20E−
    02
    57 SERPING1 Viral −2.41 0.852 0.577 0.8 0.833 0.778 2.56E−
    02
    58 SH3BGRL2 Bacterial 1.12 0.852 0.444 0.867 1 0.778 2.56E−
    02
    59 SIGLEC1 Viral −2.47 0.889 0.6 0.867 1 0.778 1.20E−
    02
    60 TMEM176A Bacterial 1.17 0.667 0.218 0.733 0.333 1 3.45E−
    01
    61 TNFAIP6 Viral −1.80 0.796 0.444 0.733 0.667 0.778 6.63E−
    02
    62 TREML4 Viral −1.64 0.833 0.6 0.8 0.833 0.778 3.60E−
    02
    63 USP18 Viral −3.12 0.926 0.6 0.867 1 0.778 7.99E−
    04
    64 UTY Viral −1.86 0.611 0.492 0.867 0.833 0.889 5.29E−
    01
    65 XAF1 Viral −1.65 0.889 0.444 0.733 0.667 0.778 1.20E−
    02
  • Next, a logistic regression was used to develop a classifier for each gene pair (2080 combinations) in order to test whether combining two RNA determinants can improve diagnostic accuracy. Table 9 summarizes the 50 RNA pairs that demonstrated the highest accuracy improvement as calculated by the difference in AUC of the pair compared to the AUC of the single RNA (out of the same pair) with the highest AUC (delta AUC). Using pairs of RNA determinants improved diagnostic accuracy up to 53% (when comparing AUC of single vs. pairs of RNA determinants), to generate highly discriminative combinations (AUCs between 0.83-1.0, average AUC 0.95, when testing 50 pairs with highest accuracy; Table 9, FIGS. 3A-3B and 4A-4B).
  • TABLE 9
    RNA pairs that presented the highest accuracy improvement compared to the
    single RNA determinants. AUC_1 and AUC_2 are the respective AUCs of RNA #1 and
    RNA #2. The accuracy of combining RNA#1 and RNA#2 is depicted in the column
    AUC pair, and the improvement of the combination over that of the individual RNA's
    is captured in the AUC delta (AUC_delta = AUC pair − max(AUC_1, AUC2).
    AUC pair
    (RNA#1
    and
    RNA #1 RNA #2 AUC_1 AUC_2 RNA#2) AUC_delta
    BMX CYP1B1 0.667 0.63 1 0.333
    C19orf59 CYP1B1 0.611 0.63 0.926 0.296
    CLEC4D CYP1B1 0.685 0.63 0.963 0.278
    CYP1B1 PLSCR1 0.63 0.704 0.981 0.277
    CYP1B1 FFAR3 0.63 0.741 1 0.259
    CYP1B1 PSTPIP2 0.63 0.611 0.87 0.24
    FFAR3 LOC100132244 0.741 0.685 0.981 0.24
    PLSCR1 TMEM176A 0.704 0.667 0.926 0.222
    ANKRD22 CYP1B1 0.648 0.63 0.87 0.222
    BMX TMEM176A 0.667 0.667 0.889 0.222
    CD177 CYP1B1 0.63 0.63 0.852 0.222
    CD177 FFAR3 0.63 0.741 0.963 0.222
    AIM2 CYP1B1 0.722 0.63 0.926 0.204
    CLEC4D TMEM176A 0.685 0.667 0.889 0.204
    CYP1B1 TNFAIP6 0.63 0.796 1 0.204
    CYP1B1 INCA 0.63 0.741 0.926 0.185
    LOC100132244 PLSCR1 0.685 0.704 0.889 0.185
    BMX LOC100132244 0.667 0.685 0.87 0.185
    CEACAM1 CYP1B1 0.796 0.63 0.981 0.185
    CLEC4D LOC100132244 0.685 0.685 0.852 0.167
    PSTPIP2 TMEM176A 0.611 0.667 0.833 0.166
    AIM2 CYBRD1 0.722 0.852 1 0.148
    BMX CYBRD1 0.667 0.852 1 0.148
    C19orf59 FFAR3 0.611 0.741 0.889 0.148
    CEACAM1 CYBRD1 0.796 0.852 1 0.148
    CYBRD1 EIF2AK2 0.852 0.833 1 0.148
    CYBRD1 EPSTI1 0.852 0.833 1 0.148
    CYBRD1 HERC5 0.852 0.833 1 0.148
    CYBRD1 IFI44 0.852 0.852 1 0.148
    CYBRD1 IFI6 0.852 0.815 1 0.148
    CYBRD1 IFIT2 0.852 0.852 1 0.148
    CYBRD1 IFIT3 0.852 0.796 1 0.148
    CYBRD1 IFITM3 0.852 0.759 1 0.148
    CYBRD1 INCA 0.852 0.741 1 0.148
    CYBRD1 OAS1 0.852 0.852 1 0.148
    CYBRD1 PLSCR1 0.852 0.704 1 0.148
    CYBRD1 PSTPIP2 0.852 0.611 1 0.148
    CYBRD1 SERPING1 0.852 0.852 1 0.148
    CYBRD1 TNFAIP6 0.852 0.796 1 0.148
    CYP1B1 RASA4 0.63 0.722 0.87 0.148
    CYP1B1 IRF7 0.63 0.741 0.889 0.148
    FFAR3 TMEM176A 0.741 0.667 0.889 0.148
    CEACAM1 TMEM176A 0.796 0.667 0.944 0.148
    LOC100132244 PPBP 0.685 0.796 0.944 0.148
    CCL2 CYBRD1 0.87 0.852 1 0.13
    CD177 LOC26010 0.63 0.87 1 0.13
    (SPATS2L)
    CMPK2 CYBRD1 0.87 0.852 1 0.13
    CYBRD1 LOC26010 0.852 0.87 1 0.13
    (SPATS2L)
    CYBRD1 MT1G 0.852 0.87 1 0.13
    CYBRD1 MX1 0.852 0.87 1 0.13
  • As illustrated in the heat maps of FIGS. 3A-3B, some RNA determinant combinations exhibit an improved diagnostic accuracy compared to that of the corresponding individual determinants, whereas other combinations exhibit a reduced accuracy.
  • Next, the present inventors performed a rigorous bioinformatics analysis of RNA determinants using an external set generated from a cohort of 91 pediatric patients with acute infectious diseases caused by influenza A virus, Escherichia coli, Staphylococcus aureus or Streptococcus pneumoniae, which were tested using AFFYMETRIX™ HGU133A GENECHIPS™ (Ramilo et al. 2007).
  • For each RNA determinant they calculated the log2 fold change between bacterial and viral patients and created a logistic regression classifier to calculate its AUC, MCC, total accuracy, sensitivity and specificity. They also created logistic regression classifiers for the pairs of RNA determinants and calculated the same measures of accuracy (Tables 10-12).
  • TABLE 10
    Single RNA determinants that differentiate between bacterial versus
    viral infected subjects using an external data set (measured using AFFYMETRIX ™
    HGU133A GENECHIPS ™ on 73 bacterial and 18 viral patients).
    Log2
    [(fold
    change
    bacterial)-
    Bacterial log2
    or (fold ranksum
    viral change Total P-
    determinant induced viral)] AUC MCC accuracy Sensitivity Specificity value
    AIM2 Viral −1.08 0.754 0.281 0.71 0.71 0.72 0.00226
    CCL2 Viral −6.12 0.628 0.162 0.66 0.63 0.78 0.003239
    CXCL10 Viral −4.00 0.768 0.094 0.67 0.64 0.78 0.017969
    CYBRD1 Bacterial 1.79 0.885 0.384 0.79 0.77 0.89 1.37E−05
    EIF2AK2 Viral −1.08 0.881 0.579 0.84 0.84 0.83 2.06E−06
    F13A1 Bacterial 1.25 0.673 0.171 0.60 0.59 0.67 0.04261
    HERC5 Viral −2.89 0.87 0.492 0.86 0.88 0.78 1.19E−05
    IFI27 Viral −7.06 0.963 0.763 0.96 0.95 1.00 1.94E−09
    IFI44 Viral −3.22 0.942 0.645 0.85 0.84 0.89 9.54E−08
    IFI44L Viral −5.23 0.943 0.686 0.89 0.89 0.89 5.18E−08
    IFI6 Viral −2.35 0.875 0.493 0.78 0.75 0.89 1.13E−06
    IFIT1 Viral −5.43 0.9 0.627 0.86 0.88 0.78 9.05E−06
    IFIT2 Viral −2.49 0.787 0.436 0.79 0.81 0.72 0.002939
    IFIT3 Viral −4.08 0.887 0.603 0.81 0.81 0.83 1.19E−05
    ISG15 Viral −3.51 0.925 0.557 0.86 0.86 0.83 1.74E−07
    JUP Viral −0.94 0.881 0.498 0.80 0.80 0.83 5.67E−06
    LAMP3 Viral −2.25 0.863 0.584 0.84 0.82 0.89 8.25E−06
    LY6E Viral −2.15 0.958 0.686 0.90 0.90 0.89 5.43E−09
    MT1G Viral −0.69 0.68 0.257 0.66 0.66 0.67 0.076983
    MX1 Viral −2.79 0.938 0.6 0.87 0.86 0.89 4.90E−08
    OAS1 Viral −1.97 0.863 0.403 0.82 0.84 0.78 8.25E−06
    OAS2 Viral −1.75 0.938 0.6 0.84 0.82 0.89 8.55E−08
    OAS3 Viral −2.77 0.937 0.622 0.88 0.88 0.89 8.55E−08
    OASL Viral −2.42 0.9 0.557 0.85 0.86 0.78 9.25E−07
    OTOF Viral −4.18 0.988 0.831 0.95 0.95 0.94 9.82E−09
    PI3 Bacterial 3.19 0.545 0.243 0.54 0.47 0.83 0.74609
    PLSCR1 Viral −0.59 0.709 0.345 0.71 0.71 0.72 0.017024
    PPBP Bacterial 0.58 0.714 0.239 0.62 0.56 0.83 0.008164
    RGS1 Viral −2.97 0.568 0.092 0.62 0.66 0.44 0.66472
    RSAD2 Viral −4.20 0.947 0.669 0.85 0.85 0.83 2.39E−06
    RTP4 Viral −1.80 0.849 0.391 0.71 0.70 0.78 2.56E−05
    SERPING1 Viral −3.29 0.863 0.374 0.77 0.73 0.94 4.92E−06
    SIGLEC1 Viral −4.33 0.948 0.695 0.88 0.86 0.94 2.35E−08
    TMEM176A Bacterial 1.48 0.642 0.194 0.58 0.55 0.72 0.093191
    USP18 Viral −3.12 0.965 0.71 0.90 0.90 0.89 3.36E−09
  • TABLE 11
    Pairs of RNA DETERMINANTS and their measures of accuracy in
    differentiating between bacterial versus viral infected subjects as tested
    using an external data set (measured using AFFYMETRIX ™ HGU133A
    GENECHIPS ™ on 73 bacterial and 18 viral patients).
    Total ranksum
    RNA#1 RNA #2 ACC MCC accuracy Sensitivity Specificity P-value
    EIF1AY OTOF 0.995 0.802 0.934 0.932 0.944 1.99E−
    10
    HLA- OTOF 0.995 0.792 0.956 0.986 0.833 1.08E−
    DQA1 05
    LAMP3 OTOF 0.995 0.831 0.956 0.973 0.889 3.26E−
    07
    OTOF UTY 0.995 0.824 0.934 0.945 0.889 5.76E−
    09
    IFI6 OTOF 0.994 0.792 0.967 1 0.833 7.47E−
    06
    CXCL10 OTOF 0.992 0.824 0.945 0.973 0.833 7.51E−
    06
    JARID1D OTOF 0.992 0.824 0.923 0.932 0.889 8.30E−
    08
    MT1G OTOF 0.991 0.861 0.967 0.973 0.944 3.79E−
    09
    OTOF RGSI 0.991 0.763 0.945 0.973 0.833 7.88E−
    07
    CCL2 OTOF 0.99 0.824 0.934 0.932 0.944 1.10E−
    08
    CEACAM1 OTOF 0.99 0.831 0.945 0.959 0.889 8.23E−
    09
    IFI44L OTOF 0.99 0.831 0.945 0.959 0.889 1.86E−
    08
    CYP1B1 OTOF 0.989 0.831 0.945 0.945 0.944 1.17E−
    08
    F13A1 OTOF 0.989 0.831 0.945 0.959 0.889 5.11E−
    09
    IFI27 OTOF 0.989 0.831 0.923 0.918 0.944 1.32E−
    08
    IFI44 OTOF 0.989 0.792 0.956 0.986 0.833 1.87E−
    06
    IRF7 OTOF 0.989 0.792 0.945 0.945 0.944 7.31E−
    09
    ISG15 OTOF 0.989 0.792 0.945 0.945 0.944 7.76E−
    09
    OAS3 OTOF 0.989 0.802 0.945 0.945 0.944 7.31E−
    09
    OASL OTOF 0.989 0.831 0.945 0.945 0.944 6.89E−
    09
    OTOF PI3 0.989 0.831 0.945 0.945 0.944 8.73E−
    09
    OTOF PLSCR1 0.989 0.802 0.956 0.973 0.889 6.94E−
    07
    OTOF PPBP 0.989 0.831 0.956 0.973 0.889 1.76E−
    08
    OTOF PSTPIP2 0.989 0.736 0.956 0.986 0.833 9.39E−
    06
    OTOF SIGLEC1 0.989 0.831 0.934 0.932 0.944 1.10E−
    08
    OTOF TMEM176A 0.989 0.831 0.945 0.945 0.944 2.80E−
    09
    AIM2 OTOF 0.988 0.792 0.934 0.945 0.889 4.74E−
    07
    BMX OTOF 0.988 0.831 0.945 0.945 0.944 1.10E−
    08
    CYBRD1 OTOF 0.988 0.792 0.945 0.959 0.889 1.64E−
    07
    EIF2AK2 OTOF 0.988 0.831 0.945 0.945 0.944 1.04E−
    08
    IFIT1 OTOF 0.988 0.792 0.934 0.945 0.889 2.66E−
    07
    IFIT2 OTOF 0.988 0.792 0.945 0.945 0.944 9.82E−
    09
    IFIT3 OTOF 0.988 0.831 0.945 0.945 0.944 8.23E−
    09
    JUP OTOF 0.988 0.831 0.923 0.918 0.944 1.24E−
    08
    LY6E OTOF 0.988 0.792 0.945 0.945 0.944 1.04E−
    08
    OAS1 OTOF 0.988 0.792 0.945 0.945 0.944 7.76E−
    09
    OTOF RSAD2 0.988 0.792 0.945 0.945 0.944 1.04E−
    08
    OTOF RTP4 0.988 0.792 0.912 0.904 0.944 1.39E−
    08
    OTOF TNFAIP6 0.988 0.831 0.945 0.945 0.944 1.10E−
    08
    OTOF USP18 0.988 0.831 0.945 0.945 0.944 1.10E−
    08
    HERC5 OTOF 0.987 0.792 0.945 0.945 0.944 7.76E−
    09
    OAS2 OTOF 0.987 0.763 0.934 0.945 0.889 3.65E−
    07
    OTOF SERPING1 0.987 0.792 0.956 0.986 0.833 9.39E−
    06
    MX1 OTOF 0.986 0.792 0.945 0.959 0.889 1.66E−
    08
    HLA- USP18 0.979 0.71 0.901 0.904 0.889 3.36E−
    DQA1 09
  • TABLE 12
    Additional pairs of RNA DETERMINANTS and their measures of accuracy
    in differentiating between bacterial versus viral infected subjects as tested using an
    external data set (measured using AFFYMETRIX ™ HGU133A GENECHIPS ™ on
    73 bacterial and 18 viral patients).
    RNA RNA Total ranksum
    #
    1 #2 ACC MCC accuracy Sensitivity Specificity P-value
    LAMP3 USP18 0.997 0.899 0.978 0.973 1 4.56E−
    10
    CXCL10 USP18 0.989 0.736 0.934 0.932 0.944 3.80E−
    10
    IFI6 LY6E 0.983 0.736 0.912 0.932 0.833 1.32E−
    07
    CCL2 IFI27 0.982 0.869 0.956 0.959 0.944 2.95E−
    08
    IFI6 USP18 0.982 0.802 0.923 0.918 0.944 6.73E−
    10

    Using a larger cohort of patients provided the required statistical power to accurately evaluating of triplets of RNA determinants. A linear logistic regression classifier was developed for each gene triplets (14190 combinations) and their diagnostic accuracy was evaluated. Tables 13 and 14 present different RNA triplets with very high accuracy levels (AUCs between 0.929-1.0; sensitivity between 0.836-1.0).
  • TABLE 13
    Accuracy measures of different RNA triplets that demonstrated very high
    accuracy levels when tested on 73 bacterial and 18 viral patients.
    RNA RNA RNA ranksum Total
    #1 #2 #3 AUC P-value MCC accuracy Sensitivity Specificity
    EIF1AY F13A1 OTOF 1 3.38E−17 0.931 0.978 0.986 0.944
    EIF1AY HERC5 OTOF 1 1.33E−16 0.894 0.967 0.986 0.889
    EIF1AY HLA- OTOF 1 2.80E−14 0.792 0.934 0.959 0.833
    DQA1
    EIF1AY OTOF RGS1 1 7.77E−15 0.82 0.945 0.986 0.778
    EIF2AK2 LAMP3 USP18 1 3.38E−17 0.894 0.967 0.986 0.889
    HLA- LAMP3 USP18 1 5.05E−17 0.936 0.978 0.973 1
    DQA1
    HLA- MT1G OTOF 1 6.19E−17 0.792 0.934 0.959 0.833
    DQA1
    HLA- OTOF UTY 1 6.49E−14 0.743 0.923 0.986 0.667
    DQA1
    IFI44L OTOF UTY 1 2.16E−18 0.967 0.989 0.986 1
    IFI6 JARID1D OTOF 1 1.19E−13 0.785 0.934 0.973 0.778
    IFI6 OTOF UTY 1 2.80E−14 0.792 0.934 0.959 0.833
    LAMP3 OAS1 USP18 1 1.15E−19 0.967 0.989 0.986 1
    LAMP3 OAS3 USP18 1 2.32E−16 0.831 0.945 0.959 0.889
    OTOF PLSCR1 UTY 1 5.86E−17 0.894 0.967 0.986 0.889
    OTOF PSTPIP2 UTY 1 2.73E−16 0.93 0.978 1 0.889
    CCL2 EIF1AY OTOF 0.999 2.17E−07 0.861 0.956 0.973 0.889
    EIF1AY EIF2AK2 OTOF 0.999 7.95E−09 0.824 0.945 0.973 0.833
    EIF1AY IFI44 OTOF 0.999 4.55E−09 0.899 0.967 0.973 0.944
    EIF1AY IFI6 OTOF 0.999 4.24E−06 0.792 0.934 0.959 0.833
    EIF1AY ISG15 OTOF 0.999 1.43E−09 0.899 0.967 0.973 0.944
    EIF1AY LAMP3 OTOF 0.999 7.68E−08 0.824 0.945 0.973 0.833
    EIF1AY OASL OTOF 0.999 7.65E−09 0.894 0.967 0.986 0.889
    LAMP3 PSTPIP2 USP18 0.999 2.80E−07 0.831 0.945 0.959 0.889
    LAMP3 TMEM176A USP18 0.999 1.99E−08 0.899 0.967 0.973 0.944
    AIM2 EIF1AY OTOF 0.998 3.57E−08 0.763 0.923 0.945 0.833
    AIM2 LAMP3 USP18 0.998 1.92E−08 0.899 0.967 0.973 0.944
    CCL2 OTOF UTY 0.998 3.53E−07 0.785 0.934 0.973 0.778
    CXCL10 EIF1AY OTOF 0.998 3.67E−05 0.723 0.912 0.945 0.778
    CYBRD1 LAMP3 USP18 0.998 1.51E−10 0.967 0.989 0.986 1
    CYP1B1 EIF1AY OTOF 0.998 2.74E−10 0.861 0.956 0.973 0.889
    EIF1AY IFI44L OTOF 0.998 8.12E−09 0.831 0.945 0.959 0.889
    EIF1AY IFIT1 OTOF 0.998 2.62E−08 0.763 0.923 0.945 0.833
    EIF1AY IFIT2 OTOF 0.998 3.06E−06 0.785 0.934 0.973 0.778
    EIF1AY IFIT3 OTOF 0.998 1.67E−07 0.792 0.934 0.959 0.833
    EIF1AY IRF7 OTOF 0.998 4.89E−09 0.831 0.945 0.959 0.889
    EIF1AY JARID1D OTOF 0.998 1.36E−06 0.792 0.934 0.959 0.833
    EIF1AY JUP OTOF 0.998 3.43E−10 0.802 0.934 0.945 0.889
    EIF1AY MT1G OTOF 0.998 2.09E−08 0.861 0.956 0.973 0.889
    IFI44 Mx1 OTOF 0.992 3.81E−07 0.831 0.956 0.973 0.889
    IFI44 OTOF RSAD2 0.992 3.43E−07 0.861 0.967 0.986 0.889
    IFI44L Mx1 OTOF 0.992 8.23E−09 0.792 0.945 0.959 0.889
    IFI44 IFI44L OTOF 0.989 1.13E−05 0.603 0.956 0.986 0.833
    IFI44L OTOF RSAD2 0.989 1.03E−05 0.824 0.945 0.973 0.833
    Mx1 OTOF RSAD2 0.958 6.77E−07 0.831 0.956 0.973 0.889
  • TABLE 14
    Accuracy measures of different RNA triplets that demonstrated very high
    accuracy levels when tested on 73 bacterial and 18 viral patients.
    RNA RNA RNA ranksum Total
    #
    1 #2 #3 AUC P-value MCC accuracy Sensitivity Specificity
    HERC5 LAMP3 USP18 1 1.25E−19 1 1 1 1
    IFI44 LAMP3 USP18 1 2.32E−16 0.831 0.945 0.959 0.889
    IFI6 LAMP3 USP18 1 6.79E−19 0.967 0.989 0.986 1
    IFIT3 LAMP3 USP18 1 2.11E−20 0.967 0.989 0.986 1
    ISG15 LAMP3 USP18 1 3.33E−18 0.894 0.967 0.986 0.889
    LAMP3 MX1 USP18 1 1.32E−16 0.907 0.967 0.959 1
    LAMP3 PLSCR1 USP18 1 8.25E−18 0.894 0.967 0.986 0.889
    LAMP3 RSAD2 USP18 1 5.86E−17 0.894 0.967 0.986 0.889
    LAMP3 SERPING1 USP18 1 8.96E−17 0.869 0.956 0.959 0.944
    LAMP3 SIGLEC1 USP18 1 3.93E−16 0.861 0.956 0.973 0.889
    IRF7 LAMP3 USP18 0.999 2.96E−08 0.869 0.956 0.959 0.944
    LAMP3 OAS2 USP18 0.999 1.80E−08 0.861 0.956 0.973 0.889
    IFI44 IFI44L RSAD2 0.948 1.61E−06 0.792 0.813 0.808 0.833
    IFI44 IFI44L Mx1 0.942 1.12E−07 0.622 0.846 0.836 0.889
    IFI44L Mx1 RSAD2 0.938 3.33E−05 0.627 0.846 0.849 0.833
    IFI44 Mx1 RSAD2 0.929 8.64E−06 0.627 0.857 0.863 0.833
    • Example 2 Whole Transcriptome Expression Analysis to Identify RNA-Based Determinants and Signature for Discriminating Between Patients with Bacterial and Viral Infections
  • Materials and Methods
  • Patient recruitment Patients were prospectively recruited as part of the ‘Curiosity’ and the ‘Tailored-Treatment’ clinical studies (NCT01917461 and NCT02025699). Informed consent was obtained from each participant or legal guardian, as applicable. Inclusion criteria for the infectious disease cohort included: clinical suspicion of an acute infectious disease, peak fever >37.5° C. since symptoms onset, and duration of symptoms <12 days. Inclusion criteria for the control group included: clinical impression of a non-infectious disease (e.g. trauma, stroke and myocardial infarction), or healthy subjects. Exclusion criteria included: evidence of any episode of acute infectious disease in the two weeks preceding enrollment; diagnosed congenital immune deficiency; current treatment with immunosuppressive or immunomodulatory therapy; active malignancy, proven or suspected human immunodeficiency virus (HIV)-1, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection. Importantly, in order to enable broad generalization, antibiotic treatment at enrollment did not cause exclusion from the study. An overview of study workflow is depicted in FIG. 1 .
  • Enrollment process and data collection: as in Example 1.
  • Microbiological investigation: as in Example 1.
  • Establishing the reference standard: as in Example 1.
  • Samples, procedures and RNA purification: Venous blood samples were collected in EDTA contained CBC tube and stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma and cell pellet. Red cells were lysed using EL buffer (QIAGEN, Cat 79217) at room temperature (RT). Leukocytes were lysed in RLT buffer (QIAGEN, Cat 79216) and Homogenized via QIAshredder homogenizer (QIAGEN, Cat 79654). Total RNA was purified from 400 μl lysed Leukocytes using RNeasy™ Micro Kit (QIAGEN, Cat. 74004) according to manufacturer recommended protocols.
  • Microarray experiments: A total of 30 of 255 ng/30 (85 ng/μl) RNA were transferred and prepare for whole transcriptome expression analysis with GeneChip™ Whole Transcript (WT) Expression Arrays. Amplified ss-cDNA was prepared from 255 ng total RNA using GeneChip™ WT PLUS Reagent Kit (902310, AFFYMETRIX™), following manufacturer protocol. Samples were hybridized to GENECHIP™ Human Transcriptome Arrays 2.0 (HTA-AFFYMETRIX™) which is the highest resolution microarray for gene expression profiling of all transcript isoforms. HTA display approximately ten probes per exon and four probes per exon-exon splice junction. This array interrogates >245,000 coding transcripts, >40,000 non coding transcripts and >339,000 probe sets covering exon-exon junctions. Arrays were scanned using the AFFYMETRIX™ GENECHIP™ Scanner 3000 7G.
  • Statistical analysis: Primary analysis was performed as in Example 1.
  • Results
  • Patients characteristics The studied group of patients included 71 children and 59 adults and was gender balanced (65 females and 65 males). The cohort included 51 patients with bacterial infection and 79 patients with viral infections as determined by the expert panel as described above. Additionally, we studied 13 non-infectious patients (as controls). The patients presented with a variety of clinical syndromes affecting different physiological systems (e.g., respiratory, urinal, central nervous system, systemic).
  • Single and combinations of RNA determinants can distinguish between bacterial and viral patients The gene expression profiles of blood leukocytes obtained from the described acute infection patients using the GeneChip Human Transcriptome Arrays 2.0 (HTA-Affymetrix) were studied. The results suggest a differential response of the immune system to bacterial and viral infections, which can potentially be used classify acute infection patients. 1089 RNA determinants, out of which 828 are coding determinants (Table 15A) and 261 are non-coding determinants (Table 15B), were identified as differentially expressed in the bacterial and viral patients tested. The present inventors further identified 41 RNA determinants, out of which 34 are coding determinants (Tables 16A-B) and 7 are non-coding determinants (Table 16C), with the highest ability to differentiate between bacterial and viral patients according to the following criteria: ttest p-value<10−14 AND absolute linear fold change >3.5. The present inventors further calculated for these RNA determinants the measures of accuracy in distinguishing between bacterial and viral patients including AUC, sensitivity, specificity and ttest P-value (Tables 15-16). The inventors further evaluated the accuracy of selected RNAs in distinguishing between patients presenting with bacterial (n=51) infections and non-bacterial infections (n=92; Table 16D), as well as between infectious (bacterial and viral; n=130) and non-infectious (n=13) patients (Table 16E).
  • TABLE 15A
    Differentially expressed coding RNA determinants and their measures of
    accuracy in differentiating between bacterial (“B”) versus viral (“V”) infected
    subjects.
    ttest p-
    value Up
    Linear (Bacterial in
    Probe Set Gene mRNA fold vs. B/
    ID Symbol Accession change Viral) V AUC Sensitivity Specificity
    TC14000584. IFI27 NM_001130080 −73.5 1.11E−16 V 0.89 0.84 0.79
    hg.1
    TC22000029. USP18 NM_017414 −34.7 <10-17 V 0.92 0.90 0.84
    hg.1
    TC01000794. IFI44L NM_006820 −31.5 <10-17 V 0.89 0.78 0.85
    hg.1
    TC10000639. IFIT1 NM_001548 −28.2 <10-17 V 0.90 0.80 0.82
    hg.1
    TC0X001155. XIST NR_001564 −27.3 0.210251 V 0.58 0.53 0.52
    hg.1
    TC02000034. RSAD2 NM_080657 −26.3 1.44E−15 V 0.86 0.75 0.85
    hg.1
    TC04000485. HERC5 NM_016323 −16.5 9.99E−16 V 0.87 0.80 0.81
    hg.1
    TC12000885. OAS3 NM_006187 −16.2 1.11E−16 V 0.88 0.78 0.80
    hg.1
    TC20000341. PI3 NM_002638 16.1 9.9E−11 B 0.80 0.69 0.76
    hg.1
    TC12000886. OAS2 NM_001032731 −15.4 <10-17 V 0.89 0.77 0.85
    hg.1
    TC01000795. IFI44 NM_006417 −15.2 1.44E−15 V 0.86 0.78 0.80
    hg.1
    TC20000575. SIGLEC1 NM_023068 −15.1 <10-17 V 0.92 0.88 0.82
    hg.1
    TC19000605. CD 177 NM_020406 14.7 2.57E−07 B 0.76 0.73 0.61
    hg.1
    TC17000383. CCL2 NM_002982 −14.3 2.76E−11 V 0.86 0.94 0.67
    hg.1
    TC19001585. CD177P1 ENST00000378007 14.1 1.98E−07 B 0.77 0.73 0.58
    hg.1
    TC22000519. USP41 ENST00000454608 −13.8 3.33E−16 V 0.91 0.92 0.82
    hg.1
    TC02005020. CMPK2 NM_207315 −13.6 <10-17 V 0.89 0.82 0.82
    hg.1
    TC04001305. CXCL10 NM_001565 −12.9 6.57E−09 V 0.80 0.80 0.67
    hg.1
    TC0X000449. OTTHUMG00000021953; ENST00000420327 11.9 0.601833 B 0.54 0.47 0.48
    hg.1 RP13-
    348B13.2
    TC08000814. LY6E NM_001127213 −11.8 <10-17 V 0.89 0.78 0.86
    hg.1
    TC04000484. HERC6 NM_001165136 −11.2 <10-17 V 0.92 0.88 0.82
    hg.1
    TC12002059. OASL NM_003733 −10.2 3.44E−15 V 0.87 0.78 0.79
    hg.1
    TC0Y000018. LINC00278 ENST00000425031 9.6 0.630938 B 0.54 0.49 0.48
    hg.1
    TC22000467. 0 uc011agg.1 −9.3 0.000022 V 0.71 0.69 0.71
    hg.1
    TC16002057. HP NM_001126102 9.2 3.01E−12 B 0.83 0.73 0.80
    hg.1
    TC0Y000053. DDX3Y NM_001122665 8.9 0.315758 B 0.59 0.53 0.52
    hg.1
    TC21000189. MX1 NM_001144925 −8.8 <10-17 V 0.90 0.84 0.82
    hg.1
    TC06002119. VNN1 NM_004666 8.7 3.45E−12 B 0.84 0.78 0.73
    hg.1
    TC20000876. SLPI NM_003064 8.2 4.89E−11 B 0.81 0.75 0.76
    hg.1
    TC0Y000160. 0 uc022cjh.1 −7.7 0.000012 V 0.72 0.69 0.68
    hg.1
    TC16002034. MT2A NM_005953 −7.7 5.05E−14 V 0.86 0.78 0.76
    hg.1
    TC03002054. LAMP3 NM_014398 −7.6 4.24E−10 V 0.83 0.78 0.67
    hg.1
    TC02004946. IGKV2-28 ENST00000482769 −7.5 0.000008 V 0.73 0.73 0.67
    hg.1
    TC02004974. IGKV1D-39 ENST00000448155 −7.4 0.000042 V 0.71 0.71 0.66
    hg.1
    TC19000134. C19orf59 NM_174918 7.1 2.3E−10 B 0.82 0.82 0.71
    hg.1
    TC04001718. DDX60 NM_017631 −7.0 5.66E−15 V 0.85 0.78 0.84
    hg.1
    TC02001159. SPATS2L NM_001100422 −6.9 1.78E−13 V 0.87 0.84 0.73
    hg.1
    TC02001866. PNPT1 NM_033109 −6.8 <10-17 V 0.93 0.90 0.82
    hg.1
    TC0Y000163. UTY NM_182660 6.8 0.438206 B 0.57 0.53 0.52
    hg.1
    TC04000362. STAPI NM_012108 −6.5 7.46E−14 V 0.85 0.80 0.79
    hg.1
    TC17001523. DHX58 NM_024119 −6.5 4.79E−14 V 0.87 0.82 0.81
    hg.1
    TC07001916. PARP12 NM_022750 −6.4 <10-17 V 0.89 0.78 0.79
    hg.1
    TC06000983. ARG1 NM_000045 6.4 2.25E−08 B 0.78 0.69 0.72
    hg.1
    TC12000884. OAS1 NM_001032409 −6.4 1.11E−16 V 0.88 0.78 0.84
    hg.1
    TC01000272. ALPL NM_000478 6.3 6.33E−13 B 0.90 0.88 0.76
    hg.1
    TC02001749. CYP1B1 OTTHUMT00000325647 6.3 1.11E−16 B 0.88 0.82 0.81
    hg.1
    TC02004979. IGKV1D-27 ENST00000558152 −6.2 0.000015 V 0.71 0.69 0.67
    hg.1
    TC02004982. IGKV1D-16 ENST00000492446 −6.2 0.000036 V 0.71 0.73 0.63
    hg.1
    TC02004978. IGKV2D-28 ENST00000453166 −6.1 0.000005 V 0.73 0.73 0.66
    hg.1
    TC02004976. IGKV1D-33 ENST00000390265 −6.1 0.000023 V 0.72 0.73 0.65
    hg.1
    TC22000191. KREMEN1 NM_001039570 5.9 1.11E−16 B 0.89 0.82 0.84
    hg.1
    TC22001449. IGLC7 ENST00000390331 −5.9 0.000006 V 0.72 0.63 0.72
    hg.1
    TC02001750. CYP1B1 NM_000104 5.8 <10-17 B 0.88 0.78 0.80
    hg.1
    TC02004975. IGKV1D−37 ENST00000509129 −5.8 0.000042 V 0.71 0.75 0.65
    hg.1
    TC10000637. IFIT3 NM_001031683 −5.8 4.29E−12 V 0.84 0.71 0.80
    hg.1
    TC06002156. OTTHUMG00000015665; ENST00000417932 −5.6 0.000008 V 0.74 0.73 0.61
    hg.1 RP11-10J5.1
    TC01002412. IFI6 NM_002038 −5.5 7.37E−14 V 0.87 0.71 0.82
    hg.1
    TC02000531. 0 ENST00000448813 −5.5 0.000007 V 0.72 0.69 0.70
    hg.1
    TC02000559. IGKV3D−7 ENST00000423080 −5.5 0.000017 V 0.72 0.69 0.65
    hg.1
    TC14002215. IGHJ5 ENST00000488476 −5.3 0.001438 V 0.67 0.67 0.58
    hg.1
    TC0Y000072. EIF1AY NM_004681 5.3 0.41431 B 0.57 0.53 0.52
    hg.1
    TC18000223. ZCCHC2 NM_017742 −5.2 6.66E−16 V 0.87 0.77 0.77
    hg.1
    TC02002481. IFIH1 NM_022168 −5.2 1.18E−11 V 0.82 0.71 0.71
    hg.1
    TC02004944. IGKV2-24 ENST00000484817 −5.2 0.000016 V 0.73 0.73 0.65
    hg.1
    TC10000640. IFIT5 NM_012420 −5.1 2.18E−13 V 0.84 0.78 0.79
    hg.1
    TC02000082. TRIB2 NM_021643 −5.1 2.78E−15 V 0.88 0.78 0.85
    hg.1
    TC02004941. IGKV1-9 ENST00000493819 −5.0 0.000014 V 0.72 0.73 0.65
    hg.1
    TC16000397. 0 uc002edk. 1 −5.0 0.001747 V 0.66 0.65 0.60
    hg.1
    TC20000363. MMP9 NM_004994 4.9 5.78E−10 B 0.81 0.78 0.71
    hg.1
    TC22000122. 0 ENST00000385098 −4.9 0.000058 V 0.70 0.63 0.67
    hg.1
    TC14002234. IGHD3-10 ENST00000390583 −4.9 0.014346 V 0.63 0.65 0.51
    hg.1
    TC02000553. IGKV2D-29 ENST00000491977 −4.9 0.000049 V 0.71 0.75 0.63
    hg.1
    TC14002257. IGHV3-23 ENST00000390609 −4.9 0.001561 V 0.67 0.63 0.66
    hg.1
    TC16000387. 0 uc002ecv.1 −4.9 0.001933 V 0.66 0.65 0.65
    hg.1
    TC22000119. IGLV2-18 ENST00000390310 −4.8 0.000062 V 0.70 0.67 0.66
    hg.1
    TC11000467. SERPING1 NM_000062 −4.8 1.4E−07 V 0.76 0.65 0.70
    hg.1
    TC11002227. 0 uc021qph.l −4.8 6.82E−12 V 0.85 0.78 0.70
    hg.1
    TC04001270. IGJ NM_144646 −4.7 0.00001 V 0.72 0.71 0.68
    hg.1
    TC02004939. IGKV1-6 ENST00000464162 −4.7 0.00003 V 0.71 0.73 0.63
    hg.1
    TC17000077. XAF1 NM_017523 −4.7 3.8E−14 V 0.86 0.69 0.82
    hg.1
    TC02000554. IGKV2D-26 ENST00000390268 −4.7 0.000015 V 0.72 0.75 0.63
    hg.1
    TC02004947. IGKV2-29 ENST00000558710 −4.7 0.000264 V 0.70 0.73 0.58
    hg.1
    TC02004945. IGKV1-27 ENST00000498435 −4.7 0.00001 V 0.73 0.73 0.63
    hg.1
    TC14002213. IGHM ENST00000390559 −4.7 0.004526 V 0.66 0.63 0.63
    hg.1
    TC11000812. DGAT2 NM_001253891 4.6 7.77E−16 B 0.89 0.88 0.80
    hg.1
    TC14002260. LOC100653243; ENST00000390612 −4.6 0.001009 V 0.67 0.67 0.62
    hg.1 LOC642131;
    IGHV4-28
    TC0X000067. BMX NM_001721 4.6 9.33E−11 B 0.82 0.75 0.72
    hg.1
    TC01000023. ISG15 NM_005101 −4.6 5.72E−13 V 0.88 0.86 0.73
    hg.1
    TC17001129. GAS7 NM_201433 4.5 <10-17 B 0.91 0.82 0.87
    hg.1
    TC14002211. IGHA1 ENST00000390547 −4.5 0.000025 V 0.72 0.63 0.71
    hg.1
    TC04000151. LAP3 NM_015907 −4.5 4.11E−09 V 0.77 0.69 0.77
    hg.1
    TC22001442. IGLV3-25 ENST00000390305 −4.5 0.003579 V 0.66 0.73 0.56
    hg.1
    TC14002254. IGHV1-18 ENST00000390605 −4.4 0.003737 V 0.65 0.69 0.58
    hg.1
    TC14002241. IGHD2-2 ENST00000390591 −4.4 0.021171 V 0.62 0.67 0.53
    hg.1
    TC10001366. PRF1 NM_001083116 −4.3 2.69E−09 V 0.79 0.78 0.80
    hg.1
    TC07000899. MGAM NM_004668 4.3 4.8E−14 B 0.89 0.86 0.79
    hg.1
    TC12001843. LTA4H NM_000895 4.3 <10-17 B 0.93 0.86 0.85
    hg.1
    TC16001073. 0 ENST00000450856 −4.2 0.003437 V 0.65 0.65 0.57
    hg.1
    TC02004943. IGKV1-17 ENST00000490686 −4.2 0.00006 V 0.71 0.75 0.63
    hg.1
    TC10000636. IFIT2 NM_001547 −4.2 1.36E−10 V 0.82 0.71 0.73
    hg.1
    TC01001140. 0 uc021ovy.1 −4.2 0.000654 V 0.67 0.65 0.67
    hg.1
    TC02004950. IGKC; IGKJ5; ENST00000390237 −4.2 0.000009 V 0.72 0.73 0.68
    hg.1 IGKJ4; IGKJ3;
    IGKJ2; IGKJ1;
    IGKV3-15;
    IGKV2-30;
    IGKV1-33;
    IGKV1-16;
    IGKV1-12;
    IGKV3-20;
    IGKV1-39;
    IGK;
    OTTHUMG00000151686;
    AC096579.7;
    OTTHUMG00000151694;
    AC096579.13
    TC22001445. IGLV3-1 ENST00000390319 −4.1 0.000276 V 0.69 0.71 0.58
    hg.1
    TC02004942. IGKV3-11 ENST00000483158 −4.1 0.000019 V 0.72 0.73 0.63
    hg.1
    TC14002261. IGHV3-30 ENST00000390613 −4.1 0.000883 V 0.67 0.65 0.66
    hg.1
    TC22000117. 0 ENST00000385099 −4.1 0.000153 V 0.69 0.67 0.63
    hg.1
    TC11002231. MMP8 NM_002424 4.1 0.000012 B 0.72 0.61 0.71
    hg.1
    TC04001373. PPM1K NM_152542 −4.0 <10-17 V 0.91 0.84 0.85
    hg.1
    TC17000396. SLFN5 NM_144975 −4.0 3.84E−13 V 0.84 0.80 0.80
    hg.1
    TC17002907. JUP NM_002230 −4.0 3.36E−11 V 0.82 0.75 0.75
    hg.1
    TC03001031. RTP4 NM_022147 −4.0 1.04E−09 V 0.79 0.67 0.72
    hg.1
    TC02001739. EIF2AK2 NM_001135651 −3.9 4.59E−14 V 0.86 0.75 0.80
    hg.1
    TC19001640. PGLYRP1 NM_005091 3.9 3.49E−14 B 0.86 0.82 0.80
    hg.1
    TC01003498. F5 NM_000130 3.9 6.05E−12 B 0.83 0.78 0.76
    hg.1
    TC01001738. CR1 NM_000573 3.9 <10-17 B 0.90 0.88 0.77
    hg.1
    TC04000145. CD38 NM_001775 −3.9 7.61E−12 V 0.84 0.82 0.79
    hg.1
    TC04001753. HPGD NM_000860 3.9 4.36E−11 B 0.82 0.69 0.80
    hg.1
    TC02002766. CXCR2P1 NR_002712 −3.9 1.02E−10 V 0.82 0.71 0.73
    hg.1
    TC02000245. GALM NM_138801 −3.8 3.55E−08 V 0.80 0.78 0.61
    hg.1
    TC05001087. 0 ENST00000410601 3.7 2.28E−07 B 0.76 0.75 0.70
    hg.1
    TC14000982. GZMB NM_004131 −3.7 2.33E−09 V 0.80 0.78 0.79
    hg.1
    TC04001267. SULT1B1 NM_014465 3.7 3.33E−16 B 0.88 0.78 0.76
    hg.1
    TC04000248. NSUN7 NM_024677 3.6 1.67E−13 B 0.86 0.77 0.82
    hg.1
    TC09000999. DDX58 NM_014314 −3.6 2.56E−10 V 0.81 0.73 0.71
    hg.1
    TC01002055. 0 ENST00000410691 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC01003865. 0 ENST00000410467 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC04001502. 0 ENST00000411276 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC09000858. 0 ENST00000411324 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC16000715. 0 ENST00000410427 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC19000980. 0 ENST00000410397 3.6 8.8E−07 B 0.76 0.71 0.67
    hg.1
    TC09000274. 0 uc004ady.3 3.6 3.81E−07 B 0.75 0.63 0.71
    hg.1
    TC14002191. TRDJ1; ENST00000390473 −3.6 1.05E−07 V 0.77 0.77 0.76
    hg.1 TRDJ4;
    TRDJ2;
    TRDJ3; TRDC
    TC18000060. IMPA2 NM_014214 3.6 <10-17 B 0.93 0.86 0.86
    hg.1
    TC07001605. SAMD9 NM_001193307 −3.6 2.6E−14 V 0.86 0.82 0.79
    hg.1
    TC14001124. PYGL NM_001163940 3.6 <10-17 B 0.91 0.90 0.80
    hg.1
    TC01002287. PADI2 NM_007365 3.5 5.71E−14 B 0.87 0.77 0.76
    hg.1
    TC02001723. NLRC4 NM_001199138 3.5 1.39E−14 B 0.87 0.80 0.77
    hg.1
    TC10000258. 0 ENST00000410382 3.5 0.000001 B 0.76 0.73 0.67
    hg.1
    TC11001227. 0 ENST00000410293 3.5 8.83E−07 B 0.76 0.75 0.67
    hg.1
    TC13000612. EPSTI1 NM_001002264 −3.5 1.58E−11 V 0.84 0.67 0.85
    hg.1
    TC05000097. 0 ENST00000517099 3.5 6.93E−08 B 0.77 0.71 0.72
    hg.1
    TC09000998. 0 uc003zqz.1 −3.5 2.79E−09 V 0.78 0.75 0.66
    hg.1
    TC04001372. 0 uc003hrl.1 −3.5 2.22E−16 V 0.89 0.82 0.80
    hg.1
    TC04000606. 0 ENST00000411293 3.5 3.41E−07 B 0.76 0.73 0.68
    hg.1
    TC06000633. CD2AP NM_012120 −3.5 <10-17 V 0.89 0.80 0.79
    hg.1
    TC12001569. GPR84 NM_020370 3.4 3.22E−10 B 0.80 0.69 0.77
    hg.1
    TC11001241. IRF7 NM_004031 −3.3 1.74E−10 V 0.81 0.77 0.71
    hg.1
    TC19000120. EMR1 NM_001256252 3.3 3.8E−12 B 0.84 0.77 0.73
    hg.1
    TC01000232. PADI4 NM_012387 3.3 5.96E−11 B 0.82 0.78 0.77
    hg.1
    TC13000828. GPR18 NM_001098200 −3.3 6.92E−11 V 0.82 0.75 0.72
    hg.1
    TC04000826. KLHL2 NM_001161522 3.3 5.55E−16 B 0.89 0.84 0.84
    hg.1
    TC07001067. 0 ENST00000469418 3.3 1.13E−11 B 0.84 0.73 0.73
    hg.1
    TC09001097. 0 uc004abr.1 3.3 1.7E−07 B 0.76 0.59 0.73
    hg.1
    TC14000810. TDRD9 NM_153046 3.3 1.95E−12 B 0.82 0.71 0.86
    hg.1
    TC20000726. APMAP NM_020531 3.3 2.31E−14 B 0.86 0.80 0.81
    hg.1
    TC20000979. ZBP1 NM_001160418 −3.3 7.7E−10 V 0.80 0.77 0.67
    hg.1
    TC0X000051. TLR7 NM_016562 −3.2 4.92E−10 V 0.82 0.77 0.76
    hg.1
    TC16000473. MT1F NM_005949 −3.2 8.19E−11 V 0.82 0.82 0.71
    hg.1
    TC09000724. TOR1B NM_014506 −3.2 1.56E−09 V 0.79 0.86 0.71
    hg.1
    TC11002226. TMEM123 NM_052932 −3.2 <10-17 V 0.92 0.82 0.86
    hg.1
    TC02005019. OTTHUMG00000151730; ENST00000366140 −3.2 2.34E−12 V 0.87 0.84 0.71
    hg.1 AC017076.5
    TC01002071. 0 ENST00000411249 3.2 9.46E−07 B 0.75 0.69 0.65
    hg.1
    TC17001917. SOCS3 NM_003955 3.2 1.42E−08 B 0.78 0.82 0.73
    hg.1
    TC06000545. FTSJD2 NM_015050 −3.1 4.23E−13 V 0.85 0.78 0.79
    hg.1
    TC12001602. STAT2 NM_005419 −3.1 3.38E−10 V 0.80 0.69 0.77
    hg.1
    TC17000965. FAM101B NM_182705 3.1 2.34E−09 B 0.79 0.71 0.70
    hg.1
    TC08000873. LOC101060626; ENST00000523795 3.1 2.67E−11 B 0.82 0.73 0.72
    hg.1 LOC100653358;
    LOC100288646;
    LOC100286914
    TC07001582. STEAP4 NM_001205315 3.1 1.74E−12 B 0.85 0.84 0.77
    hg.1
    TC05000983. 0 ENST00000363778 3.1 7.41E−07 B 0.75 0.71 0.62
    hg.1
    TC03001720. HEG1 NM_020733 −3.1 5.35E−14 V 0.87 0.82 0.81
    hg.1
    TC05003444. LOC101060626; ENST00000509192 3.1 1.17E−10 B 0.81 0.73 0.73
    hg.1 LOC100653358;
    LOC100288646;
    LOC 100286914;
    OTTHUMG00000162985;
    AC138035.1
    TC12000152. KLRF1 NM_016523 −3.1 8.77E−08 V 0.79 0.75 0.73
    hg.1
    TC01003871. TLR5 NM_003268 3.1 8.84E−13 B 0.85 0.77 0.75
    hg.1
    TC12000591. IRAK3 NM_001142523 3.1 1.33E−14 B 0.87 0.78 0.80
    hg.1
    TC16000471. MT1CP ENST00000379816 −3.1 9.9E−12 V 0.84 0.86 0.68
    hg.1
    TC17000545. IFI35 NM_005533 −3.1 8.5E−08 V 0.76 0.67 0.71
    hg.1
    TC06001204. OTTHUMG00000016090; ENST00000426839 3.1 7.09E−10 B 0.79 0.69 0.79
    hg.1 XX-
    C2158C6.1
    TC12001578. TESPA1 NM_001136030 −3.1 1.11E−07 V 0.78 0.65 0.72
    hg.1
    TC17000736. CA4 NM_000717 3.1 1.54E−13 B 0.86 0.73 0.79
    hg.1
    TC12001207. CD69 NM_001781 −3.0 2.79E−08 V 0.78 0.73 0.66
    hg.1
    TC01001408. 0 uc021pdm. 1 −3.0 1.52E−09 V 0.80 0.73 0.71
    hg.1
    TC02000445. ALMS1P NR_003683 −3.0 3.84E−07 V 0.80 0.80 0.57
    hg.1
    TC02002891. ARL4C NM_005737 −3.0 8.84E−08 V 0.76 0.73 0.77
    hg.1
    TC20000134. RIN2 NM_001242581 −3.0 4.65E−10 V 0.82 0.88 0.66
    hg.1
    TC05002100. HK3 NM_002115 3.0 1.3E−13 B 0.85 0.73 0.79
    hg.1
    TC01000008. LOC101060495; ENST00000455464 3.0 6.13E−11 B 0.82 0.75 0.73
    hg.1 LOC101060494;
    LOC101059936;
    LOC100996502;
    LOC100996328;
    LOC100287894
    TC01001050. 0 uc001eis.2 −3.0 1.68E−12 V 0.88 0.84 0.75
    hg.1
    TC02005009. SP140 NM_001005176 −3.0 1.44E−12 V 0.82 0.69 0.72
    hg.1
    TC02001556. MBOAT2 NM_138799 2.9 1.47E−12 B 0.85 0.78 0.81
    hg.1
    TC01000514. ZNF684 NM_152373 −2.9 1.05E−09 V 0.84 0.80 0.67
    hg.1
    TC19000318. HSH2D NM_032855 −2.9 7.59E−14 V 0.86 0.82 0.73
    hg.1
    TC01001347. PYHIN1 NM_152501 −2.9  1.5E−08 V 0.79 0.73 0.79
    hg.1
    TC02002644. PGAP1 NM_024989 −2.9 6.21E−12 V 0.88 0.82 0.67
    hg.1
    TC10001555. BLNK NM_001114094 −2.9 2.38E−07 V 0.76 0.73 0.66
    hg.1
    TC0X000922. OTTHUMG00000021255; ENST00000366134 −2.9 2.82E−07 V 0.75 0.84 0.58
    hg.1 RP13-
    314C10.5
    TC12001116. PARP11 NM_020367 −2.9 9.09E−11 V 0.81 0.71 0.67
    hg.1
    TC01003624. FAM129A NM_052966 2.8 1.49E−14 B 0.89 0.88 0.80
    hg.1
    TC01003878. 0 ENST00000390855 2.8 8.25E−07 B 0.75 0.71 0.71
    hg.1
    TC01004037. 0 ENST00000390928 2.8 8.25E−07 B 0.75 0.71 0.71
    hg.1
    TC07000284. 0 ENST00000391148 2.8 8.25E−07 B 0.75 0.71 0.71
    hg.1
    TC06001668. SRPK1 NM_003137 2.8 1.55E−15 B 0.88 0.82 0.79
    hg.1
    TC12000160. KLRD1 NM_002262 −2.8 0.000001 V 0.77 0.75 0.70
    hg.1
    TC01002977. KCNA3 NM_002232 −2.8 1.24E−07 V 0.78 0.69 0.81
    hg.1
    TC12000487. METTL7B NM_152637 2.8 1.98E−07 B 0.72 0.57 0.75
    hg.1
    TC20000942. NFATC2 NM_001136021 −2.8 1.4E−08 V 0.79 0.69 0.77
    hg.1
    TC01000992. MOV10 NM_001130079 −2.8 1.83E−09 V 0.80 0.69 0.65
    hg.1
    TC01002052. OTTHUMG00000001096; ENST00000453576 2.8 8.61E−12 B 0.82 0.73 0.75
    hg.1 RP11-34P13.7;
    OTTHUMG00000001097;
    RP11-34P13.8
    TC17000349. CPD NM_001199775 2.8 3.16E−10 B 0.83 0.77 0.76
    hg.1
    TC14001125. 0 ENST00000554141 2.8 1.04E−08 B 0.77 0.69 0.76
    hg.1
    TC17001924. LGALS3BP NM_005567 −2.8 1.88E−08 V 0.79 0.75 0.71
    hg.1
    TC01001074. SRGAP2B; NR_034178 −2.8  1.5E−11 V 0.86 0.82 0.68
    hg.1 LOC100505630
    TC03000634. PARP14 NM_017554 −2.8 1.19E−08 V 0.78 0.65 0.72
    hg.1
    TC01002411. OTTHUMG00000003519; ENST00000440492 −2.7 2.11E−10 V 0.84 0.88 0.67
    hg.1 RP11-288L9.1
    TC06001205. LINC00266-3 ENST00000436899 2.7 6.66E−11 B 0.81 0.73 0.73
    hg.1
    TC11001593. 0 uc021qgp.1 2.7 5.22E−11 B 0.84 0.77 0.79
    hg.1
    TC15001837. ANPEP NM_001150 2.7 1.04E−09 B 0.81 0.73 0.71
    hg.1
    TC17001311. FLOT2 NM_004475 2.7 7.24E−14 B 0.86 0.78 0.76
    hg.1
    TC19002672. LRG1 NM_052972 2.7 9.67E−09 B 0.79 0.75 0.76
    hg.1
    TC07001264. NT5C3A; NM_001002010 −2.7 8.98E−10 V 0.80 0.78 0.67
    hg.1 NT5C3
    TC01001740. CR1L NM_175710 2.7 <10-17 B 0.90 0.86 0.80
    hg.1
    TC02001713. GALNT14 NM_001253826 2.7 1.94E−13 B 0.84 0.77 0.81
    hg.1
    TC04000437. ANXA3 NM_005139 2.6 1.03E−08 B 0.80 0.78 0.68
    hg.1
    TC12001370. 0 ENST00000535163 −2.6 4.26E−08 V 0.79 0.80 0.66
    hg.1
    TC13000722. DACH1 NM_080759 2.6 3.47E−13 B 0.87 0.80 0.84
    hg.1
    TC16000374. ITGAM NM_000632 2.6 2.89E−15 B 0.89 0.78 0.79
    hg.1
    TC16000718. 0 uc002fqw.3 2.6 3.8E−11 B 0.81 0.69 0.75
    hg.1
    TC03000637. LOC100129550 NR_024618 2.6 1.92E−14 B 0.85 0.77 0.77
    hg.1
    TC16000293. IL4R NM_000418 2.6 5.85E−13 B 0.85 0.82 0.72
    hg.1
    TC21000470. OTTHUMG00000086754; ENST00000411427 −2.6 2.62E−09 V 0.83 0.82 0.62
    hg.1 AP001610.5
    TC18000436. DSC2 NM_004949 2.6 7.35E−08 B 0.78 0.69 0.71
    hg.1
    TC02000184. PLB1 NM_001170585 2.6 1.66E−12 B 0.85 0.78 0.82
    hg.1
    TC15000949. IGF1R NM_000875 2.6 1.08E−12 B 0.85 0.78 0.79
    hg.1
    TC02001034. ZAK; NM_016653 2.6 5.55E−16 B 0.88 0.80 0.86
    hg.1 OTTHUMG00000132297;
    AC013461.1
    TC03000633. PARP15 NM_001113523 −2.6 1.65E−07 V 0.77 0.69 0.70
    hg.1
    TC03000801. GYG1 NM_001184720 2.6 3.48E−12 B 0.84 0.80 0.72
    hg.1
    TC06000523. MAPK14 NM_001315 2.6 1.89E−15 B 0.88 0.77 0.77
    hg.1
    TC09001092. CNTNAP3 NM_033655 2.6 5.62E−07 B 0.75 0.61 0.75
    hg.1
    TC11000374. CD82 NM_001024844 2.6 3.05E−14 B 0.86 0.77 0.80
    hg.1
    TC02000432. DYSF NM_001130455 2.6 1.17E−11 B 0.85 0.78 0.73
    hg.1
    TC01000745. GADD45A NM_001199741 2.6 2.9E−09 B 0.81 0.63 0.79
    hg.1
    TC09001403. TRIM14 NM_033220 −2.6 3.11E−15 V 0.88 0.84 0.80
    hg.1
    TC20000164. CST7 NM_003650 2.6 1.97E−08 B 0.80 0.82 0.70
    hg.1
    TC19001088. RFX2 NM_000635 2.6 3.13E−14 B 0.86 0.80 0.81
    hg.1
    TC02002707. KLF7-IT1 ENST00000428777 2.5 0.000001 B 0.74 0.67 0.73
    hg.1
    TC12003236. CLEC2D NM_001004419 −2.5 3.4E−09 V 0.79 0.67 0.73
    hg.1
    TC11000526. CD5 NM_014207 −2.5 0.000001 V 0.75 0.63 0.71
    hg.1
    TC21000188. MX2 NM_002463 −2.5 1.7E−10 V 0.82 0.86 0.76
    hg.1
    TC02001072. OTTHUMG00000154423; ENST00000436616 −2.5 9.95E−14 V 0.92 0.92 0.82
    hg.1 AC009948.5
    TC16000455. LPCAT2 NM_017839 2.5 8.79E−12 B 0.86 0.77 0.80
    hg.1
    TC04000607. 0 uc021xrc.1 2.5 2.05E−09 B 0.81 0.77 0.72
    hg.1
    TC05000075. ROPN1L NM_001201466 2.5  6.2E−08 B 0.79 0.73 0.77
    hg.1
    TC06001065. RAB32 NM_006834 2.5 1.11E−16 B 0.89 0.77 0.80
    hg.1
    TC08001264. ASPH NM_001164750 2.5 6.56E−08 B 0.78 0.69 0.71
    hg.1
    TC04001442. OTTHUMG00000160994; ENST00000504082 2.5 2.52E−11 B 0.81 0.75 0.75
    hg.1 AC004069.2
    TC19000426. DPY19L3 NM_001172774 2.5 4.44E−16 B 0.88 0.80 0.82
    hg.1
    TC19000772. SIGLEC9 NM_001198558 2.5 1.34E−12 B 0.86 0.78 0.76
    hg.1
    TC19001275. BST2 NM_004335 −2.5 9.91E−09 V 0.77 0.67 0.66
    hg.1
    TC07001416. 0 ENST00000411398 2.5 1.92E−08 B 0.77 0.69 0.72
    hg.1
    TC07001606. SAMD9L NM_152703 −2.5 1.61E−08 V 0.78 0.59 0.80
    hg.1
    TC11001794. TIMM10 NM_012456 −2.5 2.33E−07 V 0.77 0.75 0.58
    hg.1
    TC02002827. DOCK10 NM_014689 −2.5 4.05E−08 V 0.79 0.67 0.76
    hg.1
    TC20001739. LINC00266-1 BC122537 2.5 3.04E−11 B 0.81 0.73 0.75
    hg.1
    TC16000717. 0 uc021tmw.l 2.4 1.19E−09 B 0.81 0.75 0.68
    hg.1
    TC07001920. SLC37A3 NM_032295 2.4 1.26E−10 B 0.82 0.69 0.76
    hg.1
    TC09000565. HSDL2 NM_001195822 2.4 1.86E−13 B 0.85 0.84 0.79
    hg.1
    TC10000259. OTTHUMG00000017996; ENST00000428915 2.4  5.8E−12 B 0.82 0.71 0.77
    hg.1 RP11-
    291L22.4
    TC15000930. MCTP2 NM_001159643 2.4  1.6E−10 B 0.82 0.77 0.73
    hg.1
    TC15001079. ATP10A NM_024490 −2.4 <10-17 V 0.90 0.84 0.81
    hg.1
    TC01001721. SRGAP2; NM_001042758 −2.4 6.08E−12 V 0.88 0.80 0.76
    hg.1 SRGAP2D
    TC15000671. PML NM_002675 −2.4 8.53E−08 V 0.77 0.69 0.65
    hg.1
    TC01003479. CD247 NM_000734 −2.4 9.33E−08 V 0.76 0.73 0.75
    hg.1
    TC09001228. GNAQ NM_002072 2.4 2.22E−15 B 0.89 0.82 0.85
    hg.1
    TC17001372. SLFN12L NM_001195790 −2.4  2.3E−10 V 0.82 0.77 0.75
    hg.1
    TC01002060. OTTHUMG00000002331; ENST00000414688 2.4 2.29E−11 B 0.82 0.71 0.77
    hg.1 RP5-857K21.4
    TC08000298. ADAM9 NM_003816 2.4 6.31E−14 B 0.86 0.73 0.77
    hg.1
    TC13000829. GPR183 NM_004951 −2.4 4.57E−07 V 0.76 0.61 0.73
    hg.1
    TC17001794. ICAM2 NM_000873 −2.4 7.45E−13 V 0.85 0.71 0.81
    hg.1
    TC09000490. TDRD7 NM_014290 −2.4 4.39E−07 V 0.75 0.75 0.67
    hg.1
    TC16002058. HPR NM_020995 2.4 1.68E−11 B 0.84 0.77 0.82
    hg.1
    TC01000129. PGD NM_002631 2.4 2.22E−16 B 0.89 0.82 0.84
    hg.1
    TC0X001544. MPP1 NM_001166460 2.4 <10-17 B 0.91 0.84 0.84
    hg.1
    TC10001444. 0 uc001jzc.1 2.4 <10-17 B 0.90 0.88 0.82
    hg.1
    TC16000474. MT1H NM_005951 −2.4 4.13E−12 V 0.85 0.86 0.75
    hg.1
    TC20000685. RALGAPA2 NM_020343 2.4 6.71E−09 B 0.78 0.78 0.75
    hg.1
    TC05001449. 0 uc003jyj.1 2.4  1.3E−07 B 0.78 0.75 0.67
    hg.1
    TC12001772. LIN7A NM_004664 2.4   2E−09 B 0.81 0.77 0.73
    hg.1
    TC01000127. KIF1B NM_015074 2.4 1.89E−10 B 0.82 0.73 0.75
    hg.1
    TC01001881. 0 uc001hrn.2 2.4 1.16E−09 B 0.82 0.80 0.73
    hg.1
    TC01002954. SORT1 NM_001205228 2.4 1.15E−07 B 0.78 0.71 0.77
    hg.1
    TC01003854. 0 uc010puo.1 2.4 1.16E−09 B 0.82 0.80 0.73
    hg.1
    TC03000378. PXK NM_017771 2.4 2.44E−15 B 0.87 0.80 0.80
    hg.1
    TC03000636. DIRC2 NM_032839 2.4 3.33E−16 B 0.88 0.77 0.77
    hg.1
    TC04000439. LOC100505702; NR_038303 2.4 3.72E−12 B 0.83 0.73 0.80
    hg.1 OTTHUMG00000160897;
    RP11-
    792D21.2
    TC09000712. OTTHUMG00000020775; ENST00000455981 2.4 2.13E−11 B 0.81 0.73 0.75
    hg.1 RP11-344B5.2
    TC12001941. SSH1 NM_001161330 2.4 <10-17 B 0.92 0.88 0.86
    hg.1
    TC07000843. NUP205 NM_015135 −2.3 1.11E−16 V 0.90 0.78 0.85
    hg.1
    TC09001287. AGTPBP1 NM_015239 2.3 <10-17 B 0.89 0.82 0.85
    hg.1
    TC0Y000089. 0 uc022cow.1 2.3 1.05E−09 B 0.83 0.80 0.73
    hg.1
    TC0Y000215. 0 uc022cpa.1 2.3 1.05E−09 B 0.83 0.80 0.73
    hg.1
    TC10000053. PFKFB3 NM_001145443 2.3 9.59E−08 B 0.77 0.75 0.68
    hg.1
    TC11001107. SORL1 NM_003105 2.3 2.59E−13 B 0.87 0.84 0.79
    hg.1
    TC14000781. TECPR2 NM_014844 2.3 2.89E−09 B 0.81 0.77 0.76
    hg.1
    TC15000796. TM6SF1 NM_001144903 2.3 6.81E−11 B 0.83 0.77 0.73
    hg.1
    TC17001890. ACOX1 NM_001185039 2.3 3.46E−14 B 0.86 0.82 0.80
    hg.1
    TC20001000. PPP1R3D NM_006242 2.3 2.09E−10 B 0.81 0.73 0.77
    hg.1
    TC05001766. OTTHUMG00000059494; ENST00000422204 2.3 1.08E−08 B 0.79 0.75 0.73
    hg.1 AC116366.5
    TC13000425. TMCO3 NM_017905 2.3 <10-17 B 0.91 0.84 0.86
    hg.1
    TC15000844. ABHD2 NM_007011 2.3 4.43E−13 B 0.86 0.84 0.79
    hg.1
    TC16000098. MMP25 NM_022468 2.3 1.42E−09 B 0.82 0.80 0.76
    hg.1
    TC20000552. 0 ENST00000516294 2.3 3.57E−12 B 0.82 0.73 0.75
    hg.1
    TC20000944. ATP9A NM_006045 2.3 3.92E−10 B 0.84 0.69 0.89
    hg.1
    TC02002705. KLF7 NM_003709 2.3 <10-17 B 0.89 0.78 0.82
    hg.1
    TC14000371. PRKCH NM_006255 −2.3 2.66E−08 V 0.78 0.71 0.76
    hg.1
    TC19000564. SHKBP1 NM_138392 2.3 <10-17 B 0.90 0.82 0.84
    hg.1
    TC10002945. ENTPD7 NM_020354 2.3 2.73E−09 B 0.83 0.67 0.86
    hg.1
    TC12001981. 0 ENST00000552784 −2.3 3.22E−12 V 0.87 0.80 0.73
    hg.1
    TC18000009. NDC80 NM_006101 −2.3 7.22E−15 V 0.90 0.84 0.79
    hg.1
    TC20000402. LOC284751; NR_034124 2.3 0.000001 B 0.76 0.73 0.72
    hg.1 OTTHUMG00000032719;
    RP11-
    290F20.1
    TC11001414. RNF141 NM_016422 2.3 1.82E−12 B 0.84 0.77 0.76
    hg.1
    TC12001027. GLT1D1 NM_144669 2.3 8.17E−10 B 0.83 0.78 0.76
    hg.1
    TC14000630. EVL NM_016337 −2.3  1.1E−14 V 0.87 0.77 0.82
    hg.1
    TC02001007. SSB NM_003142 −2.3 <10-17 V 0.89 0.82 0.84
    hg.1
    TC10001014. PRKCQ NM_001242413 −2.3 5.49E−08 V 0.77 0.65 0.73
    hg.1
    TC11000327. CAT NM_001752 2.3 1.96E−08 B 0.79 0.67 0.77
    hg.1
    TC13000733. KLF12 NM_007249 −2.3 5.58E−07 V 0.76 0.65 0.73
    hg.1
    TC17000902. PGS1 NM_024419 2.3 1.18E−10 B 0.81 0.75 0.75
    hg.1
    TC01004034. OTTHUMG00000039860; ENST00000420830 2.3 6.75E−11 B 0.81 0.71 0.75
    hg.1 RP11-
    261C10.2
    TC03000714. ACPP NM_001099 2.3 3.33E−16 B 0.88 0.77 0.86
    hg.1
    TC03002096. BCL6 NM_001130845 2.3 1.44E−10 B 0.84 0.78 0.77
    hg.1
    TC05001536. ANKRD34B NM_001004441 2.3 3.47E−11 B 0.82 0.65 0.84
    hg.1
    TC15000622. SMAD3 NM_001145102 −2.3   2E−15 V 0.90 0.84 0.89
    hg.1
    TC20000999. SYCP2 NM_014258 2.3 7.77E−16 B 0.90 0.80 0.85
    hg.1
    TC08000641. ATP6V1C1 NM_001695 2.3 2.55E−12 B 0.84 0.71 0.76
    hg.1
    TC12001269. PLBD1 NM_024829 2.3 9.99E−16 B 0.89 0.80 0.79
    hg.1
    TC13000336. DNAJC3 NM_006260 2.3 <10-17 B 0.91 0.86 0.80
    hg.1
    TC16002068. SPN NM_001030288 −2.3 7.68E−09 V 0.79 0.67 0.80
    hg.1
    TC02001963. TGFA NM_003236 2.3 1.86E−10 B 0.81 0.75 0.77
    hg.1
    TC07000401. 0 ENST00000363800 2.3 4.28E−07 B 0.75 0.71 0.72
    hg.1
    TC16000660. CRISPLD2 NM_031476 2.3 8.23E−08 B 0.77 0.63 0.71
    hg.1
    TC17000918. RNF213 NM_001256071 −2.3  1.8E−10 V 0.80 0.73 0.72
    hg.1
    TC19001072. PLIN3 NM_005817 2.3 6.49E−12 B 0.83 0.73 0.73
    hg.1
    TC03001490. HESX1 NM_003865 −2.3  5.8E−09 V 0.86 0.88 0.66
    hg.1
    TC07001534. HIP1 NM_001243198 2.3 2.62E−08 B 0.78 0.77 0.71
    hg.1
    TC11002211. MAML2 NM_032427 −2.3 8.43E−13 V 0.88 0.78 0.80
    hg.1
    TC15001465. RAB27A NM_004580 2.3 <10-17 B 0.90 0.84 0.81
    hg.1
    TC22000224. LIMK2 NM_001031801 2.3 1.96E−07 B 0.77 0.73 0.68
    hg.1
    TC09000253. CNTNAP3B; NM_001201380 2.2 8.31E−07 B 0.75 0.61 0.75
    hg.1 CNTNAP3
    TC16000235. METTL9 NM_001077180 2.2 6.66E−16 B 0.89 0.82 0.81
    hg.1
    TC17001824. FAM20A NM_001243746 2.2 2.09E−12 B 0.84 0.71 0.81
    hg.1
    TC01006353. MKNK1 NM_001135553 2.2 5.29E−11 B 0.82 0.75 0.75
    hg.1
    TC11000412. PTPRJ; NM_001098503 2.2 <10-17 B 0.92 0.86 0.89
    hg.1 LOC100287223
    TC20000040. CDC25B NM_004358 −2.2 6.42E−08 V 0.78 0.67 0.72
    hg.1
    TC6_mcf_ FLOT1 NM_005803 2.2 6.71E−12 B 0.83 0.80 0.77
    hap5000121.
    hg.1
    TC04000213. PGM2 NM_018290 2.2 3.33E−16 B 0.89 0.78 0.77
    hg.1
    TC04001355. WDFY3 NM_014991 2.2 1.99E−08 B 0.79 0.75 0.66
    hg.1
    TC11001064. KMT2A; MLL NM_001197104 −2.2 1.11E−07 V 0.78 0.69 0.76
    hg.1
    TC12000793. DRAM1 NM_018370 2.2 4.79E−14 B 0.86 0.73 0.82
    hg.1
    TC15000841. ISG20 NM_002201 −2.2 2.44E−12 V 0.85 0.80 0.75
    hg.1
    TC17001738. 0 ENST00000390893 −2.2 0.000001 V 0.74 0.67 0.70
    hg.1
    TC01001974. KMO NM_003679 −2.2 4.09E−07 V 0.76 0.69 0.72
    hg.1
    TC02000620. IL1R1 NM_000877 2.2 2.87E−07 B 0.77 0.67 0.76
    hg.1
    TC06000110. GMPR NM_006877 −2.2  3.6E−11 V 0.85 0.80 0.70
    hg.1
    TC06002103. SAMD3 NM_001017373 −2.2 1.38E−07 V 0.78 0.71 0.72
    hg.1
    TC11000854. C11orf82 NM_145018 2.2 1.11E−16 B 0.89 0.75 0.86
    hg.1
    TC12001845. CDK17 NM_001170464 −2.2 8.88E−16 V 0.91 0.84 0.80
    hg.1
    TC17000953. NARF NM_001038618 2.2 4.77E−15 B 0.87 0.78 0.84
    hg.1
    TC19001779. SIGLEC10 NM_001171157 2.2 1.04E−09 B 0.76 0.65 0.76
    hg.1
    TC04000710. RTN3P1 ENST00000536295 2.2 5.55E−16 B 0.90 0.86 0.80
    hg.1
    TC05000343. FCHO2 NM_138782 2.2 2.42E−09 B 0.81 0.77 0.77
    hg.1
    TC11000182. ADM NM_001124 2.2 7.99E−12 B 0.84 0.84 0.76
    hg.1
    TC16000494. GPR97 NM_170776 2.2 9.63E−07 B 0.76 0.75 0.63
    hg.1
    TC01002419. 0 ENST00000517196 2.2 3.33E−08 B 0.77 0.67 0.71
    hg.1
    TC03001998. TNIK NM_001161560 −2.2 2.06E−13 V 0.85 0.77 0.85
    hg.1
    TC06000161. TRIM38 NM_006355 −2.2 9.43E−10 V 0.80 0.75 0.63
    hg.1
    TC06002062. MAN1A1 NM_005907 2.2 3.43E−14 B 0.86 0.78 0.80
    hg.1
    TC09000560. UGCG NM_003358 2.2 8.15E−07 B 0.74 0.63 0.67
    hg.1
    TC09000857. FAM157B NM_001145249 2.2 2.09E−10 B 0.82 0.73 0.68
    hg.1
    TC08000868. 0 — 2.2 1.41E−08 B 0.78 0.67 0.75
    hg.1
    TC06004062. NQO2 NM_000904 2.2 4.63E−11 B 0.82 0.73 0.76
    hg.1
    TC07001846. IMPDHI NM_000883 2.2 <10-17 B 0.91 0.80 0.85
    hg.1
    TC12000195. 0 ENST00000542401 2.2 7.02E−13 B 0.86 0.77 0.80
    hg.1
    TC03000579. SIDT1 NM_017699 −2.2 7.86E−21 V 0.84 0.73 0.77
    hg.1
    TC14002303. FLVCR2 NM_001195283 2.2 9.85E−08 B 0.75 0.69 0.71
    hg.1
    TC18000176. SCARNA17 NR_003003 −2.2 5.82E−07 V 0.77 0.73 0.79
    hg.1
    TC01002053. LOC729737; NR_039983 2.2 3.05E−07 B 0.77 0.75 0.66
    hg.1 OTTHUMG00000002481;
    RP11-
    34P13.14
    TC02002671. 0 uc021vux.1 2.2 7.32E−11 B 0.84 0.78 0.82
    hg.1
    TC03000100. 0 uc021wtr.1 2.2 7.32E−11 B 0.84 0.78 0.82
    hg.1
    TC07000292. 0 uc003tnn.1 2.2 1.75E−08 B 0.78 0.71 0.68
    hg.1
    TC09001141. OTTHUMG00000066812; ENST00000438517 2.2 0.000001 B 0.75 0.61 0.76
    hg.1 AL953854.2
    TC10001442. 0 uc021puq.1 2.2 7.32E−11 B 0.84 0.78 0.82
    hg.1
    TC12001157. C1RL NM_016546 2.2 3.94E−11 B 0.83 0.73 0.77
    hg.1
    TC12001170. SLC2A3 NM_006931 2.2 6.34E−13 B 0.86 0.80 0.73
    hg.1
    TC19001041. AES NM_198969 −2.2 6.17E−07 V 0.76 0.71 0.76
    hg.1
    TC19001666. NAPA NM_003827 −2.2 4.88E−09 V 0.79 0.80 0.68
    hg.1
    TC22001487. THOC5 NM_001002877 2.2 <10-17 B 0.91 0.86 0.84
    hg.1
    TC03003400. PCYT1A NM_005017 2.2 <10-17 B 0.91 0.86 0.85
    hg.1
    TC04002917. TLR6 NM_006068 2.2 4.97E−07 B 0.77 0.75 0.71
    hg.1
    TC10001688. PDZD8 NM_173791 2.2 1.84E−10 B 0.82 0.73 0.77
    hg.1
    TC20000553. SIRPD NM_178460 2.2 1.12E−13 B 0.85 0.73 0.77
    hg.1
    TC0X000052. TLR8 NM_138636 2.1 1.42E−12 B 0.88 0.80 0.84
    hg.1
    TC15000870. FURIN NM_002569 2.1 1.71E−14 B 0.88 0.78 0.81
    hg.1
    TC02001300. SLC11A1 NM_000578 2.1 4.21E−09 B 0.80 0.71 0.72
    hg.1
    TC16000519. CMTM2 NM_001199317 2.1 1.32E−08 B 0.80 0.73 0.68
    hg.1
    TC16000714. 0 uc002fqr.2 2.1 5.76E−10 B 0.81 0.73 0.68
    hg.1
    TC03001550. PROK2 NM_001126128 2.1 2.08E−08 B 0.79 0.82 0.62
    hg.1
    TC03002133. ATP13A3 NM_024524 2.1 1.04E−13 B 0.85 0.75 0.79
    hg.1
    TC0X000033. TBL1X NM_001139466 2.1 4.66E−14 B 0.87 0.82 0.79
    hg.1
    TC12001253. MANSC1 NM_018050 2.1 4.01E−08 B 0.80 0.80 0.67
    hg.1
    TC01000152. MFN2 NM_001127660 2.1 3.95E−09 B 0.79 0.71 0.79
    hg.1
    TC01002059. OTTHUMG00000002261; ENST00000417636 2.1 2.69E−10 B 0.80 0.71 0.81
    hg.1 RP5-857K21.1
    TC01003260. S100A12 NM_005621 2.1 9.28E−12 B 0.84 0.78 0.73
    hg.1
    TC03000583. ATP6V1A NM_001690 2.1 <10-17 B 0.94 0.88 0.86
    hg.1
    TC06001293. ATXN1 NM_001128164 2.1 7.22E−12 B 0.83 0.71 0.77
    hg.1
    TC19000334. 0 ENST00000500836 −2.1 1.67E−08 V 0.77 0.71 0.71
    hg.1
    TC01000011. OTTHUMG00000156971; ENST00000441866 2.1 2.17E−09 B 0.79 0.71 0.81
    hg.1 RP5-
    857K21.15
    TC07000969. CUL1 NM_003592 −2.1  1.8E−10 V 0.80 0.69 0.71
    hg.1
    TC15000324. TMEM62 NM_024956 −2.1 3.37E−10 V 0.84 0.82 0.71
    hg.1
    TC17001529. STAT5B NM_012448 2.1 4.44E−16 B 0.88 0.82 0.81
    hg.1
    TC22001427. APOBEC3D NM_152426 −2.1 1.89E−11 V 0.84 0.67 0.80
    hg.1
    TC01001315. SLC25A44 NM_014655 2.1 7.65E−11 B 0.83 0.86 0.80
    hg.1
    TC02001083. ITGA4 NM_000885 −2.1 1.83E−08 V 0.78 0.65 0.75
    hg.1
    TC03002182. MIR922; NR_030627 −2.1  2.8E−10 V 0.82 0.80 0.66
    hg.1 KIAA0226
    TC06001059. UTRN NM_007124 −2.1 1.11E−16 V 0.89 0.82 0.79
    hg.1
    TC11001341. TRIM5 NM_033093 −2.1  2.9E−09 V 0.80 0.73 0.65
    hg.1
    TC20000557. OTTHUMG00000031681; ENST00000447206 2.1  2.2E−10 B 0.80 0.69 0.76
    hg.1 RP5-968J1.1
    TC20001035. HELZ2; NM_001037335 −2.1 8.67E−09 V 0.79 0.80 0.66
    hg.1 OTTHUMG00000032982;
    RP4-
    697K14.12
    TC01000462. AGO4; NM_017629 2.1 2.81E−14 B 0.88 0.80 0.77
    hg.1 EIF2C4
    TC11000647. DRAPI NM_006442 −2.1 1.11E−16 V 0.90 0.82 0.81
    hg.1
    TC22000430. KLHDC7B NM_138433 −2.1 5.97E−11 V 0.84 0.84 0.79
    hg.1
    TC6_apd_ FLOT1 NM_005803 2.1  4.7E−09 B 0.80 0.75 0.72
    hap1000078.
    hg.1
    TC01002163. TNFRSF9 NM_001561 2.1 2.17E−12 B 0.83 0.69 0.76
    hg.1
    TC07000634. SLC12A9 NM_020246 2.1 1.11E−16 B 0.88 0.80 0.84
    hg.1
    TC17001312. DHRS13 NM_144683 2.1 1.11E−09 B 0.79 0.73 0.73
    hg.1
    TC19001163. ICAM3 NM_002162 2.1 7.78E−14 B 0.87 0.80 0.82
    hg.1
    TC01002260. OTTHUMG00000002221; ENST00000442385 −2.1 1.72E−09 V 0.84 0.90 0.68
    hg.1 ANO7L1
    TC02000221. RASGRP3 NM_001139488 −2.1 3.13E−11 V 0.86 0.80 0.77
    hg.1
    TC04002922. RASGEF1B NM_152545 −2.1 4.13E−10 V 0.82 0.75 0.77
    hg.1
    TC07001842. LRRC4 NM_022143 2.1 4.14E−09 B 0.80 0.77 0.75
    hg.1
    TC11000332. CD44 NM_001001391 2.1 7.77E−16 B 0.90 0.84 0.81
    hg.1
    TC22000213. SIRPAP1 ENST00000422796 2.1 4.66E−15 B 0.88 0.80 0.81
    hg.1
    TC03001188. NUP210 NM_024923 −2.0 0.000001 V 0.76 0.63 0.72
    hg.1
    TC05001188. RNA5SP180 ENST00000516181 2.0 4.31E−11 B 0.82 0.69 0.79
    hg.1
    TC07001102. CARD11 NM_032415 −2.0 5.67E−11 V 0.82 0.73 0.80
    hg.1
    TC11000198. MICAL2 NM_014632 2.0 3.18E−08 B 0.77 0.75 0.76
    hg.1
    TC16000185. 0 uc021tdl.1 −2.0 1.26E−09 V 0.80 0.71 0.77
    hg.1
    TC16000199. 0 uc021tdw.1 −2.0 1.26E−09 V 0.80 0.71 0.77
    hg.1
    TC20000206. HCK NM_001172129 2.0 3.97E−14 B 0.88 0.84 0.75
    hg.1
    TC02000603. EIF5B NM_015904 −2.0 3.81E−07 V 0.76 0.71 0.72
    hg.1
    TC02001363. AGFG1 NM_001135187 2.0 1.59E−10 B 0.83 0.71 0.80
    hg.1
    TC02002614. SLC40A1 NM_014585 2.0 1.76E−08 B 0.78 0.73 0.70
    hg.1
    TC04000625. EXOSC9 NM_001034194 −2.0 7.65E−13 V 0.89 0.84 0.77
    hg.1
    TC05000981. HRH2 NM_001131055 2.0 2.04E−09 B 0.80 0.75 0.73
    hg.1
    TC06002238. SYNE1 NM_182961 2.0 1.56E−09 B 0.81 0.75 0.77
    hg.1
    TC17000078. FBXO39 NM_153230 −2.0 1.34E−08 V 0.80 0.78 0.65
    hg.1
    TC01003757. OTTHUMG00000042553; ENST00000429210 −2.0 2.45E−10 V 0.85 0.80 0.65
    hg.1 RP11-31207.2
    TC11002434. ST3GAL4-AS1 NR_033839 2.0 2.43E−12 B 0.83 0.77 0.79
    hg.1
    TC17000613. TBX21 NM_013351 −2.0 0.000001 V 0.77 0.78 0.65
    hg.1
    TC19001500. RASGRP4 NM_001146202 2.0 4.26E−11 B 0.85 0.78 0.75
    hg.1
    TC20001743. SIRPB2 NM_001134836 2.0 2.18E−08 B 0.78 0.73 0.68
    hg.1
    TC01000781. ST6GALNAC3 NM_001160011 2.0 9.49E−08 B 0.79 0.69 0.75
    hg.1
    TC04000075. TBC1D14 NM_001113361 2.0 4.44E−15 B 0.88 0.82 0.77
    hg.1
    TC05001455. NAIP; NM_004536 2.0 2.83E−07 B 0.77 0.71 0.67
    hg.1 LOC101060527
    TC06001508. FLOT1 NM_005803 2.0 9.42E−12 B 0.83 0.80 0.77
    hg.1
    TC11000575. RTN3 NM_006054 2.0 1.11E−16 B 0.90 0.88 0.80
    hg.1
    TC11001540. CD59; NM_000611 2.0 1.21E−07 B 0.76 0.69 0.75
    hg.1 C11orf91
    TC14001436. CCDC88C NM_001080414 −2.0  4.9E−13 V 0.85 0.75 0.85
    hg.1
    TC05000096. BASP1 NM_006317 2.0 1.17E−13 B 0.88 0.84 0.75
    hg.1
    TC16001296. CMC2 NM_020188 −2.0 2.74E−12 V 0.86 0.80 0.72
    hg.1
    TC19000677. C5AR2 NM_018485 2.0 3.48E−07 B 0.78 0.71 0.73
    hg.1
    TC20000556. LOC100289473; NR_037142 2.0 5.14E−09 B 0.78 0.63 0.73
    hg.1 OTTHUMG00000154775;
    RP4-673D20.3
    TC01003004. PHTF1 NM_006608 2.0 2.71E−07 B 0.75 0.63 0.76
    hg.1
    TC04000460. AGPAT9 NM_001256421 2.0  1.8E−14 B 0.88 0.82 0.79
    hg.1
    TC0Y000006. CSF2RA NM_001161530 2.0 1.63E−10 B 0.83 0.78 0.73
    hg.1
    TC13000199. PHF11 NM_001040444 −2.0 3.07E−09 V 0.80 0.75 0.68
    hg.1
    TC15000239. 0 uc021siw.1 2.0 1.15E−07 B 0.76 0.71 0.72
    hg.1
    TC19001383. TSHZ3 NM_020856 2.0 1.26E−11 B 0.83 0.71 0.81
    hg.1
    TC08000869. 0 — 2.0 2.39E−10 B 0.80 0.69 0.77
    hg.1
    TC02005010. SP140L NM_138402 −2.0 7.77E−16 V 0.88 0.78 0.80
    hg.1
    TC03000667. ABTB1 NM_032548 2.0 1.11E−08 B 0.81 0.82 0.73
    hg.1
    TC09001215. TRPM6 NM_017662 2.0 3.16E−07 B 0.78 0.71 0.71
    hg.1
    TC14000941. HAUS4; NM_001166269 2.0 7.29E−07 B 0.76 0.67 0.70
    hg.1 MIR4707
    TC16000469. MT1JP NR_036677 −2.0 3.93E−12 V 0.84 0.82 0.75
    hg.1
    TC16001135. MT1G NM_005950 −2.0  2.1E−10 V 0.82 0.78 0.73
    hg.1
    TC17000520. CNP NM_033133 −2.0 1.69E−14 V 0.89 0.86 0.81
    hg.1
    TC17001019. ANKFY1 NM_016376 −2.0 1.12E−09 V 0.81 0.78 0.67
    hg.1
    TC6_cox_ FLOT1 NM_005803 2.0 4.94E−12 B 0.83 0.80 0.79
    hap2000142.
    hg.1
    TC01002801. 0 ENST00000406534 −2.0 1.64E−11 V 0.82 0.75 0.75
    hg.1
    TC02000952. GPD2 NM_000408 −2.0 1.78E−09 V 0.80 0.78 0.62
    hg.1
    TC16000375. ITGAX NM_000887 2.0 2.01E−10 B 0.83 0.80 0.76
    hg.1
    TC20000019. SIRPA NM_001040022 2.0 <10-17 B 0.91 0.86 0.86
    hg.1
    TC6_dbb_ FLOT1 NM_005803 2.0 4.93E−12 B 0.84 0.80 0.77
    hap3000131.
    hg.1
    TC6_qbl_ FLOT1 NM_005803 2.0 4.93E−12 B 0.84 0.80 0.77
    hap6000132.
    hg.1
    TC6_ssto_ FLOT1 NM_005803 2.0 4.93E−12 B 0.84 0.80 0.77
    hap7000122.
    hg.1
    TC05000639. RAD50 NM_005732 −2.0 0.000001 V 0.76 0.69 0.72
    hg.1
    TC08000591. CPQ NM_016134 2.0 5.25E−07 B 0.76 0.73 0.65
    hg.1
    TC15000945. ARRDC4 NM_183376 2.0 8.38E−10 B 0.79 0.67 0.73
    hg.1
    TC19000340. COLGALT1; NM_024656 2.0 2.92E−12 B 0.82 0.69 0.77
    hg.1 GLT25D1
    TC20000303. PLCG1 NM_002660 −2.0 2.31E−07 V 0.77 0.67 0.72
    hg.1
    TC01002882. GCLM NM_002061 2.0 4.01E−12 B 0.85 0.69 0.84
    hg.1
    TC02001667. OTOF NM_194248 −2.0 1.36E−10 V 0.88 0.92 0.73
    hg.1
    TC10000189. MASTL NM_032844 −2.0 3.66E−11 V 0.86 0.80 0.73
    hg.1
    TC12000730. PLXNC1 NM_005761 2.0  2.9E−11 B 0.85 0.84 0.76
    hg.1
    TC12000947. RNF10 NM_014868 2.0  4.3E−10 B 0.79 0.69 0.70
    hg.1
    TC12001155. LPCAT3 NM_005768 2.0 <10-17 B 0.92 0.82 0.85
    hg.1
    TC17001370. SLFN12 NM_018042 −2.0 7.73E−07 V 0.76 0.78 0.63
    hg.1
    TC01001820. MARC1 NM_022746 1.9 2.43E−07 B 0.77 0.67 0.72
    hg.1
    TC01003988. 0 ENST00000549744 1.9 5.25E−07 B 0.76 0.67 0.72
    hg.1
    TC06001715. TREML3P NR_027256 1.9 8.62E−08 B 0.77 0.61 0.75
    hg.1
    TC0X000006. CSF2RA NM_001161530 1.9  5.7E−10 B 0.82 0.75 0.73
    hg.1
    TC10002943. ENTPD1; NM_001164182 1.9 4.33E−07 B 0.76 0.71 0.67
    hg.1 OTTHUMG00000018817;
    RP11-
    429G19.3
    TC11000824. ACER3 NM_018367 1.9 3.89E−15 B 0.87 0.75 0.82
    hg.1
    TC11000981. DDX10 NM_004398 −1.9 6.32E−09 V 0.80 0.69 0.80
    hg.1
    TC19000168. C19orf66 NM_018381 −1.9 4.51E−13 V 0.88 0.86 0.76
    hg.1
    TC02002670. FAM126B NM_173822 1.9 1.84E−07 B 0.78 0.77 0.70
    hg.1
    TC02002706. MIR2355 NR_036227 1.9  8.1E−10 B 0.79 0.65 0.82
    hg.1
    TC03001824. PIK3CB NM_001256045 1.9 4.44E−16 B 0.88 0.80 0.84
    hg.1
    TC04002916. TLR1 NM_003263 1.9 1.04E−07 B 0.79 0.71 0.72
    hg.1
    TC09000585. C9orf91 NM_153045 −1.9 4.69E−11 V 0.84 0.77 0.72
    hg.1
    TC0X001324. TMEM255A; NM_001104544 −1.9 2.92E−09 V 0.82 0.86 0.68
    hg.1 FAM70A
    TC11001174. ST3GAL4 NM_001254757 1.9 1.19E−08 B 0.78 0.71 0.79
    hg.1
    TC11002390. HSPA8; NM_006597 −1.9 0.000001 V 0.74 0.67 0.72
    hg.1 SNORD14D;
    SNORD14C
    TC11003512. 0 uc001lnz.2 1.9  1.3E−08 B 0.79 0.71 0.67
    hg.1
    TC16000476. MT1X NM_005952 −1.9 5.82E−10 V 0.85 0.86 0.76
    hg.1
    TC17000312. LGALS9 NM_002308 −1.9 3.78E−08 V 0.78 0.80 0.68
    hg.1
    TC20000140. XRN2 NM_012255 1.9 <10-17 B 0.91 0.77 0.86
    hg.1
    TC6_mann_ FLOT1 NM_005803 1.9 6.72E−12 B 0.83 0.80 0.77
    hap4000120.
    hg.1
    TC01000005. LOC100132287; NR_028322 1.9 8.88E−08 B 0.78 0.75 0.66
    hg.1 LOC100133331;
    LOC101060495;
    LOC101060494;
    LOC101059936;
    LOC100996502;
    LOC100996328;
    LOC100287894;
    LOC100132062;
    OTTHUMG00000156968;
    RP4-
    669L17.10
    TC01003055. LOC100132913; ENST00000412759 −1.9 3.21E−10 V 0.86 0.86 0.70
    hg.1 OTTHUMG00000041037;
    RP11-
    439A17.7
    TC04001682. FAM198B NM_001031700 1.9 5.27E−09 B 0.80 0.71 0.75
    hg.1
    TC05000412. VCAN NM_001164098 1.9 1.47E−07 B 0.77 0.71 0.70
    hg.1
    TC05001604. MCTP1 NM_001002796 1.9 1.25E−10 B 0.82 0.73 0.77
    hg.1
    TC06000817. MANEA NM_024641 −1.9 5.71E−07 V 0.75 0.65 0.72
    hg.1
    TC07000307. UPP1 NM_003364 1.9 5.26E−08 B 0.76 0.63 0.70
    hg.1
    TC11001353. FAM160A2 NM_001098794 1.9 1.11E−16 B 0.89 0.78 0.85
    hg.1
    TC11002344. AMICA1 NM_001098526 1.9    4E−13 B 0.87 0.82 0.73
    hg.1
    TC12001754. OSBPL8 NM_001003712 1.9 <10-17 B 0.94 0.84 0.86
    hg.1
    TC16002096. NPIPA5; ENST00000427999 −1.9 1.08E−07 V 0.78 0.75 0.77
    hg.1 NPIPA2;
    NPIPA3;
    PKD1P1
    TC17001822. WIPI1 NM_017983 1.9 4.09E−10 B 0.82 0.75 0.77
    hg.1
    TC19001159. DNMT1 NM_001130823 −1.9 1.98E−11 V 0.83 0.77 0.80
    hg.1
    TC07000252. 0 ENST00000516671 1.9 3.98E−07 B 0.75 0.69 0.67
    hg.1
    TC07002021. GIMAP6 NM_001244071 −1.9 1.78E−08 V 0.78 0.65 0.73
    hg.1
    TC10001562. FRAT2 NM_012083 1.9 1.45E−07 B 0.78 0.77 0.70
    hg.1
    TC10001726. OAT NM_001171814 1.9 1.11E−16 B 0.89 0.78 0.85
    hg.1
    TC11000027. TALDO1 NM_006755 1.9 3.33E−16 B 0.89 0.88 0.82
    hg.1
    TC12001544. ITGB7 NM_000889 −1.9 1.46E−07 V 0.76 0.65 0.75
    hg.1
    TC14001343. TMED8 NM_213601 1.9 2.06E−10 B 0.83 0.73 0.79
    hg.1
    TC21000128. IFNAR1 NM_000629 1.9 <10-17 B 0.90 0.84 0.81
    hg.1
    TC02001379. CAB39 NM_001130849 1.9 1.11E−16 B 0.89 0.84 0.84
    hg.1
    TC03000632. DTX3L NM_138287 −1.9 4.97E−08 V 0.77 0.59 0.72
    hg.1
    TC04001308. SCARB2 NM_001204255 −1.9 3.72E−08 V 0.76 0.71 0.60
    hg.1
    TC06001071. STXBP5; NM_001127715 1.9 5.72E−10 B 0.80 0.75 0.73
    hg.1 OTTHUMG00000015764;
    RP11-
    361F15.2
    TC10000293. ALOX5 NM_000698 1.9 7.51E−13 B 0.86 0.84 0.77
    hg.1
    TC13000111. BRCA2 NM_000059 −1.9 9.05E−11 V 0.87 0.80 0.79
    hg.1
    TC19000417. URI1 NM_001252641 −1.9 3.9E−07 V 0.76 0.65 0.73
    hg.1
    TC22001467. APOBEC3F NM_001006666 −1.9 1.88E−14 V 0.86 0.71 0.79
    hg.1
    TC01001572. CACNA1E NM_000721 1.9 7.53E−07 B 0.74 0.65 0.68
    hg.1
    TC01003677. LINC00862; NR_040064 1.9 1.99E−10 B 0.78 0.65 0.79
    hg.1 SMIM16;
    C1orf98
    TC03000976. ATP11B NM_014616 1.9 2.24E−11 B 0.83 0.69 0.75
    hg.1
    TC08000022. AGPAT5 NM_018361 −1.9 0.000001 V 0.74 0.71 0.71
    hg.1
    TC0X000066. MOSPD2 NM_001177475 1.9  1.4E−09 B 0.82 0.78 0.77
    hg.1
    TC01001838. CAPN2 NM_001146068 −1.9 5.22E−11 V 0.85 0.80 0.82
    hg.1
    TC01002057. OTTHUMG00000002553; ENST00000424429 1.9 2.89E−11 B 0.80 0.67 0.79
    hg.1 AP006222.2
    TC01003085. FAM72D; NM_207418 −1.9 2.62E−08 V 0.82 0.73 0.72
    hg.1 LOC101060656
    TC02000594. ZAP70 NM_001079 −1.9 3.32E−08 V 0.78 0.71 0.73
    hg.1
    TC03000087. SLC6A6 NM_001134367 1.9 5.21E−07 B 0.77 0.77 0.68
    hg.1
    TC04001137. RBM47 NM_001098634 1.9 5.94E−08 B 0.79 0.77 0.75
    hg.1
    TC07001537. TMEM120A NM_031925 1.9 3.69E−13 B 0.86 0.75 0.84
    hg.1
    TC09000634. NEK6 NM_001145001 1.9 2.39E−14 B 0.86 0.73 0.80
    hg.1
    TC18000213. PMAIP1 NM_021127 −1.9 6.29E−08 V 0.78 0.84 0.63
    hg.1
    TC01001956. MTR NM_000254 −1.9 0.000001 V 0.75 0.63 0.73
    hg.1
    TC01006343. RHOU NM_021205 1.9  3.6E−12 B 0.84 0.71 0.85
    hg.1
    TC04001486. CAMK2D NM_172127 −1.9 3.67E−07 V 0.76 0.69 0.72
    hg.1
    TC05001331. EMB NM_198449 1.9 2.15E−13 B 0.85 0.75 0.82
    hg.1
    TC05002111. DOK3 NM_024872 1.9 7.76E−14 B 0.86 0.78 0.79
    hg.1
    TC0X001177. CYSLTR1 NM_006639 −1.9 4.29E−10 V 0.81 0.71 0.72
    hg.1
    TC11000208. FAR1 NM_032228 1.9 <10-17 B 0.93 0.88 0.87
    hg.1
    TC16000483. CPNE2 NM_152727 1.9 1.82E−13 B 0.85 0.75 0.80
    hg.1
    TC20000401. LOC100506115; ENST00000421019 1.9 2.99E−07 B 0.78 0.71 0.66
    hg.1 OTTHUMG00000032721;
    RP11-
    290F20.3
    TC0300337 FAM157A; NM_001145248 1.9 3.04E−08 B 0.79 0.75 0.67
    1.hg.1 FAM157C;
    LOC100132858;
    FAM157B
    TC03003380. PFKFB4 NM_004567 1.9 4.48E−09 B 0.81 0.78 0.80
    hg.1
    TC07001910. ZC3HAV1 NM_024625 −1.9 1.88E−08 V 0.77 0.65 0.62
    hg.1
    TC09000941. MLLT3 NM_004529 −1.9 0.000001 V 0.75 0.73 0.71
    hg.1
    TC0X002337. ACSL4 NM_004458 1.9 1.35E−07 B 0.76 0.69 0.72
    hg.1
    TC14000417. KIAA0247 NM_014734 1.9 1.18E−09 B 0.83 0.77 0.77
    hg.1
    TC14001116. SOS2 NM_006939 1.9 6.21E−14 B 0.87 0.82 0.75
    hg.1
    TC15001552. OAZ2 NM_002537 1.9 1.13E−10 B 0.83 0.78 0.79
    hg.1
    TC19000993. R3HDM4 NM_138774 1.9 7.81E−10 B 0.82 0.84 0.71
    hg.1
    TC01001559. QSOX1; NM_001004128 1.9 5.12E−11 B 0.83 0.65 0.79
    hg.1 FLJ23867
    TC12000725. MRPL42 NM_014050 −1.9 6.44E−08 V 0.79 0.75 0.76
    hg.1
    TC14001184. DHRS7 NM_016029 1.9 1.22E−15 B 0.90 0.88 0.82
    hg.1
    TC14001462. DDX24 NM_020414 −1.9  4.2E−07 V 0.77 0.67 0.81
    hg.1
    TC19000976. FLJ45445; NR_028324 1.9 3.52E−07 B 0.76 0.75 0.65
    hg.1 LOC100653346
    TC20000701. OTTHUMG00000032052; ENST00000440798 1.9  1.6E−11 B 0.80 0.71 0.77
    hg.1 RP4-753D10.5
    TC22000547. MAPK1 NM_002745 1.9 <10-17 B 0.90 0.86 0.81
    hg.1
    TC12001946. USP30-AS1 NR_038996 −1.8 4.75E−08 V 0.77 0.69 0.67
    hg.1
    TC16000202. 0 ENST00000327792 −1.8 1.04E−07 V 0.78 0.77 0.75
    hg.1
    TC16002043. NPIPA1; NM_006985 −1.8 7.26E−08 V 0.78 0.71 0.73
    hg.1 LOC101060412;
    NPIPA8;
    NPIPA7;
    NPIPA3; NPIP
    TC17001761. USP32 NM_032582 1.8  2.1E−08 B 0.79 0.75 0.73
    hg.1
    TC19001826. NLRP12 NM_033297 1.8 2.53E−09 B 0.80 0.73 0.73
    hg.1
    TC22000682. RTCB; NM_014306 −1.8 1.52E−10 V 0.84 0.75 0.79
    hg.1 C22orf28
    TC02002740. IKZF2 NM_001079526 −1.8 8.95E−07 V 0.76 0.73 0.62
    hg.1
    TC03000534. ALCAM NM_001243280 1.8 3.53E−07 B 0.85 0.71 0.84
    hg.1
    TC03000873. SMC4 NM_001002800 −1.8 5.53E−07 V 0.77 0.75 0.60
    hg.1
    TC06002121. VNN2 NM_001242350 1.8 1.13E−08 B 0.80 0.73 0.67
    hg.1
    TC10000779. SLK NM_014720 1.8 3.37E−10 B 0.83 0.71 0.82
    hg.1
    TC10001144. SVIL NM_003174 1.8 7.79E−08 B 0.78 0.71 0.70
    hg.1
    TC13000601. KBTBD7 NM_032138 1.8 1.38E−07 B 0.79 0.73 0.76
    hg.1
    TC02001790. ZFP36L2 NM_006887 −1.8 5.96E−08 V 0.75 0.67 0.77
    hg.1
    TC04001493. 0 ENST00000441170 −1.8 6.97E−08 V 0.77 0.73 0.67
    hg.1
    TC07001016. NUB1 NM_001243351 −1.8  4.2E−08 V 0.76 0.65 0.66
    hg.1
    TC09000567. KIAA1958 NM_133465 −1.8 7.49E−08 V 0.81 0.84 0.63
    hg.1
    TC10000097. OPTN NM_001008211 −1.8 1.47E−11 V 0.83 0.78 0.81
    hg.1
    TC12000334. PCED1B NM_138371 −1.8 7.12E−07 V 0.74 0.65 0.68
    hg.1
    TC16000620. GABARAPL2 NM_007285 1.8 7.77E−16 B 0.89 0.78 0.84
    hg.1
    TC19000737. FCGRT NM_001136019 1.8 6.99E−11 B 0.82 0.78 0.80
    hg.1
    TC20000554. SIRPB1; NM_001083910 1.8 1.28E−09 B 0.82 0.80 0.75
    hg.1 LOC100653231;
    LOC100653194
    TC22000722. IL2RB NM_000878 −1.8 5.69E−07 V 0.77 0.78 0.75
    hg.1
    TC01001688. ATP2B4 NM_001001396 1.8 1.01E−10 B 0.81 0.75 0.77
    hg.1
    TC02001781. OXER1 NM_148962 1.8 3.25E−07 B 0.77 0.61 0.76
    hg.1
    TC04000181. RBPJ NM_005349 1.8 4.93E−13 B 0.84 0.77 0.80
    hg.1
    TC10001312. IPMK NM_152230 1.8 1.46E−07 B 0.77 0.73 0.68
    hg.1
    TC12001846. 0 ENST00000364565 −1.8 2.52E−07 V 0.76 0.71 0.62
    hg.1
    TC17001029. CXCL16 NM_022059 1.8 2.92E−07 B 0.80 0.77 0.70
    hg.1
    TC19001242. EMR2 NM_013447 1.8 1.55E−08 B 0.79 0.71 0.71
    hg.1
    TC22000270. NCF4 NM_000631 1.8 3.31E−09 B 0.82 0.77 0.75
    hg.1
    TC02002398. GTDC1 NM_001006636 1.8 2.47E−10 B 0.85 0.78 0.84
    hg.1
    TC03000263. NBEAL2 NM_015175 1.8 6.81E−09 B 0.79 0.75 0.71
    hg.1
    TC03001389. PRKAR2A NM_004157 1.8 2.44E−15 B 0.89 0.80 0.84
    hg.1
    TC04001171. NFXL1 NM_152995 1.8 3.05E−08 B 0.78 0.65 0.82
    hg.1
    TC10000967. IDI1 NM_004508 1.8 1.25E−08 B 0.80 0.67 0.79
    hg.1
    TC11000184. AMPD3 NM_001172431 1.8 3.21E−13 B 0.85 0.69 0.85
    hg.1
    TC12000822. TCP11L2 NM_152772 1.8 5.29E−08 B 0.77 0.73 0.70
    hg.1
    TC15000311. MGA NM_001164273 −1.8 8.05E−07 V 0.77 0.67 0.79
    hg.1
    TC16000201. 0 uc002dey.2 −1.8 2.05E−07 V 0.77 0.67 0.77
    hg.1
    TC16000237. 0 ENST00000384315 1.8 9.47E−09 B 0.78 0.67 0.77
    hg.1
    TC01002056. LOC100996554; ENST00000466557 1.8 1.62E−08 B 0.79 0.73 0.65
    hg.1 LOC100996442;
    LOC100132055;
    OTTHUMG00000002480;
    RP11-
    34P13.13
    TC01002903. DPYD NM_000110 1.8 2.54E−10 B 0.82 0.77 0.79
    hg.1
    TC06000774. CYB5R4 NM_016230 1.8 0.000001 B 0.76 0.78 0.68
    hg.1
    TC06002122. SLC18B1 NM_052831 −1.8   1E−07 V 0.79 0.73 0.70
    hg.1
    TC08000172. PPP3CC NM_001243974 −1.8 2.35E−08 V 0.79 0.69 0.76
    hg.1
    TC08001541. TRPS1 NM_014112 1.8 3.27E−11 B 0.84 0.69 0.77
    hg.1
    TC09000637. LOC100129034 NR_027406 1.8 7.34E−08 B 0.76 0.67 0.67
    hg.1
    TC12001933. CMKLR1 ENST00000397688 −1.8 1.64E−07 V 0.76 0.69 0.70
    hg.1
    TC19001653. PRKD2 NM_001079880 −1.8 8.62E−09 V 0.78 0.69 0.67
    hg.1
    TC01000826. SH3GLB1 NM_001206651 1.8  2.8E−10 B 0.81 0.75 0.79
    hg.1
    TC01001101. GPR89C NM_001097616 −1.8 5.81E−07 V 0.76 0.73 0.71
    hg.1
    TC01001314. SEMA4A NM_001193300 1.8 5.27E−13 B 0.85 0.80 0.81
    hg.1
    TC01002421. PTAFR NM_001164721 1.8 1.06E−12 B 0.85 0.82 0.81
    hg.1
    TC02002477. 0 ENST00000435109 1.8 3.15E−10 B 0.82 0.75 0.76
    hg.1
    TC04000466. ARHGAP24 NM_001025616 1.8 9.03E−09 B 0.80 0.73 0.73
    hg.1
    TC04001673. PDGFC NM_016205 1.8 1.36E−10 B 0.85 0.65 0.87
    hg.1
    TC06002152. IFNGR1 NM_000416 1.8 3.16E−10 B 0.83 0.73 0.76
    hg.1
    TC07001562. HGF NM_000601 1.8 1.77E−07 B 0.79 0.63 0.81
    hg.1
    TC13000825. DOCK9 NM_001130048 −1.8 4.07E−07 V 0.76 0.67 0.70
    hg.1
    TC14001145. DDHDI NM_001160147 −1.8 4.92E−12 V 0.87 0.77 0.84
    hg.1
    TC17000640. ABI3 NM_016428 −1.8 1.07E−08 V 0.79 0.77 0.71
    hg.1
    TC18000049. VAPA NM_003574 1.8 1.01E−14 B 0.87 0.80 0.81
    hg.1
    TC20000389. SNORD12 NR_003030 1.8 0.000001 B 0.75 0.69 0.70
    hg.1
    TC20000469. ZNF831 NM_178457 −1.8 8.59E−09 V 0.79 0.69 0.75
    hg.1
    TC20000703. CD93 NM_012072 1.8 5.05E−07 B 0.75 0.67 0.65
    hg.1
    TC22001468. APOBEC3G NM_021822 −1.8 3.07E−13 V 0.85 0.80 0.75
    hg.1
    TC01001118. GPR89C; NM_001097616 −1.8 8.85E−08 V 0.78 0.71 0.72
    hg.1 GPR89B;
    GPR89A
    TC01001581. NPL NM_001200050 1.8 1.76E−07 B 0.76 0.75 0.65
    hg.1
    TC01003868. AIDA NM_022831 −1.8 1.13E−11 V 0.82 0.75 0.76
    hg.1
    TC03001466. TKT NM_001064 1.8 1.11E−16 B 0.90 0.78 0.80
    hg.1
    TC04000174. PI4K2B; NM_018323 −1.8 7.04E−14 V 0.91 0.82 0.79
    hg.1 LOC285540
    TC0X000425. PGK1; NM_000291 1.8 <10-17 B 0.91 0.84 0.82
    hg.1 LOC100653302;
    LOC100652805
    TC0X001091. LOC92249; NR_015353 −1.8 3.21E−07 V 0.76 0.65 0.75
    hg.1 OTTHUMG00000021699;
    RP11-357C3.3
    TC12000806. 0 uc021rcw.1 1.8 2.07E−10 B 0.81 0.69 0.73
    hg.1
    TC14000429. PCNX NM_014982 1.8 5.34E−08 B 0.77 0.77 0.68
    hg.1
    TC15001841. IDH2 NM_002168 −1.8 1.37E−08 V 0.80 0.75 0.76
    hg.1
    TC16000918. GDE1 NM_016641 1.8 3.34E−14 B 0.87 0.80 0.79
    hg.1
    TC16000972. ARHGAP17 NM_001006634 −1.8 2.55E−10 V 0.82 0.78 0.76
    hg.1
    TC11001999. UNC93B1 NM_030930 −1.8 4.04E−07 V 0.77 0.77 0.67
    hg.1
    TC11003449. TMX2 NM_001144012 −1.8 1.41E−07 V 0.79 0.77 0.72
    hg.1
    TC11003501. LOC100133161; NR_028326 1.8 8.32E−08 B 0.77 0.75 0.66
    hg.1 LOC101060495;
    LOC101059936
    TC12001791. CEP290 NM_025114 −1.8 1.99E−07 V 0.76 0.69 0.72
    hg.1
    TC14000335. TMEM260; NM_017799 1.8 3.05E−07 B 0.75 0.71 0.67
    hg.1 C14orf101
    TC14001141. ERO1L NM_014584 1.8 3.44E−11 B 0.83 0.77 0.75
    hg.1
    TC14001472. DICER1 NM_177438 1.8 <10-17 B 0.90 0.84 0.85
    hg.1
    TC15002774. MIR628; NR_030358 1.8 6.92E−11 B 0.83 0.77 0.71
    hg.1 CCPG1
    TC16000236. 0 ENST00000384519 1.8  1.1E−09 B 0.80 0.65 0.79
    hg.1
    TC16002042. LOC642799; ENST00000329793 −1.8 3.48E−07 V 0.77 0.77 0.73
    hg.1 NPIPA2;
    NPIPA3
    TC17001798. PECAM1 NM_000442 1.8 7.48E−09 B 0.76 0.61 0.71
    hg.1
    TC22000362. TSPO NM_000714 1.8 <10-17 B 0.90 0.86 0.82
    hg.1
    TC01001414. ATF6 NM_007348 1.8 2.38E−10 B 0.83 0.75 0.82
    hg.1
    TC01003970. OTTHUMG00000039487; ENST00000416221 1.8 1.66E−07 B 0.75 0.65 0.71
    hg.1 RP11-
    295G20.2
    TC04001455. PAPSS1 NM_005443 1.8 9.03E−10 B 0.81 0.65 0.81
    hg.1
    TC05000084. FAM105A NM_019018 1.8 1.91E−11 B 0.85 0.75 0.84
    hg.1
    TC06001990. SCML4 NM_198081 −1.8  6.9E−11 V 0.82 0.69 0.76
    hg.1
    TC0X000118. PDK3 NM_001142386 1.8 7.72E−14 B 0.89 0.82 0.81
    hg.1
    TC10000533. 0 uc001jyo.2 1.8 8.02E−08 B 0.79 0.77 0.76
    hg.1
    TC13000330. GPR180 NM_180989 −1.8 2.65E−13 V 0.87 0.77 0.81
    hg.1
    TC13000472. ZDHHC20 NM_153251 1.8 1.37E−09 B 0.78 0.67 0.81
    hg.1
    TC14001310. AREL1; NM_001039479 1.8 3.22E−15 B 0.87 0.75 0.81
    hg.1 KIAA0317
    TC16000499. USB1; NM_001195302 1.8 1.72E−13 B 0.84 0.69 0.75
    hg.1 C16orf57
    TC19000773. SIGLEC7 NM_016543 1.8  1.3E−08 B 0.77 0.63 0.73
    hg.1
    TC19001280. JAK3 NM_000215 1.8 2.45E−14 B 0.86 0.77 0.80
    hg.1
    TC19001501. MAP4K1 NM_001042600 −1.8 6.82E−07 V 0.75 0.65 0.72
    hg.1
    TC01001907. GALNT2 NM_004481 1.7 1.18E−11 B 0.85 0.67 0.82
    hg.1
    TC05000398. ZFYVE16 NM_001105251 1.7 1.01E−07 B 0.77 0.73 0.73
    hg.1
    TC06000120. RNF144B NM_182757 1.7 2.73E−07 B 0.76 0.77 0.71
    hg.1
    TC06001754. GTPBP2 NM_019096 −1.7 3.56E−08 V 0.78 0.84 0.66
    hg.1
    TC07003328. RABGEF1; NM_014504 1.7 6.02E−09 B 0.80 0.65 0.80
    hg.1 KCTD7
    TC15001264. ZNF106; NM_022473 1.7 <10-17 B 0.91 0.82 0.86
    hg.1 ZFP106
    TC16000178. NPIPA3 ENST00000413283 −1.7 3.77E−07 V 0.77 0.75 0.71
    hg.1
    TC20000587. RASSF2 NM_014737 1.7    2E−13 B 0.87 0.80 0.82
    hg.1
    TC01001053. EMBP1 NR_003955 1.7 8.29E−14 B 0.86 0.80 0.81
    hg.1
    TC01001587. RGL1 NM_015149 −1.7    5E−07 V 0.77 0.80 0.63
    hg.1
    TC02000280. PRKCE NM_005400 −1.7 1.26E−13 V 0.92 0.88 0.75
    hg.1
    TC04000320. TMEM165 NM_018475 1.7 3.42E−14 B 0.85 0.78 0.79
    hg.1
    TC04000553. SGMS2 NM_001136257 1.7 1.72E−08 B 0.79 0.69 0.77
    hg.1
    TC06000697. PTP4A1 NM_003463 −1.7 2.97E−07 V 0.75 0.78 0.66
    hg.1
    TC06001217. SERPINB1 NM_030666 1.7 1.81E−08 B 0.78 0.67 0.72
    hg.1
    TC06001994. SNX3 NM_003795 1.7 3.35E−09 B 0.81 0.75 0.81
    hg.1
    TC07003292. LSMEM1; NM_001134468 1.7 0.000001 B 0.74 0.65 0.75
    hg.1 C7orf53
    TC08000835. GRINA NM_000837 1.7 1.32E−11 B 0.83 0.82 0.81
    hg.1
    TC13000197. CDADC1 NM_001193478 1.7 8.07E−08 B 0.78 0.67 0.73
    hg.1
    TC01002072. LOC100288069; NR_033908 1.7  1.8E−08 B 0.79 0.71 0.67
    hg.1 OTTHUMG00000002409;
    RP11-206L10.5;
    OTTHUMG00000002406;
    RP11-206L10.2
    TC02000500. TMSB10 NM_021103 −1.7 1.07E−12 V 0.84 0.77 0.77
    hg.1
    TC02001192. BMPR2 NM_001204 −1.7 6.86E−11 V 0.83 0.77 0.72
    hg.1
    TC03000331. PPM1M NM_001122870 1.7 2.22E−16 B 0.89 0.82 0.81
    hg.1
    TC04001809. ACSL1 NM_001995 1.7 7.34E−08 B 0.79 0.73 0.68
    hg.1
    TC05000389. JMY NM_152405 −1.7 8.31E−07 V 0.76 0.67 0.72
    hg.1
    TC05003443. LOC100132287; NR_028322 1.7 6.51E−07 B 0.76 0.75 0.65
    hg.1 LOC100133331;
    LOC100653346;
    LOC100653241;
    LOC100652945;
    LOC100508632;
    LOC100132050;
    LOC100128326;
    LOC100996769;
    LOC100132062
    TC09001580. NR6A1 NM_001489 1.7 3.72E−11 B 0.82 0.67 0.80
    hg.1
    TC12001688. 0 ENST00000536412 1.7 1.04E−09 B 0.81 0.69 0.80
    hg.1
    TC18000205. MALT1 NM_006785 −1.7 5.93E−07 V 0.77 0.73 0.73
    hg.1
    TC19000262. 0 ENST00000510342 1.7 <10-17 B 0.90 0.78 0.81
    hg.1
    TC19000454. GRAMD1A; NM_020895 1.7 1.33E−08 B 0.78 0.75 0.75
    hg.1 OTTHUMG00000044564;
    AC020907.6
    TC22001428. MTMR3 NM_021090 1.7  1.6E−07 B 0.77 0.75 0.71
    hg.1
    TC01001348. IFI16 NM_001206567 −1.7 1.18E−09 V 0.80 0.67 0.75
    hg.1
    TC01004036. LOC731275; NR_029401 1.7 9.71E−07 B 0.75 0.71 0.62
    hg.1 LOC100506479;
    LOC100288102;
    OTTHUMG00000039861;
    RP11-
    261C10.3
    TC05003427. FNIP1; NM_001008738 1.7 1.97E−07 B 0.78 0.77 0.72
    hg.1 OTTHUMG00000162684;
    AC005593.1
    TC06000950. SMPDL3A NM_006714 1.7 2.51E−08 B 0.76 0.65 0.71
    hg.1
    TC09000024. OTTHUMG00000019457; ENST00000457566 −1.7 1.53E−08 V 0.79 0.75 0.67
    hg.1 RP11-
    509J21.1
    TC12001567. NFE2 NM_001136023 1.7 7.17E−07 B 0.75 0.67 0.67
    hg.1
    TC12001678. GNS NM_002076 1.7 5.62E−11 B 0.82 0.73 0.72
    hg.1
    TC13000744. MYCBP2 ENST00000544440 −1.7 1.67E−15 V 0.88 0.82 0.79
    hg.1
    TC14001288. NUMB NM_001005743 1.7 7.35E−09 B 0.80 0.71 0.72
    hg.1
    TC17000770. MAP3K3 NM_002401 1.7 6.52E−11 B 0.83 0.77 0.77
    hg.1
    TC19000195. LPPR2 NM_001170635 1.7 4.85E−08 B 0.78 0.69 0.72
    hg.1
    TC19001181. RAB3D NM_004283 1.7 7.16E−10 B 0.83 0.77 0.75
    hg.1
    TC01002553. PPT1 NM_000310 1.7 1.97E−11 B 0.83 0.75 0.73
    hg.1
    TC01002678. YIPF1 NM_018982 1.7 7.81E−10 B 0.81 0.73 0.73
    hg.1
    TC01003114. GPR89A; NM_001097612 −1.7 1.59E−07 V 0.77 0.71 0.72
    hg.1 LOC101060636;
    LOC101060247;
    GPR89B
    TC01003800. LPGAT1 NM_014873 1.7 1.11E−16 B 0.90 0.86 0.85
    hg.1
    TC02001021. CYBRD1 NM_001127383 1.7 1.91E−10 B 0.80 0.71 0.76
    hg.1
    TC02001769. SLC8A1 NM_001112800 1.7 7.54E−09 B 0.79 0.73 0.70
    hg.1
    TC02002853. SP110 NM_001185015 −1.7 1.16E−07 V 0.77 0.71 0.70
    hg.1
    TC02005052. PECR NM_018441 1.7 1.05E−07 B 0.80 0.63 0.80
    hg.1
    TC03001906. P2RY13 NM_176894 1.7 1.04E−08 B 0.83 0.80 0.76
    hg.1
    TC03002156. ZDHHC19 NM_001039617 1.7 2.21E−12 B 0.82 0.73 0.81
    hg.1
    TC04001501. FLJ14186 NR_037596 1.7 5.97E−07 B 0.75 0.71 0.61
    hg.1
    TC07000437. GTF2IP1; NR_002206 1.7 3.42E−12 B 0.83 0.75 0.71
    hg.1 LOC100093631
    TC07001970. LOC100507507; ENST00000429630 1.7 2.49E−09 B 0.81 0.73 0.77
    hg.1 OTTHUMG00000152694;
    AC093673.5
    TC0X000785. 0 ENST00000453508 1.7 8.94E−08 B 0.77 0.69 0.71
    hg.1
    TC0X001552. TMLHE-ASI; NR_039991 1.7 2.54E−07 B 0.76 0.67 0.73
    hg.1 OTTHUMG00000022662;
    RP13-
    228J13.1
    TC10000092. CDC123 NM_006023 1.7 1.58E−13 B 0.89 0.82 0.84
    hg.1
    TC11000316. HIPK3 NM_001048200 1.7 4.19E−12 B 0.84 0.80 0.82
    hg.1
    TC15000871. FES NM_001143783 1.7 2.61E−14 B 0.85 0.75 0.82
    hg.1
    TC16000472. MT1B NM_005947 −1.7 8.61E−10 V 0.79 0.84 0.70
    hg.1
    TC16000893. NPIPA8; ENST00000358815 −1.7 2.27E−07 V 0.78 0.78 0.75
    hg.1 PKD1P6
    TC16002075. MT1L NR_001447 −1.7 2.43E−11 V 0.83 0.82 0.75
    hg.1
    TC05001491. COL4A3BP NM_001130105 1.7 <10-17 B 0.91 0.88 0.90
    hg.1
    TC14001372. SEL1L NM_001244984 1.7 <10-17 B 0.89 0.82 0.81
    hg.1
    TC12002073. CLIP1 NM_002956 1.6 <10-17 B 0.90 0.86 0.80
    hg.1
    TC15000864. IQGAP1 NM_003870 1.6 1.11E−1 B 0.88 0.84 0.82
    hg.1 6
    TC08001218. LYPLA1 NM_006330 1.6 <10-17 B 0.89 0.78 0.81
    hg.1
    TC11001915. EHD1 NM_006795 1.6 <10-17 B 0.91 0.84 0.87
    hg.1
    TC17000725. CLTC NM_004859 1.6 <10-17 B 0.90 0.84 0.84
    hg.1
    TC04000257. TMEM33 NM_018126 1.6 <10-17 B 0.91 0.77 0.84
    hg.1
    TC09000428. SYK NM_001135052 1.6 2.22E−16 B 0.89 0.77 0.81
    hg.1
    TC02001555. KIDINS220 NM_020738 1.6 5.55E−16 B 0.88 0.78 0.79
    hg.1
    TC19001231. PRKACA NM_002730 1.6 3.33E−16 B 0.87 0.73 0.81
    hg.1
    TC17000804. PRKAR1A NM_002734 1.6 <10-17 B 0.91 0.82 0.85
    hg.1
    TC05000664. PHF15 NM_015288 −1.6 <10-17 V 0.89 0.77 0.84
    hg.1
    TC10001623. ACTR1A NM_005736 1.6 <10-17 B 0.90 0.80 0.82
    hg.1
    TC03001074. LINC00884; NR_033929 1.6 5.55E−16 B 0.90 0.78 0.86
    hg.1 OTTHUMG00000156037;
    AC046143.7
    TC01003712. CYB5R1 NM_016243 1.5 3.33E−16 B 0.88 0.78 0.82
    hg.1
    TC10000240. CCNY NM_145012 1.5 1.11E−16 B 0.89 0.77 0.80
    hg.1
    TC17000057. RNF167 NM_015528 1.5 7.77E−16 B 0.89 0.84 0.85
    hg.1
    TC03001293. ACAA1; NM_001130410 1.5 <10-17 B 0.90 0.77 0.85
    hg.1 OTTHUMG00000155978;
    AP006309.4
    TC10000010. WDR37 NM_014023 1.5 <10-17 B 0.92 0.84 0.87
    hg.1
    TC21000538. ITGB2 NM_001127491 1.5 8.88E−16 B 0.88 0.78 0.81
    hg.1
    TC02000378. ACTR2 NM_001005386 1.5 <10-17 B 0.91 0.86 0.81
    hg.1
    TC03000816. SELT; NM_016275 1.4 6.66E−16 B 0.87 0.80 0.80
    hg.1 OTTHUMG00000159825;
    RP11-
    392O18.1
    TC02002035. TGOLN2 NM_001206840 1.4 <10-17 B 0.90 0.80 0.82
    hg.1
    TC05001553. TMEM167A NM_174909 1.4 5.55E−16 B 0.89 0.75 0.82
    hg.1
    TC12001797. POC1B; NM_001199777 1.4 9.99E−16 B 0.89 0.86 0.85
    hg.1 POC1B-
    GALNT4;
    GALNT4
    TC17000862. UNK NM_001080419 −1.4 <10-17 V 0.89 0.82 0.82
    hg.1
    TC07003288. ARPC1B NM_005720 1.4 <10-17 B 0.89 0.78 0.87
    hg.1
    TC12000379. TMBIM6 NM_001098576 1.3 <10-17 B 0.89 0.77 0.80
    hg.1
    TC13000421. LAMP1 NM_005561 1.3 3.33E−16 B 0.88 0.82 0.81
    hg.1
    TC15000840. AEN NM_022767 −1.3 2.22E−16 V 0.89 0.78 0.79
    hg.1
    TC12003225. ARF3 NM_001659 1.3 3.33E−16 B 0.89 0.82 0.81
    hg.1
    TC17001755. TBC1D3P1- NR_002924 1.3 3.33E−16 B 0.88 0.77 0.80
    hg.1 DHX40P1
    TC02002826. CUL3 NM_003590 1.3 <10-17 B 0.90 0.88 0.79
    hg.1
    TC08000486. TMEM70 NM_001040613 1.3 1.11E−16 B 0.89 0.78 0.82
    hg.1
    TC09000550. AKAP2; NM_001004065 −1.2 7.77E−16 V 0.87 0.84 0.79
    hg.1 PALM2;
    PALM2-
    AKAP2
    TC0X000256. WDR13 NM_001166426 1.2 3.33E−16 B 0.88 0.82 0.82
    hg.1
    TC03000282. TREX1; NM_016381 −1.2 <10-17 V 0.91 0.82 0.86
    hg.1 ATRIP
    TC01002089. UBE2J2 NM_058167 1.2 5.55E−16 B 0.88 0.73 0.82
    hg.1
    TC07001120. ACTB; NM_001101 1.2 <10-17 B 0.89 0.84 0.82
    hg.1 LOC100505829
    TC01003383. AIM2 NM_004833 −1.31 0.251342 V 0.57 0.49 0.66
    hg.1
    TC10000698. ANKRD2 NM_020349 1.03 0.032655 B 0.61 0.59 0.60
    hg.1
    TC11003507. CARD17 NM_001007232 −2.06 0.05858 V 0.60 0.63 0.60
    hg.1
    TC19001575. CEACAM1 NM_001712 −1.02 0.751956 V 0.50 0.55 0.54
    hg.1
    TC12000130. CLEC4D NM_080387 1.67 0.003066 B 0.65 0.63 0.63
    hg.1
    TC06001246. F13A1 NM_000129 1.68 0.000269 B 0.68 0.73 0.58
    hg.1
    TC07000004. FAM20C NM_020223 1.1 0.000007 B 0.75 0.75 0.66
    hg.1
    TC19000467. FFAR3 NM_005304 1.03 0.740673 B 0.51 0.45 0.66
    hg.1
    TC6_cox_ HLA-DQA1 uc011fnq.1 −1.96 0.010798 V 0.62 0.71 0.49
    hap2000091.
    hg.1
    TC11001230. IFITM3 NM_021034 −1.18 0.001761 V 0.70 0.82 0.58
    hg.1
    TC0Y000185. KDM5D NM_001146705 2.64 0.847674 B 0.54 0.53 0.67
    hg.1
    TC17001661. PHOSPHO1 NM_001143804 1.2 0.131061 B 0.57 0.53 0.54
    hg.1
    TC03001869. PLSCR1 NM_021105 −1.69 0.004736 V 0.67 0.75 0.60
    hg.1
    TC04001282. PPBP NM_002704 1.31 0.174717 B 0.57 0.63 0.54
    hg.1
    TC18000477. PSTPIP2 NM_024430 2.08 0.000182 B 0.70 0.67 0.70
    hg.1
    TC07003373. RASA4B ENST00000306682 −1.13 0.005496 V 0.64 0.78 0.53
    hg.1
    TC01001622. RGS1 NM_002922 −2.07 0.000003 V 0.75 0.73 0.65
    hg.1
    TC06000758. SH3BGRL2 NM_031469 1.36 0.04134 B 0.60 0.57 0.62
    hg.1
    TC02001159. SPATS2L NM_001100422 −6.94 1.78E−13 V 0.87 0.84 0.75
    hg.1
    TC07001007. TMEM176A NM_018487 −1.05 0.541629 V 0.52 0.57 0.52
    hg.1
    TC02000937. TNFAIP6 NM_007115 −1.45 0.086888 V 0.60 0.67 0.51
    hg.1
    TC06000565. TREML4 NM_198153 −1 0.763806 V 0.54 0.55 0.62
    hg.1
    TC01003963. MIR1182 NR_031593 1.18 0.000057 B 0.68 0.65 0.70
    hg.1 (FAM89A)
    TC02001333. STK11IP NM_052902 1.1 <10-17 B 0.88 0.78 0.81
    hg.1
  • TABLE 15B
    Differentially expressed non-coding RNA determinants and their
    measures of accuracy in differentiating between bacterial (“B”) versus viral (“V”)
    infected subjects that met the following criteria: (ttest p-value < 10−15) OR (absolute
    linear fold change > 4) OR (ttest p-value < 10−6 AND absolute linear fold change > 1.7).
    ttest
    p-
    value
    (Bac- Up
    Probe Linear terial in
    Set mRNA Chro- fold vs. B/ Sensi- Speci-
    ID Accession mosome Strand Start Stop change Viral) V AUC tivity ficity
    TC0X002125.hg.1 n337756 chrX − 73040450 73072588 −18.6 0.195207 V 0.57 0.53 0.52
    TC02003396.hg.1 n334260 chr2 + 89901316 89901798 −9.0 0.000036 V 0.70 0.67 0.70
    TC02003397.hg.1 n336673 chr2 + 89952882 89953388 −7.0 0.00001 V 0.72 0.73 0.63
    TC02003398.hg.1 n387119 chr2 + 89976190 89976490 −7.0 0.000048 V 0.72 0.73 0.66
    TC02003395.hg.1 n335962 chr2 + 89890582 89891332 −6.9 0.000006 V 0.73 0.73 0.68
    TC02003400.hg.1 n336675 chr2 + 89998820 89999595 −6.9 0.000004 V 0.73 0.77 0.67
    TC02003399.hg.1 n383671 chr2 + 89986780 89987080 −6.8 0.000124 V 0.71 0.73 0.61
    TC02003402.hg.1 n337071 chr2 + 90198907 90199183 −6.8 0.000044 V 0.70 0.69 0.66
    TC02003394.hg.1 n335964 chr2 + 89185097 89185706 −6.8 0.0001 V 0.70 0.67 0.65
    TC02003403.hg.1 n386251 chr2 + 90273948 90274233 −6.7 0.000005 V 0.73 0.71 0.65
    TC02004397.hg.1 n335966 chr2 − 89416834 89417284 −5.9 0.000013 V 0.72 0.69 0.63
    TC02003401.hg.1 n33570l chr2 + 90077752 90078323 −5.7 0.000017 V 0.72 0.69 0.66
    TC02004396.hg.1 n336205 chr2 − 89160711 89326952 −5.5 0.000017 V 0.72 0.71 0.66
    TC06003881.hg.1 TCONS_ chr6 − 138264216 138266939 −5.2 0.000015 V 0.73 0.71 0.61
    00011573-
    XLOC_
    005851
    TC02004395.hg.1 n346551 chr2 − 89109725 89619842 −5.1 0.000007 V 0.72 0.73 0.70
    TC22001009.hg.1 n387241 chr22 + 23261707 23262023 −4.4 0.000019 V 0.72 0.63 0.68
    TC01004103.hg.1 TCONS_ chr1 + 329784 342806 2.9 6.87E−11 B 0.81 0.69 0.75
    12_
    00001926−
    XLOC_
    12_000004
    TC01004106.hg.1 TCONS_ chr1 + 459656 461954 2.1 1.25E−09 B 0.79 0.69 0.80
    00000121-
    XLOC_
    000003
    TC01004233.hg.1 TCONS_ chr1 + 21912965 21917680 1.7 5.73E−09 B 0.79 0.65 0.77
    00000166-
    XLOC_
    000087
    TC01004314.hg.1 n337860 chr1 + 36321683 36323276 2.3 3.34E−12 B 0.85 0.80 0.82
    TC01004517.hg.1 n338053 chr1 + 89647181 89647394 −3.2 0.000001 V 0.73 0.67 0.67
    TC01004674.hg.1 n345870 chr1 + 121107156 121124603 −2.8 3.91E−12 V 0.87 0.84 0.76
    TC01004676.hg.1 n406301 chr1 + 121260910 121313686 1.8 4.27E−14 B 0.87 0.80 0.81
    TC01004687.hg.1 n346374 chrl + 143913797 144094477 −2.7 1.81E−11 V 0.86 0.82 0.67
    TC01004799.hg.1 n338331 chr1 + 161929297 161929914 2.3 1.51E−13 B 0.87 0.84 0.81
    TC01005153.hg.1 n407167 chr1 + 245133284 245288530 2.0 0.000001 B 0.72 0.61 0.71
    TC01005184.hg.1 n326766 chr1 − 131337 140566 2.2 1.42E−07 B 0.77 0.73 0.66
    TC01005186.hg.1 n326763 chr1 − 154268 163727 2.5 4.16E−07 B 0.76 0.73 0.68
    TC01005187.hg.1 n387107 chr1 − 222732 373960 1.8 3.15E−08 B 0.77 0.71 0.75
    TC01005190.hg.1 TC0NS_ chr1 − 521369 523833 1.8 5.06E−09 B 0.78 0.69 0.80
    00000442-
    XLOC_
    000663
    TC01005192.hg.1 TC0NS_ chr1 − 637316 659930 2.8 1.19E−11 B 0.82 0.71 0.75
    12_
    00002386-
    XLOC_
    12_000726
    TC01005194.hg.1 n326751 chr1 − 684275 703869 3.1 5.67E−07 B 0.76 0.73 0.67
    TC01005199.hg.1 n381032 chr1 − 803453 812182 1.8 3.23E−07 B 0.75 0.71 0.71
    TC01005259.hg.1 n335242 chr1 − 9794131 9796076 −2.0 6.82E−07 V 0.77 0.65 0.72
    TC01005339.hg.1 n337573 chr1 − 25245804 25254104 −2.7 6.07E−12 V 0.83 0.80 0.81
    TC01005358.hg.1 TC0NS_ chr1 − 27986839 27989233 −2.0 4.06E−10 V 0.84 0.86 0.68
    00000492-
    XLOC_
    000755
    TC01005362.hg.1 n337807 chr1 − 28476184 28520663 1.9  5.9E−13 B 0.87 0.78 0.84
    TC01005450.hg.1 n406645 chr1 − 47023090 47069966 2.3 1.74E−11 B 0.83 0.77 0.73
    TC01005498.hg.1 n410164 chr1 − 54317392 54355487 1.9 4.17E−10 B 0.81 0.73 0.72
    TC01005541.hg.1 TC0NS_ chr1 − 65450881 65451399 2.0 1.77E−09 B 0.80 0.75 0.68
    12_
    00001274-
    XLOC_
    2_000942
    TC01005698.hg.1 n338128 chr1 − 111196209 111197947 −3.1 1.09E−07 V 0.79 0.67 0.80
    TC01005699.hg.1 n338129 chr1 − 111197952 111199662 −3.2 5.73E−07 V 0.76 0.61 0.75
    TC01005765.hg.1 n345681 chr1 − 143906069 143912903 −2.0 2.72E−08 V 0.82 0.78 0.70
    TC01005770.hg.1 n337415 chr1 − 144521639 144522054 −2.4 9.33E−15 V 0.86 0.78 0.79
    TC01005782.hg.1 n410110 chr1 − 145764595 145827103 −1.8 2.22E−07 V 0.77 0.67 0.71
    TC01006029.hg.1 n344753 chr1 − 200298333 200343319 1.8 3.81E−10 B 0.78 0.63 0.76
    TC01006030.hg.1 n338446 chr1 − 200374083 200375228 1.9 1.24E−09 B 0.81 0.71 0.77
    TC01006060.hg.1 n338489 chr1 − 205578844 205581144 −2.2 0.000001 V 0.75 0.69 0.71
    TC01006141.hg.1 TC0NS_ chr1 − 222683786 222685126 1.8 1.51E−07 B 0.76 0.71 0.65
    12_
    00001794-
    XLOC_
    12_001325
    TC01006194.hg.1 TC0NS_ chr1 − 231658134 231664321 1.8 9.56E−08 B 0.76 0.65 0.71
    00000756-
    XLOC_
    001257
    TC01006221.hg.1 n342369 chr1 − 237167403 237167718 −2.2 1.85E−10 V 0.82 0.88 0.67
    TC01006237.hg.1 TC0NS_ chr1 − 243192814 243215554 2.6 2.71E−11 B 0.82 0.71 0.71
    12_
    00002811-
    XLOC_
    12_001398
    TC01006238.hg.1 n381375 chr1 − 243219131 243265070 1.8 0.000001 B 0.75 0.73 0.63
    TC01006418.hg.1 TC0NS_ chr1 − 89295 173864 2.2 8.17E−12 B 0.82 0.73 0.75
    12_
    00002367-
    XLOC_
    12_000720
    TC01006419.hg.1 TC0NS_ chr1 − 227615 267253 2.5 1.32E−10 B 0.80 0.65 0.79
    12_
    00002379-
    XLOC_
    12_000720
    TC02003008.hg.1 n332762 chr2 + 7037597 7037917 −36.0 2.22E−16 V 0.87 0.75 0.82
    TC02003047.hg.1 n407780 chr2 + 12856998 12882860 −4.4 6.55E−15 V 0.88 0.80 0.82
    TC02003330.hg.1 n406230 chr2 + 73872046 73912694 −2.8 0.000001 V 0.79 0.78 0.57
    TC02003660.hg.1 TC0NS_ chr2 + 163175544 163176543 −2.5 8.45E−08 V 0.76 0.78 0.61
    00004935-
    XLOC_
    001732
    TC02003715.hg.1 n383778 chr2 + 179278390 179303866 −2.7 3.91E−14 V 0.92 0.90 0.82
    TC02003877.hg.1 n335493 chr2 + 231090476 231110628 −3.2 3.39E−12 V 0.82 0.67 0.71
    TC02003946.hg.1 n346494 chr2 + 243030784 243102469 1.9 9.16E−11 B 0.80 0.65 0.76
    TC02004017.hg.1 TC0NS_ chr2 − 6968645 6973662 −8.1 5.22E−15 V 0.88 0.82 0.77
    00003184-
    XLOC_
    001966
    TC02004035.hg.1 n338647 chr2 − 8992820 8994376 2.9 2.78E−09 B 0.80 0.75 0.77
    TC02004123.hg.1 n332362 chr2 − 26680851 26683576 −9.8 1.15E−11 V 0.88 0.92 0.73
    TC02004161.hg.1 n332938 chr2 − 37334617 37347277 −4.0 1.35E−14 V 0.86 0.78 0.81
    TC02004178.hg.1 n338783 chr2 − 40324916 40328505 2.1 5.25E−12 B 0.84 0.77 0.77
    TC02004676.hg.1 n334856 chr2 − 167159708 167163041 1.8 4.12E−07 B 0.75 0.63 0.72
    TC02004815.hg.1 n405559 chr2 218923878 218926013 −2.8 7.48E−09 V 0.79 0.73 0.70
    TC02004860.hg.1 n339176 chr2 − 235401690 235403788 −3.7 9.58E−09 V 0.78 0.71 0.79
    TC02004915.hg.1 TC0NS_ chr2 − 243173849 243176661 2.0 1.54E−09 B 0.79 0.69 0.76
    00003525-
    XLOC_
    002561
    TC03002496.hg.1 n407114 chr3 + 122605360 122611263 2.5 6.45E−14 B 0.85 0.73 0.76
    TC03002524.hg.1 n409261 chr3 + 127391781 127399769 2.5 7.05E−10 B 0.83 0.80 0.75
    TC03002862.hg.1 n406516 chr3 − 38164201 38178733 1.9 0 B 0.91 0.80 0.84
    TC03003206.hg.1 n408012 chr3 − 171064723 171178197 −2.3 2.72E−11 V 0.83 0.75 0.81
    TC03003297.hg.1 n384393 chr3 − 195961240 195964785 2.3 0 B 0.90 0.82 0.86
    TC03003311.hg.1 n339763 chr3 − 197398856 197476259 −2.5 3.06E−11 V 0.83 0.77 0.66
    TC04001945.hg.1 n342579 chr4 + 26322301 26432628 2.1 4.35E−14 B 0.87 0.80 0.80
    TC04001946.hg.1 n384435 chr4 + 26434615 26436541 2.0 5.15E−12 B 0.84 0.80 0.81
    TC04001986.hg.1 n409153 chr4 + 38869354 38947365 1.4 9.99E−16 B 0.87 0.73 0.85
    TC04002081.hg.1 n344493 chr4 + 79567001 79611023 2.5 1.01E−12 B 0.83 0.71 0.77
    TC04002168.hg.1 TC0NS_ chr4 + 120299287 120326770 2.3 6.11E−15 B 0.87 0.86 0.75
    12_
    00021682-
    XLOC_
    12_010810
    TC04002243.hg.1 n342622 chr4 + 146298061 146299109 2.3 7.77E−16 B 0.89 0.84 0.81
    TC04002305.hg.1 n342624 chr4 + 166143201 166144207 2.7 9.13E−09 B 0.79 0.67 0.86
    TC04002425.hg.1 n335597 chr4 − 3514297 3520638 2.1 1.42E−13 B 0.86 0.80 0.80
    TC04002523.hg.1 n339952 chr4 − 40434720 40631861 1.9 1.33E−07 B 0.79 0.80 0.67
    TC04002626.hg.1 n384478 chr4 − 89178768 89180508 −3.2 4.44E−16 V 0.89 0.82 0.81
    TC04002660.hg.1 n346125 chr4 − 106058437 106061776 1.9 5.55E−12 B 0.82 0.71 0.73
    TC04002680.hg.1 n337482 chr4 − 114421622 114434514 −1.9 3.94E−08 V 0.78 0.71 0.75
    TC04002689.hg.1 TC0NS_ chr4 − 120314433 120316288 1.8 0.000001 B 0.76 0.77 0.67
    12_
    00021261-
    XLOC_
    12_011236
    TC04002799.hg.1 n410166 chr4 − 157682763 157892546 2.0 1.21E−10 B 0.84 0.65 0.86
    TC04002840.hg.1 n407800 chr4 − 175411328 175444044 8.2 2.48E−11 B 0.82 0.71 0.76
    TC05002362.hg.1 n335106 chr5 + 44809946 44811233 −1.8 1.55E−07 V 0.77 0.73 0.76
    TC05002634.hg.1 n340631 chr5 + 133861798 133918918 −2.1 2.22E−16 V 0.88 0.77 0.82
    TC05002675.hg.1 n340670 chr5 + 142605733 142608561 2.1 4.47E−09 B 0.81 0.77 0.73
    TC05002912.hg.1 n377768 chr5 − 17130137 17217531 1.9 4.27E−13 B 0.84 0.77 0.77
    TC05002971.hg.1 TC0NS_ chr5 − 39721152 39721560 1.8 4.88E−09 B 0.80 0.73 0.73
    12_
    00022861-
    XLOC_
    12_012011
    TC05002988.hg.1 n334545 chr5 − 49692315 49692643 1.7 7.43E−09 B 0.80 0.73 0.75
    TC05003028.hg.1 n340484 chr5 − 64961757 64965240 1.7 5.65E−13 B 0.85 0.69 0.80
    TC06002755.hg.1 TC0NS_ chr6 + 36084167 36091301 3.0 1.37E−08 B 0.77 0.75 0.76
    00011799-
    XLOC_
    005273
    TC06002848.hg.1 n334582 chr6 + 64288842 64290046 −2.0 7.44E−08 V 0.77 0.78 0.68
    TC06003117.hg.1 n385033 chr6 + 147708804 147711601 2.0  2.2E−09 B 0.79 0.75 0.73
    TC06003248.hg.1 n377578 chr6 − 130803 148170 2.5 1.72E−10 B 0.81 0.71 0.73
    TC06003606.hg.1 n408222 chr6 − 33259431 33267176 1.7  1.4E−08 B 0.79 0.80 0.77
    TC06003619.hg.1 n409669 chr6 − 35800811 35888957 3.1 6.66E−16 B 0.88 0.80 0.77
    TC06003642.hg.1 n384933 chr6 − 41176294 41185685 1.9 5.42E−07 B 0.76 0.57 0.79
    TC06003793.hg.1 n342100 chr6 − 108018503 108021422 −1.7 4.81E−09 V 0.79 0.71 0.72
    TC06003855.hg.1 n409772 chr6 − 133065009 133079033 1.9 1.58E−08 B 0.80 0.77 0.68
    TC07002132.hg.1 n385079 chr7 + 7284225 7288212 −1.8 9.47E−09 V 0.78 0.73 0.66
    TC07002222.hg.1 n341216 chr7 + 28863333 28865505 2.2 3.18E−09 B 0.82 0.77 0.71
    TC07002377.hg.1 n334125 chr7 + 64838819 64863612 −2.1 4.56E−08 V 0.79 0.77 0.72
    TC07002398.hg.1 n406149 chr7 + 72569012 72621336 1.7 4.01E−12 B 0.82 0.73 0.70
    TC07002401.hg.1 n410558 chr7 + 73097898 73112542 −1.7 0.000001 V 0.75 0.69 0.68
    TC07002501.hg.1 n341285 chr7 + 101898320 101901513 1.8 1.05E−14 B 0.86 0.73 0.81
    TC07002605.hg.1 n341331 chr7 + 135242688 135333497 −2.5 1.11E−16 V 0.89 0.78 0.87
    TC07002618.hg.1 n408329 chr7 + 139528952 139720125 1.9 2.73E−12 B 0.87 0.82 0.80
    TC07002675.hg.1 n334779 chr7 + 150270658 150271040 −1.9 5.09E−11 V 0.82 0.71 0.79
    TC07002730.hg.1 TC0NS_ chr7 − 26392 35472 4.1 7.08E−13 B 0.84 0.73 0.76
    00013664-
    XLOC_
    006324
    TC07002731.hg.1 n385322 chr7 − 29246 31980 2.0 4.36E−07 B 0.75 0.67 0.67
    TC07002879.hg.1 n408312 chr7 − 33053742 33102409 −3.2 1.41E−10 V 0.81 0.75 0.70
    TC07003028.hg.1 n345780 chr7 − 74607898 74616892 1.8 6.39E−12 B 0.82 0.75 0.73
    TC07003054.hg.1 n341270 chr7 − 87900207 87903065 4.4 1.23E−13 B 0.86 0.82 0.79
    TC07003216.hg.1 n341349 chr7 − 142917561 142919360 3.6  5.5E−13 B 0.83 0.75 0.76
    TC07003239.hg.1 n406596 chr7 − 150322464 150329680 −2.0 2.22E−08 V 0.78 0.65 0.76
    TC08001887.hg.1 n407940 chr8 + 38854505 38962779 2.6 2.81E−14 B 0.86 0.78 0.77
    TC08001978.hg.1 n409175 chr8 + 74888377 74895018 1.6 0 B 0.91 0.77 0.84
    TC08002026.hg.1 n332977 chr8 + 97797356 97847419 2.3 1.08E−07 B 0.78 0.78 0.66
    TC08002146.hg.1 n346557 chr8 + 144112674 144113822 −4.2 1.49E−12 V 0.87 0.84 0.70
    TC08002165.hg.1 TCONS_ chr8 − 19134 20808 2.5 1.02E−10 B 0.80 0.71 0.77
    00014550-
    XLOC_
    006964
    TC08002237.hg.1 n342832 chr8 − 15966968 16050148 −2.2 4.75E−07 V 0.75 0.73 0.65
    TC08002245.hg.1 n406527 chr8 − 19261672 19540261 1.9 2.56E−07 B 0.74 0.67 0.62
    TC08002386.hg.1 TCONS_ chr8 − 74817620 74827134 3.0 9.91E−12 B 0.83 0.80 0.75
    12_
    00028242-
    XLOC_
    12_014551
    TC08002477.hg.1 n341587 chr8 − 116580684 116581247 1.8 0.000001 B 0.73 0.63 0.72
    TC09001920.hg.1 TCONS_ chr9 + 40308216 40324812 3.4 8.55E−07 B 0.74 0.63 0.70
    12_
    00028738-
    XLOC_
    12_014777
    TC09001921.hg.1 TCONS_ chr9 + 40332917 40337603 3.3 7.07E−07 B 0.75 0.61 0.72
    12_
    00028740-
    XLOC_
    12_014778
    TC09001933.hg.1 n385531 chr9 + 42669321 42714943 −1.7 3.23E−09 V 0.80 0.73 0.71
    TC09001936.hg.1 TCONS_ chr9 + 43313921 43314368 1.9 2.84E−12 B 0.84 0.78 0.76
    12_
    00028743-
    XLOC_
    12014791
    TC09001937.hg.1 n385540 chr9 + 43685063 43890529 2.6  3.7E−07 B 0.76 0.61 0.73
    TC09001960.hg.1 TCONS_ chr9 + 65629315 65635808 3.3 0.000001 B 0.74 0.63 0.70
    12_
    00028757-
    XLOC_
    12_014812
    TC09002184.hg.1 n410169 chr9 + 115142189 115234685 2.5 1.91E−13 B 0.85 0.86 0.80
    TC09002185.hg.1 n342875 chr9 + 115390750 115391983 −2.6 7.38E−09 V 0.80 0.78 0.68
    TC09002192.hg.1 n341760 chr9 + 116304733 116306551 −1.7 4.88E−07 V 0.78 0.80 0.75
    TC09002248.hg.1 n407851 chr9 + 127115752 127121465 1.7 1.31E−07 B 0.75 0.67 0.67
    TC09002501.hg.1 TCONS_ chr9 − 39722426 39817486 3.3 3.51E−07 B 0.75 0.59 0.76
    12_
    00029273-
    XLOC_
    12_015163
    TC09002504.hg.1 n346129 chr9 − 40501701 40503928 3.5 2.67E−07 B 0.75 0.63 0.72
    TC09002505.hg.1 n346251 chr9 − 40610094 40633391 2.2  3.8E−07 B 0.76 0.59 0.76
    TC09002572.hg.1 n332826 chr9 − 70216593 70484472 −1.8  6.4E−12 V 0.84 0.80 0.77
    TC09002865.hg.1 n335572 chr9 − 139889137 139890155 −2.6 0.000001 V 0.77 0.80 0.71
    TC0X001567.hg.1 n408005 chrX + 1387693 1428828 2.2 6.01E−11 B 0.84 0.80 0.73
    TC0X001610.hg.1 n337638 chrX + 17168618 17171104 2.7 2.15E−08 B 0.79 0.71 0.70
    TC0X001627.hg.1 n337646 chrX + 24564003 24568583 2.0 8.53E−14 B 0.86 0.73 0.80
    TC0X001767.hg.1 n344547 chrX + 73164159 73290875 −1.9 5.09E−08 V 0.77 0.77 0.67
    TC0X002006.hg.1 n335186 chrX − 18959675 18972456 1.8 5.31E−13 B 0.83 0.71 0.77
    TC0X002012.hg.1 TCONS_ chrX − 23791109 23801073 −2.4 7.26E−07 V 0.75 0.82 0.56
    00017047-
    XLOC_
    008133
    TC0X002294.hg.1 n334213 chrX − 153764207 153775148 1.8 3.17E−12 B 0.84 0.77 0.77
    TC0Y000339.hg.1 TCONS_ chrY − 27524447 27540866 2.1 6.15E−10 B 0.80 0.75 0.70
    12_
    00031062-
    XLOC_
    12_015962
    TC10001860.hg.1 n334819 chr10 + 6266130 6275053 2.5 2.73E−07 B 0.75 0.77 0.63
    TC10001982.hg.1 TCONS_ chr10 + 30964483 30980403 1.8 2.39E−08 B 0.79 0.73 0.70
    12_
    00002951-
    XLOC_
    12_001529
    TC10002016.hg.1 TCONS_ chr10 + 38692121 38712064 1.8 2.15E−08 B 0.79 0.77 0.68
    12_
    00003945-
    XLOC_
    12_001559
    TC10002018.hg.1 TCONS_ chr10 + 38742109 38764837 2.4 5.43E−11 B 0.81 0.71 0.71
    12_
    00003949-
    XLOC_
    12_001561
    TC10002216.hg.1 n340102 chr10 + 93041765 93044015 −1.9 4.57E−10 V 0.80 0.69 0.71
    TC10002454.hg.1 n339954 chr10 − 7200572 7203727 −2.1 2.41E−10 V 0.81 0.69 0.77
    TC11002664.hg.1 n341031 chr11 + 48191599 48192391 2.6 1.11E−16 B 0.89 0.86 0.84
    TC11002677.hg.1 n333961 chr11 + 57373517 57381926 −5.7 1.11E−07 V 0.76 0.65 0.73
    TC11002743.hg.1 n335245 chr11 + 67807246 67810288 1.8 1.33E−08 B 0.81 0.78 0.68
    TC11002900.hg.1 n334248 chr11 + 118355620 118360888 −2.4 2.67E−07 V 0.77 0.67 0.76
    TC11002916.hg.1 n341141 chr11 + 121500203 121504387 3.1 5.55E−16 B 1.89 0.86 0.81
    TC11002977.hg.1 n408184 chr11 − 126987 139099 1.7 6.65E−07 B 0.75 0.75 0.63
    TC11002978.hg.1 TCONS_ chr11 − 141453 180409 2.0 4.66E−09 B 0.80 0.71 0.70
    12_
    00005353-
    XLOC_
    12_002537
    TC11002997.hg.1 TCONS_ chr11 − 1792623 1793111 2.4 8.95E−13 B 0.84 0.73 0.77
    00019559-
    XLOC_
    009354
    TC11003030.hg.1 n342750 chr11 − 5699550 5959849 −1.9 1.16E−08 V 0.78 0.71 0.66
    TC11003402.hg.1 n341149 chr11 − 126210853 126225482 2.0  3.1E−12 B 0.83 0.78 0.77
    TC12002184.hg.1 TCONS_ chr12 + 4207356 4208695 −1.9 2.41E−07 V 0.76 0.73 0.70
    12_
    00005430-
    XLOC_
    12_002852
    TC12002236.hg.1 n410190 chr12 + 9822308 9852151 −2.8 2.96E−09 V 0.80 0.69 0.73
    TC12002266.hg.1 n335556 chr12 + 16507188 16516972 1.9 2.57E−07 B 0.75 0.71 0.66
    TC12002425.hg.1 n341954 chr12 + 66645119 66647711 4.1 2.44E−14 B 0.86 0.82 0.77
    TC12002517.hg.1 n410728 chr12 + 93861266 93897548 −2.0 5.49E−09 V 0.86 0.75 0.77
    TC12002519.hg.1 n333487 chr12 + 94542725 94543688 2.3 3.51E−13 B 0.85 0.77 0.77
    TC12002520.hg.1 n410532 chr12 + 94656297 94701451 1.9 1.17E−11 B 0.86 0.86 0.76
    TC12002538.hg.1 n333357 chr12 + 102108381 102120187 1.8 2.39E−07 B 0.75 0.65 0.75
    TC12002557.hg.1 TCONS_ chr12 + 106642520 106646559 1.7 1.47E−11 B 0.83 0.71 0.77
    00021206-
    XLOC_
    009869
    TC12002572.hg.1 n332510 chr12 + 113357284 113369947 −11.0 0 V 0.88 0.80 0.84
    TC12002717.hg.1 n345165 chr12 − 3900217 3910010 −2.1 3.98E−08 V 0.78 0.71 0.68
    TC12002734.hg.1 n333279 chr12 − 8074143 8078504 2.1 2.55E−11 B 0.84 0.75 0.71
    TC12002752.hg.1 n407205 chr12 − 9906735 9913497 −3.0 3.68E−08 V 0.78 0.71 0.65
    TC12002854.hg.1 n380970 chr12 − 47599681 47610239 −1.8 4.85E−08 V 0.77 0.67 0.72
    TC12002894.hg.1 n407142 chr12 − 54104902 54121307 1.8 8.93E−09 B 0.79 0.75 0.71
    TC12002904.hg.1 n345754 chr12 − 55803574 55808808 −1.8  3.6E−08 V 0.79 0.73 0.76
    TC12002978.hg.1 n341975 chr12 − 81186299 81189685 2.9 3.17E−11 B 0.83 0.80 0.75
    TC12003061.hg.1 n342007 chr12 − 106631814 106632649 2.3 8.97E−07 B 0.74 0.67 0.65
    TC12003076.hg.1 n411168 chr12 − 109489846 109491770 −2.0 7.05E−08 V 0.77 0.71 0.67
    TC12003131.hg.1 n335251 chr12 − 122826057 122839031 1.9 7.77E−16 B 0.88 0.86 0.79
    TC13001082.hg.1 n410011 chr13 + 49822047 49867622 1.8 2.64E−08 B 0.78 0.69 0.75
    TC13001241.hg.1 n333122 chr13 + 96444953 96445186 2.5 0 B 0.90 0.80 0.84
    TC13001352.hg.1 n381983 chr13 − 21946716 21948651 1.8 3.85E−08 B 0.76 0.67 0.73
    TC13001566.hg.1 n338763 chr13 − 72431194 72434285 1.8 3.22E−10 B 0.79 0.63 0.79
    TC13001567.hg.1 n332831 chr13 − 72440078 72440376 3.2 2.38E−13 B 0.86 0.78 0.85
    TC14001755.hg.1 n338370 chr14 + 71581486 71582098 2.0 1.29E−07 B 0.76 0.69 0.75
    TC14001821.hg.1 n334829 chr14 + 94577084 94582176 −79.5 0 V 0.89 0.84 0.79
    TC14001850.hg.1 n335717 chr14 + 100610216 100610573 −3.2 6.49E−13 V 0.85 0.73 0.82
    TC14002012.hg.1 n338309 chr14 − 53503463 53506755 −1.7 1.04E−09 V 0.81 0.77 0.72
    TC14002131.hg.1 n335642 chr14 − 94517267 94517543 −1.9 8.32E−08 V 0.77 0.67 0.77
    TC14002137.hg.1 n335477 chr14 − 95599758 95624347 1.8 4.44E−16 B 0.88 0.80 0.85
    TC15002250.hg.1 n337905 chr15 + 67401665 67403023 −2.1 2.58E−08 V 0.79 0.73 0.71
    TC15002251.hg.1 n332456 chr15 + 67457682 67473649 −3.7 3.11E−15 V 0.89 0.84 0.87
    TC15002275.hg.1 n333159 chr15 + 74325510 74325718 −3.0 3.84E−08 V 0.77 0.73 0.66
    TC15002276.hg.1 TCONS_ chr15 + 74342347 74346215 −2.3 5.28E−07 V 0.75 0.67 0.62
    00023979-
    XLOC_
    011308
    TC15002352.hg.1 n335102 chr15 + 89631707 89695017 2.0 1.02E−12 B 0.86 0.82 0.77
    TC15002368.hg.1 n334242 chr15 + 91422951 91423375 2.1 4.59E−13 B 0.86 0.75 0.79
    TC15002576.hg.1 n335709 chr15 − 64979775 64980426 1.8 7.51E−08 B 0.77 0.65 0.67
    TC16001471.hg.1 n339145 chr16 + 28333040 28335170 −2.4 3.53E−11 V 0.83 0.78 0.80
    TC16001575.hg.1 n342483 chr16 + 56651373 56652729 −2.1 7.77E−12 V 0.84 0.80 0.75
    TC16001576.hg.1 n382996 chr16 + 56669651 56670998 −1.7 8.34E−12 V 0.83 0.80 0.73
    TC16001663.hg.1 n339307 chr16 + 81994217 81996288 2.2 9.92E−09 B 0.78 0.69 0.75
    TC16001724.hg.1 TCONS_ chr16 + 90168702 90204399 2.1 6.08E−10 B 0.81 0.73 0.67
    12_
    00010437-
    XLOC_
    12_005350
    TC16001727.hg.1 TCONS_ chr16 + 90244125 90289178 2.4 5.65E−11 B 0.80 0.69 0.73
    12_
    00010440-
    XLOC_
    12_005352
    TC16001933.hg.1 n333730 chr16 − 67472406 67472900 1.7 5.11E−15 B 0.88 0.78 0.79
    TC16001982.hg.1 n342493 chr16 − 81009699 81040500 −2.3 4.03E−12 V 0.85 0.80 0.73
    TC16002024.hg.1 n339330 chr16 − 88781755 88802659 −1.8 2.9E−11 V 0.83 0.73 0.80
    TC17002123.hg.1 n335565 chr17 + 21188234 21217539 1.7 4.78E−10 B 0.83 0.78 0.76
    TC17002136.hg.1 n406530 chr17 + 25958174 25976586 −2.0 7.29E−08 V 0.78 0.78 0.71
    TCI7002137.hg.1 TCONS_ chr17 + 26075314 26081148 −2.0 1.44E−07 V 0.77 0.77 0.71
    12_
    00010642-
    XLOC_
    12_005719
    TC17002330.hg.1 n336256 chr17 + 66508142 66518982 1.8 5.77E−15 B 0.89 0.86 0.84
    TC17002331.hg.1 n334200 chr17 + 66528681 66528910 1.9 1.53E−11 B 0.83 0.75 0.76
    TC17002417.hg.1 n332377 chr17 + 80439022 80441618 2.0 1.77E−12 B 0.85 0.77 0.82
    TC17002426.hg.1 n340411 chr17 − 289773 292354 2.9 5.86E−09 B 0.78 0.67 0.73
    TC17002741.hg.1 TCONS_ chr17 − 60363989 60374387 2.0 4.35E−10 B 0.82 0.77 0.76
    12_
    00011393-
    XLOC_
    12_006157
    TC17002760.hg.1 n407998 chr17 − 66531257 66554568 3.2 3.11E−13 B 0.84 0.73 0.81
    TC17002840.hg.1 n410075 chr17 − 80400463 80408707 2.0 7.25E−10 B 0.80 0.75 0.72
    TC18000741.hg.1 n342580 chr18 + 57567237 57571537 −2.3 2.71E−08 V 0.79 0.84 0.66
    TC18000749.hg.1 n383341 chr18 + 60249038 60253962 −2.7 2.58E−09 V 0.80 0.80 0.66
    TC18000932.hg.1 n342577 chr18 − 52890054 52891798 −2.3 2.09E−07 V 0.76 0.63 0.71
    TC18000950.hg.1 n345365 chr18 − 60181761 60186196 −3.9 1.43E−09 V 0.82 0.86 0.67
    TC18000987.hg.1 n339927 chr18 − 74072230 74207146 1.8 3.44E−09 B 0.79 0.77 0.72
    TC19001991.hg.1 TCONS_ chr19 + 12670361 12671726 1.9 4.44E−16 B 0.88 0.75 0.79
    12_
    00012321-
    XLOC_
    12_006609
    TC19002006.hg.1 TCONS_ chr19 + 14903114 14903565 1.7 1.11E−16 B 0.89 0.73 0.81
    00026907-
    XLOC_
    012980
    TC19002021.hg.1 n386206 chr19 + 17516503 17526545 −2.2 1.84E−08 V 0.77 0.69 0.71
    TC19002275.hg.1 n408181 chr19 − 197016 202209 1.9 7.74E−07 B 0.76 0.69 0.66
    TC19002278.hg.1 n332372 chr19 − 868460 891007 1.5 6.66E−16 B 0.87 0.78 0.81
    TC19002293.hg.1 n335978 chr19 − 3053866 3061276 −2.6 0.000001 V 0.76 0.69 0.75
    TC19002498.hg.1 TCONS_ chr19 − 43877100 43878596 15.6 2.88E−07 B 0.76 0.73 0.62
    12_
    00013127-
    XLOC_
    12_007062
    TC19002531.hg.1 n410995 chr19 − 47990891 48018515 −2.4  2.6E−09 V 0.79 0.77 0.71
    TC20001131.hg.1 n339591 chr20 + 19867165 19981449 −3.5 4.95E−10 V 0.82 0.88 0.67
    TC20001169.hg.1 TCONS_ chr20 + 24911303 24912191 2.1 6.96E−08 B 0.77 0.67 0.75
    00028139-
    XLOC_
    013499
    TC20001204.hg.1 n407149 chr20 + 35234137 35240960 1.7 1.85E−09 B 0.80 0.71 0.75
    TC20001277.hg.1 n333665 chr20 + 48808287 48808546 2.1 0.000001 B 0.76 0.75 0.65
    TC20001280.hg.1 TCONS_ chr20 + 48917632 48918579 2.0 4.45E−08 B 0.77 0.65 0.73
    00028564-
    XLOC_
    013561
    TC20001365.hg.1 TCONS_ chr20 + 62921738 62944485 2.4 7.42E−11 B 0.81 0.69 0.72
    12_
    00016828-
    XLOC_
    12_008724
    TC20001380.hg.1 n325088 chr20 − 1732662 1746252 2.3 7.45E−10 B 0.78 0.63 0.77
    TC20001381.hg.1 n410198 chr20 − 1754011 1760392 2.0 2.35E−09 B 0.79 0.67 0.76
    TC20001463.hg.1 n383870 chr20 − 20370273 20372205 2.3 3.57E−09 B 0.80 0.75 0.77
    TC21000739.hg.1 n333319 chr21 + 42823118 42824743 −15.5 0 V 0.91 0.82 0.84
    TC21000820.hg.1 n346212 chr21 − 16133566 16135557 −2.1 2.46E−09 V 0.85 0.90 0.73
    TC21000987.hg.1 n335673 chr21 − 40714244 40714822 −1.7 1.58E−08 V 0.79 0.78 0.72
    TC22000937.hg.1 n384074 chr22 + 17593918 17596583 2.5 2.22E−16 B 0.90 0.82 0.84
    TC22001279.hg.1 n337998 chr22 − 26917964 26920144 2.1 3.42E−08 B 0.77 0.69 0.70
    TC22001419.hg.1 TCONS_ chr22 − 50981206 50983413 −2.2 5.55E−08 V 0.78 0.77 0.65
    00029745-
    XLOC_
    014418
    TC0Y000317.hg.1 n337555 chrY − 15470990 15471751 4.1 0.37108 B 0.57 0.53 0.52
    TCUn_ TCONS_ chrUn_ − 21807 48108 −1.7 1.14E−09 V 0.81 0.75 0.68
    gl000211000004.hg.1 00030021- gl000211
    XLOC_
    014504
    TC0Y000231.hg.1 TCONS_ chrY + 2870953 2970313 10.2 0.598582 B 0.56 0.49 0.48
    00017606-
    XLOC_
    008276
    TC0X001784.hg.1 TCONS_ chrX + 88678737 88701982 10.7 0.655978 B 0.53 0.47 0.48
    00017000-
    XLOC_
    008022
  • TABLE 16A
    Differentially expressed coding RNA determinants and their measures of
    accuracy in differentiating between bacterial (“B”) versus viral (“V”) infected
    subjects that met the following criteria: ttest p-value < 10−14 AND absolute linear fold
    change > 3.5.
    Linear UP
    Probe Set mRNA Gene fold ttest p-value in
    ID Accession Symbol change (Bacterial vs. Viral) B/V AUC Sensitivity Specificity
    TC17001129.hg.1 NM_201433 GAS7 4.5 <10-17 B 0.91 0.82 0.87
    TC04000485.hg.1 NM_016323 HERC5 16.5 9.99E−16 V 0.87 0.80 0.82
    TC04000484.hg.1 NM_001165136 HERC6 11.2 <10-17 V 0.92 0.88 0.84
    TC14000584.hg.1 NM_001130080 IFI27 73.5 1.11E−16 V 0.89 0.77 0.89
    TCO1000795.hg.1 NM_006417 IFI44 15.2 1.44E−15 V 0.86 0.80 0.80
    TCO1000794.hg.1 NM_006820 IFI44L 31.5 <10-17 V 0.89 0.84 0.81
    TCI0000639.hg.1 NM_001548 IFIT1 28.2 <10-17 V 0.90 0.80 0.86
    TCI8000060.hg.1 NM_014214 IMPA2 3.6 <10-17 B 0.93 0.88 0.85
    TC22000191.hg.1 NM_001039570 KREMEN1 5.9 1.11E−16 B 0.89 0.80 0.89
    TC12001843.hg.1 NM_000895 LTA4H 4.3 <10-17 B 0.93 0.86 0.92
    TC08000814.hg.1 NM_001127213 LY6E 11.8 <10-17 V 0.89 0.80 0.85
    TC21000189.hg.1 NM_001144925 MXI −8.8 <10-17 V 0.90 0.84 0.84
    TC12000884.hg.1 NM_001324090 OAS1 −6.4 1.11E−16 V 0.88 0.78 0.85
    TC12000886.hg.1 NM_001032731 OAS2 15.4 <10-17 V 0.89 0.82 0.84
    TC12000885.hg.1 NM_006187 OAS3 16.2 1.11E−16 V 0.88 0.86 0.76
    TCI2002059.hg.1 NM_003733 OASL 10.2 3.44E−15 V 0.87 0.78 0.81
    TC04001373.hg.1 NM_152542 PPM1K −4.0 <10-17 V 0.91 0.84 0.87
    TC02000034.hg.1 NM_080657 RSAD2 26.3 1.44E−15 V 0.86 0.77 0.85
    TC20000575.hg.1 NM_023068 SIGLEC1 15.1 <10-17 V 0.92 0.86 0.89
    TC22000029.hg.1 NM_017414 USP18 34.7 <10-17 V 0.92 0.92 0.84
  • TABLE 16B
    Differentially expressed coding RNA determinants and their measures of
    accuracy in differentiating between bacterial (“B”) versus viral (“V”) infected
    subjects that met the following criteria: ttest p-value<10−14 AND absolute linear fold
    change > 3.5.
    Linear ttest p-value UP
    Probe Set mRNA Gene fold (Bacterial vs. in
    ID Accession Symbol change Viral) B/V AUC Sensitivity Specificity
    TC04001372.hg.1 uc003hrl.1 — −3.5 2.22E−16 V 0.89 0.90 0.79
    TC02005020.hg.1 NM_207315 CMPK2 −13.6 <10-17 V 0.89 0.86 0.81
    TC01001738.hg.1 NM_000573 CR1 3.9 <10-17 B 0.90 0.88 0.81
    TC02001749.hg.1 OTTHUMT00000325647 CYP1B1 6.3 1.11E−16 B 0.88 0.82 0.81
    TC02001750.hg.1 NM_000104 CYP1B1 5.8 <10-17 B 0.88 0.77 0.85
    TC04001718.hg.1 NM_017631 DDX60 −7.0 5.66E−15 V 0.85 0.80 0.84
    TC11000812.hg.1 NM_001253891 DGAT2 4.6 7.77E−16 B 0.89 0.88 0.80
    TC07001916.hg.1 NM_022750 PARP12 −6.4 <10-17 V 0.89 0.78 0.89
    TC02001866.hg.1 NM_033109 PNPT1 −6.8 <10-17 V 0.93 0.90 0.90
    TC14001124.hg.1 NM_001163940 PYGL 3.6 <10-17 B 0.91 0.88 0.84
    TC04001267.hg.1 NM_014465 SULT1B1 3.7 3.33E−16 B 0.88 0.77 0.86
    TC02000082.hg.1 NM_021643 TRIB2 −5.1 2.78E−15 V 0.88 0.77 0.91
    TC22000519.hg.1 ENST00000454608 USP41 −13.8 3.33E−16 V 0.91 0.92 0.82
    TC18000223.hg.1 NM_017742 ZCCHC2 −5.2 6.66E−16 V 0.87 0.80 0.76
  • TABLE 16C
    Differentially expressed non-coding RNA determinants and their
    measures of accuracy in differentiating between bacterial (“B”) versus viral (“V”)
    infected subjects that met the following criteria: ttest p-value<10−14 AND absolute
    linear fold change > 3.5.
    ttest p-
    value
    Linear (Bacterial Up
    mRNA fold vs. in
    Accession Chromosome Strand Start Stop change Viral) B/V AUC Sensitivity Specificity
    n332762 chr2 + 7037 7037 −36.0 2.22E−16 V 0.87 0.80 0.80
    597 917
    n407780 chr2 + 1285 1288 −4.4 6.55E−15 V 0.88 0.80 0.85
    6998 2860
    TCONS_00003184- chr2 6968 6973 −8.1 5.22E−15 V 0.88 0.82 0.89
    XLOC_001966 645 662
    n332510 chrl2 + 1133 1133 −11.0 0 V 0.88 0.80 0.86
    5728 6994
    4 7
    n334829 chrl4 + 9457 9458 −79.5 0 V 0.89 0.86 0.79
    7084 2176
    n332456 chrl5 + 6745 6747 −3.7 3.11E−15 V 0.89 0.84 0.89
    7682 3649
    n333319 chr21 + 4282 4282 −15.5 0 V 0.91 0.84 0.84
    3118 4743
  • TABLE 16D
    Accuracy measures of selected RNAs in distinguishing between patients
    presenting with bacterial infections (“B”; n=51) or non-bacterial infections (“NB”;
    n = 92)
    ttest p-
    Transcript Transcript Linear value Up
    Cluster Cluster Gene mRNA fold (Bacterial in
    ID ID Symbol Accession change vs. Viral) B/NB AUC Sensitivity Specificity
    TC01000272.hg.1 TC01000272.hg.1 ALPL NM_000478 6.02 6.10E−14 B 0.91 0.86 0.87
    TC01001738.hg.1 TC01001738.hg.1 CRI NM_000573 3.88 <10-17 B 0.91 0.88 0.83
    TC01002287.hg.1 TC01002287.hg.1 PAD12 NM_007365 3.44 4.88E−15 B 0.88 0.88 0.73
    TC01003871.hg.1 TC01003871.hg.1 TLR5 NM_003268 3.61 3.11E−15 B 0.87 0.77 0.82
    TC02000082.hg.1 TC02000082.hg.1 TRIB2 NM_021643 −4.32 5.00E−15 V 0.87 0.77 0.92
    TC02001723.hg.1 TC02001723.hg.1 NLRC4 NM_001199138 3.96 <10-17 B 0.88 0.78 0.88
    TC02001749.hg.1 TC02001749.hg.1 CYP1B1 OTTHUMTO0000325647 6.43 <10-17 B 0.89 0.80 0.84
    TC02001750.hg.1 TC02001750.hg.1 CYP1B1 NM_000104 6.01 <10-17 B 0.89 0.77 0.87
    TC02001866.hg.1 TC02001866.hg.1 PNPT1 NM_033109 −5.10 1.92E−14 V 0.90 0.88 0.82
    TC02003047.hg.1 TC02003047.hg.1 — n407780 −3.83 1.33E−14 V 0.87 0.77 0.90
    TC04000248.hg.1 TC04000248.hg.1 NSUN7 NM_024677 3.51 4.11E−15 B 0.86 0.82 0.80
    TC04000484.hg.1 TC04000484.hg.1 HERC6 NM_001165136 −8.14 8.22E−15 V 0.88 0.90 0.72
    TC04000826.hg.1 TC04000826.hg.1 KLHL2 NM_001161522 3.36 <10-17 B 0.90 0.88 0.85
    TC04001267.hg.1 TC04001267.hg.1 SULTIB1 NM_014465 3.64 <10-17 B 0.89 0.88 0.77
    TC04001372.hg.1 TC04001372.hg.1 — uc003hrl.1 −3.08 5.11E−15 V 0.87 0.80 0.79
    TC04001373.hg.1 TC04001373.hg.1 PPM1K NM_152542 −3.45 1.11E−16 V 0.90 0.78 0.88
    TC05002100hg.1 TC05002100.hg.1 HK3 NM_002115 3.32 2.22E−16 B 0.87 0.77 0.80
    TC06002119.hg.1 TC06002119.hg.1 VNN1 NM_004666 9.55 4.06E−14 B 0.85 0.73 0.86
    TC06003619.hg.1 TC06003619.hg.1 — n409669 3.03 <10-17 B 0.89 0.92 0.76
    TC07000899.hg.1 TC07000899.hg.1 MGAM NM_004668 4.07 3.66E−15 B 0.90 0.86 0.85
    TC07002730.hg.1 TC07002730.hg.1   TCONS_00013664- 3.91 5.26E−14 B 0.85 0.71 0.86
    XLOC_006324
    TC07003216.hg.1 TC07003216.hg.1   n341349 3.55 4.25E−14 B 0.83 0.78 0.74
    TC08002386.hg.1 TC08002386.hg.1   TCONS_12_00028242- 3.57 1.98E−14 B 0.85 0.80 0.80
    XLOC_12_014551
    TC11000812.hg.1 TC11000812.hg.1 DGAT2 NM_001253891 3.86 6.77E−15 B 0.88 0.88 0.75
    TCI2000591.hg.1 TC12000591.hg.1 IRAK3 NM_001142523 3.32 <10-17 B 0.88 0.80 0.83
    TC12001843.hg.1 TC12001843.hg.1 LTA4H NM_000895 4.05 <10-17 B 0.93 0.86 0.91
    TCI2002425.hg.1 TC12002425.hg.1   n341954 4.42 1.11E−16 B 0.88 0.80 0.84
    TC13001567.hg.1 TC13001567.hg.1   n332831 3.13 3.33E−15 B 0.87 0.78 0.86
    TC14000810.hg.1 TC14000810.hg.1 TDRD9 NM_153046 3.39 1.30E−14 B 0.83 0.77 0.85
    TC14001124.hg.1 TC14001124.hg.1 PYGL NM_001163940 3.34 <10-17 B 0.92 0.88 0.87
    TC14001850.hg.1 TC14001850.hg.1   n335717 −3.20 9.55E−15 V 0.86 0.88 0.76
    TCI5002251.hg.1 TC15002251.hg.1   n332456 −3.41 3.33E−16 V 0.89 0.84 0.88
    TC16002057.hg.1 TC16002057.hg.1 HP NM_001126102 10.51 7.22E−15 B 0.85 0.75 0.84
    TC17001129.hg.1 TC17001129.hg.1 GAS7 NM_201433 4.74 <10-17 B 0.92 0.90 0.84
    TCI7002760.hg.1 TC17002760.hg.1   n407998 3.63 3.33E−16 B 0.86 0.78 0.77
    TC18000060.hg.1 TC18000060.hg.1 IMPA2 NM_014214 3.19 <10-17 B 0.91 0.90 0.79
    TC19001640.hg.1 TC19001640.hg.1 PGLYRP1 NM_005091 3.45 6.76E−14 B 0.85 0.84 0.77
    TC22000191.hg.1 TC22000191.hg.1 KREMEN1 NM_001039570 6.25 <10-17 B 0.90 0.82 0.89
  • TABLE 16E
    Accuracy measures of selected RNAs in distinguishing between infectious
    (“I”; bacterial and viral; n = 130) and non-infectious (“NI”; n = 13) patients
    Linear ttest p-value Up
    Transcript mRNA fold (Bacterial vs. in
    Cluster ID Gene Symbol Accession change Viral) I/NI AUC Sensitivity Specificity
    TC03001869.hg.1 PLSCR1 NM_021105 11.00 1.92E−14 I 0.97 0.96 0.92
    TC06002126.hg.1 SGK1 NM_001143676 −3.94 2.48E−13 N 0.97 0.88 0.92
    TC17001661.hg.1 PHOSPHO1 NM_001143804 −11.51 7.55E−15 N 0.97 0.90 0.92
    TC04001476.hg.1 TIFA NM_052864 3.74 1.01E−08 I 0.96 0.90 0.92
    TC01003057.hg.1 FCGR1B; FCGR1C NM_001004340 8.10 1.39E−11 I 0.96 0.95 0.92
    TC17000545.hg.1 IFI35 NM_005533 6.78 1.59E−09 I 0.96 0.93 0.92
    TC11000117.hg.1 OR56B1; TRIM22 NM_001005180 4.76 1.21E−13 I 0.96 0.93 1.00
    TC17000807.hg.1 MAP2K6 NM_002758 4.66 1.40E−09 I 0.96 0.93 0.92
    TC11003507.hg.1 CARD17 NM_001007232 18.65 3.18E−09 I 0.96 0.92 0.92
    TC01003383.hg.1 AIM2 NM_004833 8.24 1.93E−11 I 0.96 0.89 1.00
    TC18000477.hg.1 PSTPIP2 NM_024430 4.57 1.24E−08 I 0.96 0.90 1.00
    TC05000604.hg.1 LMNB1 NM_001198557 4.01 1.35E−11 I 0.96 0.88 0.92
    TC03001705.hg.1 PARP9 NM_001146102 4.51 4.73E−12 I 0.96 0.93 0.92
    TC10001497.hg.1 ANKRD22 NM_144590 12.83 1.48E−08 I 0.96 0.93 0.92
    TC01001172.hg.1 FCGR1A; FCGR1B NM_000566 8.01 5.80E−11 I 0.96 0.92 0.92
    TC04001719.hg.1 DDX60L NM_001012967 4.71 4.25E−11 I 0.96 0.91 1.00
    TC12001202.hg.1 KLRB1 NM_002258 −6.57 3.24E−11 N 0.96 0.88 0.92
    TC03000584.hg.1 GRAMD1C NM_001172105 −3.82 1.11E−16 N 0.96 0.94 0.85
    TC20000429.hg.1 CASS4 NM_020356 −5.08 1.18E−10 N 0.95 0.89 1.00
    TC08002364.hg.1 n341520 −5.22 5.07E−11 N 0.95 0.89 0.92
    TC01004727.hg.1 TCONS_12_00002197- 7.91 4.63E−10 I 0.95 0.92 0.92
    XLOC_12_000423
    TC13000383.hg.1 TNFSF13B NM_001145645 3.88 5.24E−10 I 0.95 0.89 0.92
    TC01001161.hg.1 FCGR1C NR_027484 7.99 6.93E−10 I 0.95 0.91 0.92
    TC11000506.hg.1 MS4A4A NM_001243266 5.03 2.87E−09 I 0.95 0.85 0.92
    TC15002563.hg.1 n337816 −3.99 2.45E−09 N 0.95 0.87 0.92
    TC09000037.hg.1 CD274 NM_014143 10.84 1.09E−08 I 0.95 0.86 1.00
    TC19000134.hg.1 C19orf59 NM_174918 16.14 4.72E−09 I 0.95 0.88 0.85
    TC08001286.hg.1 MYBL1 NM_001080416 −4.56 2.44E−11 N 0.95 0.87 0.92
    TC19002498.hg.1 TCONS_12_00013127- 52.67 9.85E−08 I 0.94 0.89 0.92
    XLOC_12_007062
    TC14002303.hg.1 FLVCR2 NM_001195283 3.53 3.51E−08 I 0.94 0.89 0.92
    TC12002572.hg.1 n332510 12.24 6.00E−08 I 0.94 0.88 0.92
    TC19001585.hg.1 CD177P1 ENST00000378007 102.17 6.64E−09 I 0.94 0.86 0.92
    TC11000467.hg.1 SERPING1 NM_000062 43.61 6.66E−11 I 0.94 0.89 1.00
    TC12000143.hg.1 ENST00000538219 −3.58 3.75E−12 N 0.94 0.89 0.85
    TC11002677.hg.1 n333961 93.37 7.22E−11 I 0.93 0.89 1.00
    TC12000884.hg.1 OAS1 NM_001032409 10.52 3.76E−09 I 0.93 0.89 1.00
    TC13000612.hg.1 EPSTI1 NM_001002264 23.09 3.14E−11 I 0.93 0.89 1.00
    TC20001235.hg.1 TCONS_00028171- −27.32 2.95E−09 N 0.93 0.86 0.92
    XLOC_013531
    TC02000937.hg.1 TNFAIP6 NM_007115 7.79 9.93E−10 I 0.93 0.85 0.92
    TC01003871.hg.1 TLR5 NM_003268 4.90 8.27E−09 I 0.93 0.87 0.92
    TC14002019.hg.1 n336571 4.72 3.98E−08 I 0.93 0.89 0.92
    TC07001606.hg.1 SAMD9L NM_152703 7.34 3.11E−11 I 0.93 0.85 1.00
    TC01001351.hg.1 FCER1A NM_002001 −4.69 1.43E−10 N 0.93 0.85 0.85
    TC19002719.hg.1 LILRA5 NM_021250 5.56 2.41E−09 I 0.92 0.86 0.92
    TC10001153.hg.1 ZNF438 NM_001143766 3.52 4.78E−08 I 0.92 0.90 0.92
    TC10002542.hg.1 n407148 3.64 3.04E−08 I 0.92 0.89 0.92
    TC12000130.hg.1 CLEC4D NM_080387 7.13 3.07E−08 I 0.92 0.88 0.92
    TC19002491.hg.1 n334140 8.48 4.90E−09 I 0.92 0.78 0.92
    TC11003322.hg.1 n410125 9.67 2.29E−08 I 0.92 0.89 0.85
    TC02001739.hg.1 EIF2AK2 NM_001135651 5.88 1.19E−08 I 0.91 0.89 0.92
    TC12001171.hg.1 C3AR1 NM_004054 5.13 6.07E−08 I 0.91 0.86 0.92
    TC04000437.hg.1 ANXA3 NM_005139 6.07 2.18E−08 I 0.91 0.85 0.85
    TC09002677.hg.1 n336823 −3.93 1.58E−09 N 0.91 0.75 0.92
    TC02004639.hg.1 n338981 9.00 7.19E−09 I 0.91 0.90 0.77
    TC02004161.hg.1 n332938 6.17 1.75E−08 I 0.91 0.89 0.92
    TC12000138.hg.1 KLRG1 NM_005810 −4.84 1.23E−08 N 0.90 0.86 0.77
    TC01000795.hg.1 IFI44 NM_006417 30.99 7.33E−08 I 0.90 0.86 0.92
    TC17000077.hg.1 XAF1 NM_017523 9.71 1.50E−08 I 0.90 0.87 0.92
    TC04001718.hg.1 DDX60 NM_017631 11.50 7.74E−08 I 0.90 0.86 0.92
    TC11003279.hg.1 n334838 −3.54 3.71E−08 N 0.90 0.90 0.85
    TC01003718.hg.1 CHI3L1 NM_001276 −6.57 7.97E−08 N 0.89 0.86 0.85
    TC02000038.hg.1 OTTHUMG00000151591; ENST00000417930 −3.52 3.47E−09 N 0.89 0.93 0.85
    AC092580.4
    TC01000794.hg.1 IFI44L NM_006820 68.37 6.19E−08 I 0.89 0.86 0.92
    TC02000034.hg.1 RSAD2 NM_080657 67.82 7.86E−08 I 0.89 0.86 0.92
    TC07002894.hg.1 n336551 −5.68 8.44E−08 N 0.89 0.90 0.77
    TC06002127.hg.1 ENST00000458970 −4.04 2.95E−08 N 0.88 0.76 0.85
  • Next, a logistic regression was used to develop a classifier for each RNA pair (of the RNAs included in Tables 16A-C; 861 combinations) in order to test whether combining two RNA determinants can improve diagnostic accuracy. Table 17 summarizes the 50 RNA pairs with the highest sensitivity in distinguishing between patients with bacterial and viral infections. Table 18 presents the 50 RNA pairs that demonstrated the highest accuracy improvement as calculated by the difference in AUC of the pair compared to the AUC of the single RNA (out of the same pair) with the highest AUC (delta AUC). Indeed using pairs of RNA determinants improved diagnostic accuracy to generate highly discriminative combinations (AUCs between 0.93-0.96, average AUC 0.94, sensitivity between 0.92-0.96, sensitivity 0.93 Tables 17-18).
  • TABLE 17
    50 RNA pairs that presented the highest sensitivity in distinguishing
    between patients with bacterial or viral infections. The columns of Feature #1 and
    Feature #2 include the probe set ID and either the gene symbol (for coding RNAs) or
    mRNA accession (for non-coding RNAs) in brackets. Logistic regression model
    parameters (constant and single RNAs coefficient) are depicted.
    Feature# 1 Feature# 2
    Feature #1 Feature #2 AUC Sensitivity Specificity Constant coefficient coefficient
    TC02000034.hg.1 TC22000191.hg.1 0.95 0.96 0.87 −7.44 −0.57 1.30
    (RSAD2 ) (KREMEN1 )
    TC14000584.hg.1 TC18000060.hg.1 0.94 0.96 0.80 −17.95 −0.25 1.93
    (IFI27 ) (IMPA2 )
    TC02003008.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.86 −0.54 1.28
    (n332762 ) (KREMEN1 )
    TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −0.46 −1.12 1.23
    (ZCCHC2 ) (KREMEN1 )
    TC04000485.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −6.42 −0.62 1.19
    (HERC5 ) (KREMEN1 )
    TC18000060.hg.1 TC21000739.hg.1 0.95 0.94 0.82 −10.5 1.89 −0.60
    (IMPA2 ) (n333319 )
    TC02004017.hg.1 TC22000191.hg.1 0.94 0.94 0.91 −5.80 −0.80 1.03
    (TCONS_00003184- (KREMEN1 )
    XLOC_001966 )
    TC01000795.hg.1 TC01001738.hg.1 0.94 0.94 0.84 −14.9 −0.57 1.44
    (IFI44 ) (CR1 )
    TC02003047.hg.1 TC04000485.hg.1 0.94 0.94 0.80 16.8 −1.29 −0.60
    (n407780 ) (HERC5 )
    TC02003008.hg.1 TC02003047.hg.1 0.94 0.94 0.84 15.4 −0.46 −1.26
    (n332762 ) (n407780 )
    TC14001124.hg.1 TC17001129.hg.1 0.93 0.94 0.86 −28.0 1.23 0.95
    (PYGL ) (GAS7 )
    TC01003963.hg.1_ TC14000584.hg.1 0.90 0.94 0.77 −6.90 2.07 −0.58
    MIR1182_FAM89A_ (IFI27 )
    NM_198552 (FAM89A )
    TC12001843.hg.1 TC18000060.hg.1 0.96 0.92 0.89 −38.5 1.41 1.80
    (LTA4H ) (IMPA2 )
    TC12000884.hg.1 TC12001843.hg.1 0.96 0.92 0.84 −20.2 −0.90 2.07
    (IFIT1 ) (LTA4H )
    TC17001129.hg.1 TC21000189.hg.1 0.96 0.92 0.87 −5.44 1.53 −0.96
    (GAS7 ) (MX1 )
    TC12002059.hg.1 TC22000191.hg.1 0.95 0.92 0.89 −4.88 −0.82 1.24
    (OASL ) (KREMEN1 )
    TC10000639.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −6.17 −0.63 1.09
    (IFIT1 ) (KREMEN1 )
    TC12001843.hg.1 TC14001821.hg.1 0.95 0.92 0.82 −22.7 1.86 −0.40
    (LTA4H ) (n334829 )
    TC02000034.hg.1 TC17001129.hg.1 0.95 0.92 0.87 −10.6 −0.48 1.60
    (RSAD2 ) (GAS7 )
    TC12000885.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −6.46 −0.68 1.22
    (OAS3 ) (KREMEN1 )
    TC12002572.hg.1 TC17001129.hg.1 0.95 0.92 0.87 −8.09 −0.76 1.51
    (n332510 ) (GAS7 )
    TC12000884.hg.1 TC17001129.hg.1 0.95 0.92 0.90 −6.75 −0.93 1.57
    (IFIT1 ) (GAS7 )
    TC01000795.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −5.57 −0.67 1.23
    (IFI44 ) (KREMEN1 )
    TC01001738.hg.1 TC21000739.hg.1 0.95 0.92 0.87 −8.03 1.30 −0.79
    (CR1 ) (n333319 )
    TC02001749.hg.1 TC04001373.hg.1 0.95 0.92 0.89 3.61 0.87 −1.61
    (CYP1B1 ) (PPM1K )
    TC04000484.hg.1 TC18000060.hg.1 0.95 0.92 0.87 −12.3 −0.71 1.82
    (HERC6 ) (IMPA2 )
    TC04001718.hg.1 TC22000191.hg.1 0.95 0.92 0.89 −3.94 −0.85 1.25
    (DDX60 ) (KREMEN1 )
    TC01000795.hg.1 TC17001129.hg.1 0.95 0.92 0.90 −9.46 −0.58 1.60
    (IFI44 ) (GAS7 )
    TC04000485.hg.1 TC17001129.hg.1 0.95 0.92 0.89 −10.1 −0.57 1.57
    (HERC5 ) (GAS7 )
    TC02001750.hg.1 TC04001373.hg.1 0.95 0.92 0.89 5.03 0.92 −1.60
    (CYP1B1 ) (PPM1K )
    TC04000484.hg.1 TC22000191.hg.1 0.95 0.92 0.91 −1.27 −0.91 0.85
    (HERC6 ) (KREMEN1 )
    TC11000812.hg.1 TC21000189.hg.1 0.94 0.92 0.84 −3.89 1.16 −0.91
    (DGAT2 ) (MX1 )
    TC01001738.hg.1 TC02001866.hg.1 0.94 0.92 0.90 −6.55 1.08 −1.07
    (CR1 ) (PNPT1 )
    TC01001738.hg.1 TC04000484.hg.1 0.94 0.92 0.90 −8.88 1.12 −0.91
    (CR1 ) (HERC6 )
    TC04001267.hg.1 TC21000739.hg.1 0.94 0.92 0.85 −2.05 1.16 −0.79
    (SULT1B1 ) (n333319 )
    TC01001738.hg.1 TC22000029.hg.1 0.94 0.92 0.89 −13.0 1.15 −0.56
    (CR1 ) (USP18 )
    TC02001749.hg.1 TC04001372.hg.1 0.94 0.92 0.89 0.32 0.97 −1.48
    (CYP1B1 ) (uc003hrl.1 )
    TC02004017.hg.1 TC17001129.hg.1 0.94 0.92 0.87 −10.1 −0.77 1.50
    (TCONS_00003184- (GAS7 )
    XLOC_001966 )
    TC12000885.hg.1 TC18000060.hg.1 0.94 0.92 0.84 −17.1 −0.39 2.06
    (OAS3 ) (IMPA2 )
    TC01001738.hg.1 TC04001373.hg.1 0.94 0.92 0.90 −5.27 1.05 −1.34
    (CR1 ) (PPM1K )
    TC01001738.hg.1 TC12002059.hg.1 0.94 0.92 0.89 −14.4 1.43 −0.68
    (CR1 ) (OASL )
    TC01001738.hg.1 TC22000519.hg.1 0.94 0.92 0.89 −13.4 1.23 −0.71
    (CR1 ) (USP41 )
    TC02001749.hg.1 TC18000060.hg.1 0.94 0.92 0.86 −27.5 0.58 2.05
    (CYP1B1 ) (IMPA2 )
    TC04001372.hg.1 TC18000060.hg.1 0.94 0.92 0.86 −15.8 −0.74 2.04
    (uc003hrl.1 ) (IMPA2 )
    TC02003047.hg.1 TC21000739.hg.1 0.94 0.92 0.84 19.6 −1.02 −0.77
    (n407780 ) (n333319 )
    TC01001738.hg.1 TC04000485.hg.1 0.94 0.92 0.87 −15.6 1.43 −0.55
    (CR1 ) (HERC5 )
    TC02000082.hg.1 TC02003008.hg.1 0.94 0.92 0.84 14.6 −1.19 −0.47
    (TRIB2 ) (n332762 )
    TC02000082.hg.1 TC02004017.hg.1 0.94 0.92 0.90 14.4 −1.12 −0.84
    (TRIB2 ) (TCONS_00003184-
    XLOC_001966 )
    TC02000034.hg.1 TC02000082.hg.1 0.94 0.92 0.82 15.2 −0.49 −1.21
    (RSAD2 ) (TRIB2 )
  • TABLE 18
    50 RNA pairs that presented the highest accuracy improvement compared
    to the single RNA determinants. AUC_1 and AUC_2 are the respective AUCs of RNA
    #
    1 and RNA #2. The accuracy of combining RNA#1 and RNA#2 is depicted in the
    column AUC pair, and the improvement of the combination over that of the individual
    RNA's is captured in the AUC delta (AUC_delta = AUC pair − max(AUC_1, AUC_2).
    The columns of Feature #1 and Feature #2 include the probe set ID and either the
    gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs) in
    brackets.
    AUC Delta
    Feature #1 Feature #2 AUC_1 AUC_2 pair AUC
    TC02003047.hg.1 (n407780 ) TC18000223.hg.1 (ZCCHC2 ) 0.88 0.87 0.95 0.07
    TC02000082.hg.1 (TRIB2 ) TC18000223.hg.1 (ZCCHC2 ) 0.88 0.87 0.95 0.07
    TC02005020.hg.1 (CMPK2 ) TC22000191.hg.1 (KREMEN1 ) 0.89 0.89 0.96 0.06
    TC12002059.hg.1 (OASL ) TC22000191.hg.1 (KREMEN1 ) 0.87 0.89 0.95 0.06
    TC02001749.hg.1 (CYP1B1 ) TC14000584.hg.1 (IFI27 ) 0.88 0.89 0.95 0.06
    TC02003008.hg.1 (n332762 ) TC22000191.hg.1 (KREMEN1 ) 0.87 0.89 0.95 0.06
    TC01000794.hg.1 (IFI44L ) TC22000191.hg.1 (KREMEN1 ) 0.89 0.89 0.95 0.06
    TC01000795.hg.1 (IFI44 ) TC02003047.hg.1 (n407780 ) 0.86 0.88 0.94 0.06
    TC01000795.hg.1 (IFI44 ) TC02000082.hg.1 (TRIB2 ) 0.86 0.88 0.94 0.06
    TC18000223.hg.1 (ZCCHC2 ) TC22000191.hg.1 (KREMEN1 ) 0.87 0.89 0.95 0.06
    TC02001749.hg.1 (CYP1B1 ) TC14001821.hg.1 (n334829 ) 0.88 0.89 0.95 0.06
    TC12002572.hg.1 (n332510 ) TC22000191.hg.1 (KREMEN1 ) 0.88 0.89 0.95 0.06
    TC02001749.hg.1 (CYP1B1 ) TC02003047.hg.1 (n407780 ) 0.88 0.88 0.94 0.06
    TC02000034.hg.1 (RSAD2 ) TC22000191.hg.1 (KREMEN1 ) 0.86 0.89 0.95 0.06
    TC12000885.hg.1 (OAS3 ) TC22000191.hg.1 (KREMEN1 ) 0.88 0.89 0.95 0.06
    TC02001750.hg.1 (CYP1B1 ) TC14000584.hg.1 (IFI27 ) 0.88 0.89 0.95 0.06
    TC02001750.hg.1 (CYP1B1 ) TC14001821.hg.1 (n334829 ) 0.88 0.89 0.95 0.06
    TC02000082.hg.1 (TRIB2 ) TC02005020.hg.1 (CMPK2 ) 0.88 0.89 0.94 0.06
    TC02003047.hg.1 (n407780 ) TC04000485.hg.1 (HERC5 ) 0.88 0.87 0.94 0.06
    TC04001267.hg.1 (SULT1B1 ) TC18000223.hg.1 (ZCCHC2 ) 0.88 0.87 0.94 0.06
    TC10000639.hg.1 (IFIT1 ) TC22000191.hg.1 (KREMEN1 ) 0.90 0.89 0.95 0.06
    TC07001916.hg.1 (PARP12 ) TC22000191.hg.1 (KREMEN1 ) 0.89 0.89 0.95 0.06
    TC02005020.hg.1 (CMPK2 ) TC04001267.hg.1 (SULT1B1 ) 0.89 0.88 0.94 0.06
    TC02001749.hg.1 (CYP1B1 ) TC02004017.hg.1 0.88 0.88 0.94 0.06
    (TCONS_00003184-
    XLOC_001966 )
    TC02003008.hg.1 (n332762 ) TC02003047.hg.1 (n407780 ) 0.87 0.88 0.94 0.06
    TC02003047.hg.1 (n407780 ) TC12000885.hg.1 (OAS3 ) 0.88 0.88 0.94 0.06
    TC02000034.hg.1 (RSAD2 ) TC02003047.hg.1 (n407780 ) 0.86 0.88 0.94 0.06
    TC02000082.hg.1 (TRIB2 ) TC02003008.hg.1 (n332762 ) 0.88 0.87 0.94 0.06
    TC02000082.hg.1 (TRIB2 ) TC02004017.hg.1 0.88 0.88 0.94 0.06
    (TCONS_00003184-
    XLOC_001966 )
    TC01000795.hg.1 (IFI44 ) TC22000191.hg.1 (KREMEN1 ) 0.86 0.89 0.95 0.06
    TC02000082.hg.1 (TRIB2 ) TC02001749.hg.1 (CYP1B1 ) 0.88 0.88 0.94 0.06
    TC12000884.hg.1 (OAS1 ) TC22000191.hg.1 (KREMEN1 ) 0.88 0.89 0.95 0.06
    TC02004017.hg.1 TC04001267.hg.1 (SULT1B1 ) 0.88 0.88 0.94 0.06
    (TCONS_00003184-
    XL0C_001966 )
    TC02003047.hg.1 (n407780 ) TC02005020.hg. 1 (CMPK2 ) 0.88 0.89 0.94 0.06
    TC02000082.hg.1 (TRIB2 ) TC12002059.hg.1 (OASL ) 0.88 0.87 0.94 0.06
    TC02001750.hg.1 (CYP1B1 ) TC02003047.hg.1 (n407780 ) 0.88 0.88 0.94 0.06
    TC02000034.hg.1 (RSAD2 ) TC02000082.hg. 1 (TRIB2 ) 0.86 0.88 0.94 0.06
    TC02003047.hg.1 (n407780 ) TC02004017.hg.1 0.88 0.88 0.94 0.06
    (TCONS_00003184-
    XLOC_001966 )
    TC02000082.hg.1 (TRIB2 ) TC12000885.hg.1 (OAS3 ) 0.88 0.88 0.94 0.06
    TC04001267.hg.1 (SULT1B1 ) TC12002059.hg.1 (OASL ) 0.88 0.87 0.94 0.06
    TC02003047.hg.1 (n407780 ) TC12002059.hg.1 (OASL ) 0.88 0.87 0.93 0.06
    TC04001718.hg.1 (DDX60 ) TC22000191.hg.1 (KREMEN1 ) 0.85 0.89 0.95 0.05
    TC02000082.hg.1 (TRIB2 ) TC02001750.hg.1 (CYP1B1 ) 0.88 0.88 0.94 0.05
    TC02001750.hg.1 (CYP1B1 ) TC02004017.hg.1 0.88 0.88 0.94 0.05
    (TCONS_00003184-
    XLOC_001966 )
    TC04000485.hg.1 (HERC5 ) TC22000191.hg.1 (KREMEN1 ) 0.87 0.89 0.95 0.05
    TC21000189.hg.1 (MX1 ) TC22000191.hg.1 (KREMEN1 ) 0.90 0.89 0.95 0.05
    TC11000812.hg.1 (DGAT2 ) TC18000223.hg.1 (ZCCHC2 ) 0.89 0.87 0.94 0.05
    TC02000082.hg.1 (TRIB2 ) TC12002572.hg.1 (n332510 ) 0.88 0.88 0.94 0.05
    TC01000795.hg.1 (IFI44 ) TC04001267.hg.1 (SULT1B1 ) 0.86 0.88 0.93 0.05
  • The evaluated cohort of patients provided the required statistical power to accurately evaluating of triplets of RNA determinants. A linear logistic regression classifier was developed for each gene triplets (of the RNAs included in Tables 16A-C; 11480 combinations) and their diagnostic accuracy was evaluated. Tables 18 and 19 present different RNA triplets with best sensitivity and the highest accuracy improvement respectively.
  • TABLE 19
    50 RNA triplets that presented the highest sensitivity in distinguishing
    between patients with bacterial or viral infections. The columns of Feature #1,
    Feature #2 and Feature #3 include the probe set ID and either the gene symbol (for
    coding RNAs) or mRNA accession (for non-coding RNAs) in brackets. Logistic
    regression model parameters (constant and single RNAs coefficient) are depicted.
    Feature#1 Feature#2 Feature#3
    Feature #1 Feature #2 Feature #3 AUC Sensitivity Specificity Constant coefficient coefficient coefficient
    TC01000795.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.94 0.92 −17.60 −0.49 1.32 0.90
    (IFI44 ) (IMPA2 ) (KREMEN1 )
    TC01003963.hg.1_ TC04000484.hg.1 TC22000191.hg.1 0.95 0.94 0.91 −6.18 0.97 −0.91 0.81
    MIR1182_ (HERC6 ) (KREMEN1 )
    FAM89A_
    NM_198552
    (FAM89A)
    TC02001749.hg.1 TC21000189.hg.1 TC22000191.hg.1 0.96 0.94 0.91 −7.29 0.60 −0.87 0.93
    (CYP1B1 ) (MX1 ) (KREMEN1 )
    TC02004017.hg.1 TC04001267.hg.1 TC22000191.hg.1 0.95 0.94 0.91 −10.68 −0.77 0.69 0.70
    (TCONS_ (SULT1B1 ) (KREMEN1 )
    00003184-
    XLOC_001966 )
    TC01001738.hg.1 TC04001372.hg.1 TC04001373.hg.1 0.94 0.94 0.91 −4.65 1.05 0.75 -2.09
    (CR1 ) (uc003hrl.1 ) (PPM1K )
    TC01003963.hg.1_ TC02005020.hg.1 TC22000191.hg.1 0.96 0.94 0.90 −10.04 0.78 −0.82 1.17
    MIR1182_ (CMPK2 ) (KREMEN1 )
    FAM89A_NM_
    198552
    (FAM89A)
    TC01003963.hg.1_ TC12002059.hg.1 TC22000191.hg.1 0.95 0.94 0.90 −8.28 0.67 −0.81 1.20
    MIR1182_ (OASL ) (KREMEN1 )
    FAM89A_NM_
    198552
    (FAM89A)
    TC01003963.hg.1_ TC22000029.hg.1 TC22000191.hg.1 0.95 0.94 0.91 −11.28 1.28 −0.59 0.79
    MIR1182_ (USP18 ) (KREMEN1 )
    FAM89A_NM_
    198552
    (FAM89A)
    TC02000034.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.92 0.92 −18.28 −0.43 1.26 0.96
    (RSAD2 ) (IMPA2 ) (KREMEN1 )
    TC02000082.hg.1 TC02001750.hg.1 TC02001866.hg.1 0.95 0.94 0.91 5.79 −0.51 0.86 −0.99
    (TRIB2 ) (CYP1B1 ) (PNPT1 )
    TC02001749.hg.1 TC08000814.hg.1 TC22000191.hg.1 0.95 0.94 0.91 −7.30 0.67 −0.74 0.73
    (CYP1B1 ) (LY6E ) (KREMEN1 )
    TC02001750.hg.1 TC21000189.hg.1 TC22000191.hg.1 0.96 0.94 0.90 −5.67 0.62 −0.88 0.90
    (CYP1B1 ) (MX1 ) (KREMEN1 )
    TC02001866.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.94 0.92 −16.63 −0.62 1.57 0.55
    (PNPT1 ) (IMPA2 ) (KREMEN1 )
    TC02003008.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.94 0.90 −18.50 −0.41 1.24 0.96
    (n332762 ) (IMPA2 ) (KREMEN1 )
    TC04000485.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.96 0.90 −19.39 −0.47 1.47 0.84
    (HERC5 ) (IMPA2 ) (KREMEN1 )
    TC10000639.hg.1 TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.90 −4.03 −0.42 −0.39 1.14
    (IFIT1 ) (ZCCHC2 ) (KREMEN1 )
    TC12000885.hg.1 TC18000060.hg.1 TC22000191.hg.1 0.96 0.94 0.90 −17.69 −0.51 1.27 0.89
    (OAS3 ) (IMPA2 ) (KREMEN1 )
    TC01000795.hg.1 TC02005020.hg.1 TC22000191.hg.1 0.96 0.94 0.89 −5.98 −0.05 −0.77 1.20
    (IFI44 ) (CMPK2 ) (KREMEN1 )
    TC01000795.hg.1 TC04000485.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −5.60 −0.62 −0.04 1.23
    (IFI44 ) (HERC5 ) (KREMEN1 )
    TC01000795.hg.1 TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −1.84 −0.23 −0.76 1.22
    (IFI44 ) (ZCCHC2 ) (KREMEN1 )
    TC01001738.hg.1 TC02000034.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −12.17 0.46 −0.54 1.06
    (CR1 ) (RSAD2 ) (KREMEN1 )
    TC01001738.hg.1 TC02000082.hg.1 TC04001373.hg.1 0.94 0.94 0.90 −6.00 1.09 0.17 −1.50
    (CR1 ) (TRIB2 ) (PPM1K )
    TC01001738.hg.1 TC020030O8.hg.1 TC22000191.hg.1 0.96 0.94 0.89 −12.56 0.46 −0.51 1.05
    (CR1 ) (n332762 ) (KREMEN1 )
    TC01001738.hg.1 TC02003047.hg.1 TC04001373.hg.1 0.94 0.94 0.90 −6.02 1.09 0.17 −1.49
    (CR1 ) (n407780 ) (PPM1K )
    TC01003963.hg.1_ TC12000885.hg.1 TC22000191.hg.1 0.95 0.94 0.90 −10.18 0.76 −0.68 1.17
    MIR1182_ (OAS3 ) (KREMEN1 )
    FAM89A_
    NM_198552
    (FAM89A)
    TC01003963.hg.1_ TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −1.71 0.23 −1.11 1.22
    MIR1182_ (ZCCHC2 ) (KREMEN1 )
    FAM89A_
    NM_198552
    (FAM89A)
    TC02000034.hg.1 TC02003008.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −8.05 0.21 −0.74 1.28
    (RSAD2 ) (n332762 ) (KREMEN1 )
    TC02000034.hg.1 TC02005020.hg.1 TC22000191.hg.1 0.96 0.94 0.89 −6.62 −0.28 −0.43 1.25
    (RSAD2 ) (CMPK2 ) (KREMEN1 )
    TC02000034.hg.1 TC04000485.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −8.17 −0.86 0.34 1.35
    (RSAD2 ) (HERC5 ) (KREMEN1 )
    TC02000034.hg.1 TC04001267.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −10.16 −0.55 0.39 1.10
    (RSAD2 ) (SULT1B1 ) (KREMEN1 )
    TC02000034.hg.1 TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −3.94 −0.33 −0.51 1.26
    (RSAD2 ) (ZCCHC2 ) (KREMEN1 )
    TC02000034.hg.1 TC20000575.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −6.60 −0.50 −0.12 1.23
    (RSAD2 ) (SIGLEC1 ) (KREMEN1 )
    TC02000082.hg.1 TC02001749.hg.1 TC02001866.hg.1 0.95 0.90 0.92 4.45 −0.53 0.81 −0.98
    (TRIB2 ) (CYP1B1 ) (PNPT1 )
    TC02000082.hg.1 TC02001749.hg.1 TC04000484.hg.1 0.96 0.92 0.92 5.66 −0.79 0.75 −0.84
    (TRIB2 ) (CYP1B1 ) (HERC6 )
    TC02001749.hg.1 TC02003047.hg.1 TC12002059.hg.1 0.96 0.94 0.89 7.62 0.77 −1.13 −0.63
    (CYP1B1 ) (n407780 ) (OASL )
    TC02001866.hg.1 TC02003008.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.42 −0.04 −0.52 1.26
    (PNPT1 ) (n332762 ) (KREMEN1 )
    TC02001866.hg.1 TC02005020.hg.1 TC22000191.hg.1 0.96 0.94 0.89 −7.01 0.11 −0.88 1.25
    (PNPT1 ) (CMPK2 ) (KREMEN1 )
    TC02001866.hg.1 TC04000485.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −5.12 −0.14 −0.56 1.12
    (PNPT1 ) (HERC5 ) (KREMEN1 )
    TC02003008.hg.1 TC02005020.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.15 −0.36 −0.29 1.26
    (n332762 ) (CMPK2 ) (KREMEN1 )
    TC02003008.hg.1 TC04001372.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.18 −0.53 −0.07 1.25
    (n332762 ) (uc003hrl.1 ) (KREMEN1 )
    TC02003008.hg.1 TC04001373.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −6.64 −0.52 −0.11 1.23
    (n332762 ) (PPM1K ) (KREMEN1 )
    TC02003008.hg.1 TC10000639.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.21 −0.37 −0.21 1.22
    (n332762 ) (IFIT1 ) (KREMEN1 )
    TC02003008.hg.1 TC18000223.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −5.15 −0.37 −0.38 1.26
    (n332762 ) (ZCCHC2 ) (KREMEN1 )
    TC02003008.hg.1 TC20000575.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −7.65 −0.53 −0.03 1.27
    (n332762 ) (SIGLEC1 ) (KREMEN1 )
    TC02003008.hg.1 TC22000029.hg.1 TC22000191.hg.1 0.95 0.94 0.89 −8.41 −0.60 0.08 1.34
    (n332762 ) (USP18 ) (KREMEN1 )
    TC02004017.hg.1 TC14001124.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −16.96 −0.66 0.94 0.68
    (TCONS_ (PYGL ) (KREMEN1 )
    00003184-
    XLOC_001966)
    TC02005020.hg.1 TC04000485.hg.1 TC22000191.hg.1 0.96 0.90 0.92 −6.10 −1.32 0.41 1.19
    (CMPK2 ) (HERC5 ) (KREMEN1 )
    TC02005020.hg.1 TC04001372.hg.1 TC22000191.hg.1 0.96 0.92 0.90 −5.41 −0.80 −0.07 1.17
    (CMPK2 ) (uc003hrl.1 ) (KREMEN1 )
    TC02005020.hg.1 TC04001373.hg.1 TC22000191.hg.1 0.96 0.92 0.90 −5.60 −0.81 −0.04 1.18
    (CMPK2 ) (PPM1K ) (KREMEN1 )
    TC02005020.hg.1 TC20000575.hg.1 TC22000191.hg.1 0.95 0.92 0.90 −6.57 −0.89 0.09 1.24
    (CMPK2 ) (SIGLEC1 ) (KREMEN1 )
  • TABLE 20
    50 RNA triplets that presented the highest accuracy improvement
    compared to the single RNA determinants. AUC_1, AUC_2 and AUC_3 are the
    respective AUCs of RNA #1, RNA #2 and RNA #3. The accuracy of combining
    RNA#1, RNA#2 and RNA#3 is depicted in the column AUC triplet, and the
    improvement of the combination over that of the individual RNA's is captured in the
    AUC delta (AUC_delta = AUC triplet − max(AUC_1, AUC_2, AUC_3). The columns
    of Feature #1 and Feature #2 include the probe set ID and either the gene symbol (for
    coding RNAs) or mRNA accession (for non-coding RNAs) in brackets.
    AUC Delta
    Feature #1 Feature #2 Feature #3 AUC_1 AUC_2 AUC_3 triplet AUC
    TC01000794.hg.1 TC02001749.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.07
    (IFI44L ) (CYP1B1 ) (KREMEN1 )
    TC01000794.hg.1 TC02001750.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.07
    (IFI44L ) (CYP1B1 ) (KREMEN1 )
    TC01000794.hg.1 TC02000082.hg.1 TC02001749.hg.1 0.89 0.88 0.88 0.96 0.07
    (IFI44L ) (TRIB2 ) (CYP1B1 )
    TC01000794.hg.1 TC02000082.hg.1 TC02001750.hg.1 0.89 0.88 0.88 0.96 0.07
    (IFI44L ) (TRIB2 ) (CYP1B1 )
    TC01000794.hg.1 TC02001749.hg.1 TC02003047.hg.1 0.89 0.88 0.88 0.96 0.07
    (IFI44L ) (CYP1B1 ) (n407780 )
    TC01000795.hg.1 TC01003963.hg.1_ TC02000082.hg.1 0.86 0.68 0.88 0.95 0.06
    (IFI44 ) MIR1182_FAM89A_ (TRIB2 )
    NM_198552
    (FAM89A)
    TC01000794.hg.1 TC02000082.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.06
    (IFI44L ) (TRIB2 ) (KREMEN1 )
    TC01000794.hg.1 TC02003047.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.06
    (IFI44L ) (n407780 ) (KREMEN1 )
    TC01000794.hg.1 TC02001750.hg.1 TC02003047.hg.1 0.89 0.88 0.88 0.96 0.06
    (IFI44L ) (CYP1B1 ) (n407780 )
    TC01000794.hg.1 TC04001267.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.06
    (IFI44L ) (SULT1B1 ) (KREMEN1 )
    TC01000794.hg.1 TC11000812.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.96 0.06
    (IFI44L ) (DGAT2 ) (KREMEN1 )
    TC01000794.hg.1 TC04001718.hg.1 TC22000191.hg.1 0.89 0.85 0.89 0.96 0.06
    (IFI44L ) (DDX60 ) (KREMEN1 )
    TC01000794.hg.1 TC12002059.hg.1 TC22000191.hg.1 0.89 0.87 0.89 0.96 0.06
    (IFI44L ) (OASL ) (KREMEN1 )
    TC01000794.hg.1 TC12002572.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.96 0.06
    (IFI44L ) (n332510 ) (KREMEN1 )
    TC01000794.hg.1 TC02000082.hg.1 TC18000223.hg.1 0.89 0.88 0.87 0.95 0.06
    (IFI44L ) (TRIB2 ) (ZCCHC2 )
    TC01000794.hg.1 TC02003008.hg.1 TC22000191.hg.1 0.89 0.87 0.89 0.95 0.06
    (IFI44L ) (n332762 ) (KREMEN1 )
    TC01000794.hg.1 TC02005020.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (CMPK2 ) (KREMEN1 )
    TC01000794.hg.1 TC08000814.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (LY6E ) (KREMEN1 )
    TC01000794.hg.1 TC14000584.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (IFI27 ) (KREMEN1 )
    TC01000794.hg.1 TC14001821.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (n334829 ) (KREMEN1 )
    TC01000794.hg.1 TC18000223.hg.1 TC22000191.hg.1 0.89 0.87 0.89 0.95 0.06
    (IFI44L ) (ZCCHC2 ) (KREMEN1 )
    TC01000794.hg.1 TC02003047.hg.1 TC18000223.hg.1 0.89 0.88 0.87 0.95 0.06
    (IFI44L ) (n407780 ) (ZCCHC2 )
    TC01000794.hg.1 TC01001738.hg.1 TC22000191.hg.1 0.89 0.90 0.89 0.96 0.06
    (IFI44L ) (CR1 ) (KREMEN1 )
    TC01000794.hg.1 TC01000795.hg.1 TC22000191.hg.1 0.89 0.86 0.89 0.95 0.06
    (IFI44L ) (IFI44 ) (KREMEN1 )
    TC01000794.hg.1 TC01003963.hg.1_ TC22000191.hg.1 0.89 0.68 0.89 0.95 0.06
    (IFI44L ) MIR1182_FAM89A_ (KREMEN1 )
    NM_198552
    (FAM89A)
    TC01000794.hg.1 TC04000485.hg.1 TC22000191.hg.1 0.89 0.87 0.89 0.95 0.06
    (IFI44L ) (HERC5 ) (KREMEN1 )
    TC01000794.hg.1 TC04001372.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (uc003hrl.1 ) (KREMEN1 )
    TC01000794.hg.1 TC07001916.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (PARP12 ) (KREMEN1 )
    TC01000794.hg.1 TC12000885.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (OAS3 ) (KREMEN1 )
    TC01000794.hg.1 TC12000886.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (OAS2 ) (KREMEN1 )
    TC01000794.hg.1 TC15002251.hg.1 TC22000191.hg.1 0.89 0.89 0.89 0.95 0.06
    (IFI44L ) (n332456 ) (KREMEN1 )
    TC01000794.hg.1 TC02001749.hg.1 TC14001821.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (n334829 )
    TC01000794.hg.1 TC02000034.hg.1 TC22000191.hg.1 0.89 0.86 0.89 0.95 0.06
    (IFI44L ) (RSAD2 ) (KREMEN1 )
    TC01000794.hg.1 TC02004017.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (TCONS_00003184- (KREMEN1 )
    XLOC_001966)
    TC01000794.hg.1 TC12000884.hg.1 TC22000191.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (OAS1 ) (KREMEN1 )
    TC01000794.hg.1 TC02001749.hg.1 TC15002251.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (n332456 )
    TC01000794.hg.1 TC02001749.hg.1 TC14000584.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (IFI27 )
    TC01000794.hg.1 TC02001750.hg.1 TC15002251.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (n332456 )
    TC01000794.hg.1 TC02001749.hg.1 TC04001372.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (uc003hrl.1 )
    TC01000794.hg.1 TC02001749.hg.1 TC11000812.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (DGAT2 )
    TC01000794.hg.1 TC02001750.hg.1 TC04001372.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (uc003hrl.1 )
    TC01000794.hg.1 TC02001750.hg.1 TC14000584.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (IFI27 )
    TC01000794.hg.1 TC02001750.hg.1 TC14001821.hg.1 0.89 0.88 0.89 0.95 0.06
    (IFI44L ) (CYP1B1 ) (n334829 )
    TC01000794.hg.1 TC10000639.hg.1 TC22000191.hg.1 0.89 0.90 0.89 0.95 0.06
    (IFI44L ) (IFIT1 ) (KREMEN1 )
    TC01000795.hg.1 TC01001738.hg.1 TC22000191.hg.1 0.86 0.90 0.89 0.95 0.06
    (IFI44 ) (CR1 ) (KREMEN1 )
    TC01000794.hg.1 TC21000189.hg.1 TC22000191.hg.1 0.89 0.90 0.89 0.95 0.06
    (IFI44L ) (MX1 ) (KREMEN1 )
    TC01000795.hg.1 TC01001738.hg.1 TC02000082.hg.1 0.86 0.90 0.88 0.95 0.06
    (IFI44 ) (CR1 ) (TRIB2 )
    TC01000794.hg.1 TC02000082.hg.1 TC04001267.hg.1 0.89 0.88 0.88 0.95 0.06
    (IFI44L ) (TRIB2 ) (SULT1B1 )
    TC01000794.hg.1 TC01001738.hg.1 TC02000082.hg.1 0.89 0.90 0.88 0.95 0.05
    (IFI44L ) (CR1 ) (TRIB2 )
    TC01000795.hg.1 TC01001738.hg.1 TC02003047.hg.1 0.86 0.90 0.88 0.95 0.05
    (IFI44 ) (CR1 ) (n407780 )
  • C-reactive protein (CRP) and TNF-related apoptosis-inducing ligand (TRAIL) are known to be potential useful biomarkers for distinguishing between patients with bacterial versus viral infections (see for example WO2013/117746, the contents of which is incorporated herein by reference). The investors further combined either CRP or TRAIL proteins with various RNAs (included in Tables 16A-C) in order to generate a synergistic effect and improve their diagnostics accuracy using logistic regression models. The accuracy measures of the best performing combinations are depicted in Table 21.
  • TABLE 21
    pairs of either CRP or TRAIL proteins and RNAs that presented the best
    sensitivity. The columns of Feature #1 and Feature #2 include the probe set ID and
    either the gene symbol (for coding RNAs) or mRNA accession (for non-coding RNAs)
    in brackets. Logistic regression model parameters (constant and single RNAs
    coefficient) are depicted.
    Feature#1 Feature#2
    Feature #1 Feature #2 AUC Sensitivity Specificity Constant coefficient coefficient
    CRP TC15002251.hg.1 0.99 0.97 0.92 33.37 0.06 −4.55
    (n332456 )
    CRP TC04001372.hg.1 0.97 0.95 0.93 15.99 0.04 −2.44
    (uc003hrl.1 )
    TC17001129.hg.1 TRAIL 0.96 0.95 0.88 −15.42 1.71 −0.03
    (GAS7 )
    TC04001372.hg.1 TRAIL 0.96 0.95 0.87 18.30 −2.21 −0.03
    (uc003hrl.1 )
    CRP TC14000584.hg.1 0.96 0.95 0.85 2.24 0.04 −0.56
    (IFI27 )
    CRP TC02003047.hg.1 0.98 0.92 0.97 21.42 0.04 −2.95
    (n407780 )
    TC15002251.hg.1 TRAIL 0.97 0.92 0.97 22.34 −2.42 −0.04
    (n332456 )
    CRP TC02000082.hg.1 0.98 0.92 0.95 20.10 0.04 −2.88
    (TRIB2 )
    CRP TC04001373.hg.1 0.97 0.92 0.95 17.78 0.03 −2.42
    (PPM1K )
    TC02003047.hg.1 TRAIL 0.97 0.92 0.95 20.76 −2.15 −0.04
    (n407780 )
    CRP TC08000814.hg.1 0.95 0.92 0.93 9.11 0.04 −1.04
    (LY6E )
    TC02000082.hg.1 TRAIL 0.97 0.92 0.93 20.49 −2.18 −0.04
    (TRIB2 )
    CRP TC12001843.hg.1 0.97 0.92 0.92 −33.90 0.03 2.25
    (LTA4H )
    CRP TC22000191.hg.1 0.96 0.92 0.92 −15.52 0.03 1.21
    (KREMEN1 )
    CRP TC04000484.hg.1 0.96 0.92 0.90 6.68 0.03 −0.95
    (HERC6 )
    CRP TC18000060.hg.1 0.98 0.92 0.90 −36.13 0.04 3.34
    (IMPA2 )
    TC18000060.hg.1 TRAIL 0.97 0.92 0.90 −27.03 2.86 −0.03
    (IMPA2 )
    CRP TC12000884.hg.1 0.95 0.92 0.88 7.83 0.04 −1.00
    (OAS1 )
    TC04001267.hg.1 TRAIL 0.96 0.92 0.88 −16.01 1.59 −0.04
    (SULT1B1 )
    CRP TC02003008.hg.1 0.94 0.92 0.87 2.98 0.04 −0.45
    (n332762 )
    CRP TC02005020.hg.1 0.95 0.92 0.87 3.85 0.04 −0.69
    (CMPK2 )
    CRP TC07001916.hg.1 0.95 0.92 0.87 8.00 0.04 −1.03
    (PARP12 )
    CRP TC12002059.hg.1 0.95 0.92 0.87 5.53 0.04 −0.73
    (OASL )
    TC20000575.hg.1 TRAIL 0.95 0.89 0.95 7.31 −0.68 −0.03
    (SIGLEC1 )
    CRP TC20000575.hg.1 0.95 0.89 0.93 3.61 0.04 −0.78
    (SIGLEC1 )
    CRP TC12000886.hg.1 0.95 0.89 0.92 8.49 0.04 −0.81
    (OAS2 )
    TC04001373.hg.1 TRAIL 0.97 0.89 0.92 21.21 −2.38 −0.02
    (PPM1K )
    CRP TC21000189.hg.1 0.95 0.89 0.90 7.16 0.04 −0.87
    (MX1 )
    CRP TC21000739.hg.1 0.96 0.89 0.90 9.76 0.04 −0.80
    (n333319 )
    TC11000812.hg.1 TRAIL 0.96 0.89 0.90 −12.28 1.30 −0.04
    (DGAT2 )
    CRP TC11000812.hg.1 0.97 0.89 0.88 −19.84 0.04 1.50
    (DGAT2 )
    CRP TC12000885.hg.1 0.95 0.89 0.88 4.14 0.04 −0.62
    (OAS3 )
    TC02001866.hg.1 TRAIL 0.96 0.89 0.88 13.69 −1.30 −0.02
    (PNPT1 )
    CRP TC01001738.hg.1 0.96 0.89 0.87 −26.80 0.04 1.61
    (CR1 )
    CRP TC04000485.hg.1 0.94 0.89 0.85 3.40 0.04 −0.52
    (HERC5 )
    TC14000584.hg.1 TRAIL 0.95 0.89 0.85 5.91 −0.36 −0.04
    (IFI27 )
    TC14001821.hg.1 TRAIL 0.95 0.89 0.85 6.30 −0.37 −0.04
    (n334829 )
    TC18000223.hg.1 TRAIL 0.93 0.89 0.85 8.87 −0.52 −0.03
    (ZCCHC2 )
    CRP TC02001866.hg.1 0.96 0.87 0.98 10.75 0.03 −1.36
    (PNPT1 )
    CRP TC02001749.hg.1 0.96 0.87 0.97 −14.86 0.05 1.15
    (CYP1B1 )
    CRP TC14001821.hg.1 0.96 0.87 0.97 2.82 0.04 −0.58
    (n334829 )
    CRP TC22000029.hg.1 0.96 0.87 0.97 3.10 0.04 −0.63
    (USP18 )
    CRP TC14001124.hg.1 0.97 0.87 0.95 −34.73 0.04 2.21
    (PYGL )
    TC22000029.hg.1 TRAIL 0.95 0.87 0.95 6.23 −0.51 −0.03
    (USP18 )
    CRP TC02004017.hg.1 0.95 0.87 0.93 4.03 0.04 −0.90
    (TCONS_00003184-
    XLOC_001966 )
    CRP TC22000519.hg.1 0.96 0.87 0.93 3.69 0.04 −0.75
    (USP41 )
    TC01001738.hg.1 TRAIL 0.95 0.87 0.93 −17.18 1.30 −0.03
    (CR1 )
    TC04000484.hg.1 TRAIL 0.95 0.87 0.93 9.08 −0.78 −0.03
    (HERC6 )
    TC22000191.hg.1 TRAIL 0.96 0.87 0.93 −10.08 1.15 −0.03
    (KREMEN1 )
    TC22000519.hg.1 TRAIL 0.94 0.87 0.93 6.59 −0.54 −0.03
    (USP41 )
  • Sub-Group Analysis
  • The inventors evaluated the accuracy measures of various RNAs in distinguishing between patients presenting with viral and gram negative bacterial infections (Table 22), and between Pneumonia caused by viral or bacterial infections (Table 23).
  • TABLE 22
    Accuracy measures of selected RNAs in distinguishing between viral
    (n = 79) and gram negative bacteria (n = 16)
    Gene
    Feature #
    1 Symbol mRNA Accession AUC Sen Spe
    TC20000575.hg.1 SIGLEC1 NM_023068 0.96 0.94 0.89
    TC02001866.hg.1 PNPT1 NM_033109 0.96 0.94 0.94
    TC04000484.hg.1 HERC6 NM_001165136 0.96 0.94 0.82
    TC12001843.hg.1 LTA4H NM_000895 0.95 0.94 0.92
    TC22000029.hg.1 USP18 NM_017414 0.94 0.94 0.84
    TC22000519.hg.1 USP41 ENST00000454608 0.94 0.94 0.84
    TC02004017.hg.1 — TCONS_00003184- 0.93 0.88 0.92
    XLOC_001966
    TC04001373.hg.1 PPM1K NM_152542 0.93 0.94 0.86
    TC14001821.hg.1 — n334829 0.93 0.88 0.90
    TC17001129.hg.1 GAS7 NM_201433 0.92 0.88 0.87
    TC21000739.hg.1 — n333319 0.92 0.88 0.84
    TC12000886.hg.1 OAS2 NM_001032731 0.92 0.94 0.79
    TC14000584.hg.1 IFI27 NM_001130080 0.92 0.88 0.89
    TC18000060.hg.1 IMPA2 NM_014214 0.92 0.88 0.84
    TC08000814.hg.1 LY6E NM_001127213 0.92 0.81 0.94
    TC21000189.hg.1 MX1 NM_001144925 0.92 0.88 0.85
    TC01000794.hg.1 IFI44L NM_006820 0.91 0.88 0.79
    TC10000639.hg.1 IFIT1 NM_001548 0.91 0.81 0.91
    TC07001916.hg.1 PARP12 NM_022750 0.91 0.81 0.87
    TC22000191.hg.1 KREMEN1 NM_001039570 0.91 0.88 0.85
    TC02005020.hg.1 CMPK2 NM_207315 0.91 0.88 0.85
    TC15002251.hg.1 — n332456 0.91 0.88 0.89
    TC04001372.hg.1 — uc003hrl.1 0.90 0.94 0.79
    TC12002059.hg.1 OASL NM_003733 0.90 0.81 0.89
    TC14001124.hg.1 PYGL NM_001163940 0.90 0.81 0.85
  • TABLE 23
    Accuracy measures of selected RNAs in distinguishing between patients
    presenting with Pneumonia caused by viral (n = 7) or bacterial (n = 16) infections
    Gene
    Feature #
    1 Symbol mRNA Accession AUC Sensitivity Specificity
    TC01000794.hg.1 IFI44L NM_006820 1.00 0.94 1.00
    TC01000795.hg.1 IFI44 NM_006417 0.99 0.94 0.86
    TC01001738.hg.1 CR1 NM_000573 1.00 1.00 1.00
    TC02000034.hg.1 RSAD2 NM_080657 0.98 0.94 1.00
    TC02000082.hg.1 TRIB2 NM_021643 0.94 0.81 0.86
    TC02001749.hg.1 CYP1B1 OTTHUMT00000325647 0.98 0.94 0.86
    TC02001750.hg.1 CYP1B1 NM_000104 0.97 0.94 0.86
    TC02001866.hg.1 PNPT1 NM_033109 0.97 0.94 1.00
    TC02003008.hg.1 — n332762 0.98 0.94 1.00
    TC02003047.hg.1 — n407780 0.93 0.81 0.86
    TC02004017.hg.1 — TCONS_00003184- 0.95 0.94 1.00
    XLOC_001966
    TC02005020.hg.1 CMPK2 NM_207315 0.97 0.94 1.00
    TC04000484.hg.1 HERC6 NM_001165136 0.99 0.94 0.86
    TC04000485.hg.1 HERC5 NM_016323 0.98 0.94 1.00
    TC04001267.hg.1 SULT1B1 NM_014465 0.98 0.88 0.86
    TC04001372.hg.1 — uc003hrl.1 0.89 0.75 0.86
    TC04001373.hg.1 PPM1K NM_152542 1.00 1.00 1.00
    TC04001718.hg.1 DDX60 NM_017631 0.96 0.88 1.00
    TC07001916.hg.1 PARP12 NM_022750 0.99 0.94 0.86
    TC08000814.hg.1 LY6E NM_001127213 0.97 0.94 1.00
    TC10000639.hg.1 IFIT1 NM_001548 0.99 0.94 0.86
    TC11000812.hg.1 DGAT2 NM_001253891 0.96 0.94 1.00
    TC12000884.hg.1 OAS1 NM_001032409 0.99 0.94 0.86
    TC12000885.hg.1 OAS3 NM_006187 0.99 0.94 0.86
    TC12000886.hg.1 OAS2 NM_001032731 0.97 0.94 1.00
    TC12001843.hg.1 LTA4H NM_000895 1.00 1.00 1.00
    TC12002059.hg.1 OASL NM_003733 0.98 0.94 1.00
    TC12002572.hg.1 — n332510 1.00 0.94 1.00
    TC14000584.hg.1 IFI27 NM_001130080 0.99 0.94 0.86
    TC14001124.hg.1 PYGL NM_001163940 1.00 1.00 1.00
    TC14001821.hg.1 — n334829 0.99 0.94 0.86
    TC15002251.hg.1 — n332456 0.94 0.94 0.71
    TC17001129.hg.1 GAS7 NM_201433 0.98 0.94 0.86
    TC18000060.hg.1 IMPA2 NM_014214 1.00 1.00 1.00
    TC18000223.hg.1 ZCCHC2 NM_017742 0.98 0.94 1.00
    TC20000575.hg.1 SIGLEC1 NM_023068 0.98 0.94 1.00
    TC21000189.hg.1 MX1 NM_001144925 0.99 0.94 0.86
    TC21000739.hg.1 — n333319 1.00 0.94 1.00
    TC22000029.hg.1 USP18 NM_017414 0.97 0.94 1.00
    TC22000191.hg.1 KREMEN1 NM_001039570 0.93 0.88 1.00
    TC22000519.hg.1 USP41 ENST00000454608 0.97 0.94 1.00
    • Diagnosing Early Bacterial Infections
  • Delayed or no antibiotic treatment in cases of bacterial disease is very common (24%-40% of all bacterial infections), and can lead to disease-related complications resulting in increased rates of morbidity and mortality. Thus, timely identification of patients with bacterial infection is of great importance to guide correct patient management. Indeed, the inventors identified specific RNAs that are highly accurate at disease onset and thus might be used as biomarkers for early diagnosis of bacterial infections (for example CYP1B land HERC6; Table 24).
  • TABLE 24
    RNAs that are highly accurate at disease onset
    Gene AUC 0-1 AUC 3-10 Delta
    Probe Set ID mRNA Accession Symbol days days AUC
    TC02004017.hg.1 TCONS_00003184- — 0.94 0.86 0.08
    XLOC_001966
    TC02001750.hg.1 NM_000104 CYP1B1 0.92 0.85 0.08
    TC02001749.hg.1 OTTHUMT00000325647 CYP1B1 0.91 0.84 0.07
    TC04000484.hg.1 NM_001165136 HERC6 0.97 0.90 0.06
    TC02005020.hg.1 NM_207315 CMPK2 0.94 0.87 0.06
    TC18000223.hg.1 NM_017742 ZCCHC2 0.94 0.88 0.06
    TC12002059.hg.1 NM_003733 OASL 0.92 0.87 0.05
    TC04001718.hg.1 NM_017631 DDX60 0.90 0.85 0.05
    TC07001916.hg.1 NM_022750 PARP12 0.92 0.88 0.04
    TC04000485.hg.1 NM_016323 HERC5 0.91 0.87 0.03
    TC10000639.hg.1 NM_001548 IFIT1 0.93 0.90 0.03
    TC01000795.hg.1 NM_006417 IFI44 0.90 0.87 0.03
    TC22000519.hg.1 ENST00000454608 USP41 0.92 0.89 0.03
    TC21000189.hg.1 NM_001144925 MX1 0.92 0.89 0.03
  • The expression levels of selected RNAs in patients presenting with different strains of viruses as compared to patients with bacterial infection or non-infectious patients were evaluated (including healthy; FIGS. 7A-7I). Interestingly, specific RNAs/genes were more differently expressed in certain types of viruses, and thus might server as specific indicators for this type of virus as part of a diagnostic assay. Examples of specific RNAs and the viral strain/s in which it is mostly differentially expressed are included in Table 25.
  • TABLE 25
    RNAs that might serve as potential indicators for a specific
    type of viral strain.
    Gene symbol/mRNA accession Virus type differentiation
    CYP1B1 Parainfluenza
    SIGLEC1 Influenza
    GAS7 Parainfluenza, Influenza
    n332456 Influenza
    DDX60 Influenza
    SLUT1B1 CMV/EBV
    HER6 Influenza
    TCGNS_00003184-XLOC_001966 Parainfluenza, Influenza, RSV
  • Additional RNA determinants that were found to be discriminative for distinguishing between viral and bacterial infections are summarized in Table 26 herein below.
  • TABLE 26
    Fold
    Change ANOVA
    (linear) p-value
    Gene mRNA (Bacterial (Bacterial Up in
    Symbol Accession AUC Sensitivity Specificity vs. Viral) vs. Viral) B/V
    TTC21A NM_001105513 0.89 0.88 0.80 −1.53 9.96E−09 V
    SDCCAG3 NM_001039707 0.68 0.59 0.75 −1.16 0.000961 V
    RABGAP1L NM_001035230 0.85 0.77 0.87 −1.36 2.39E−11 V
    PRR5 NM_181333 0.88 0.75 0.87 −1.08 1.44E−12 V
    NEXN NM_001172309 0.77 0.73 0.70 −1.6 4.16E−08 V
    NCOA7 NM_001122842 0.62 0.53 0.76 −1.13 0.084484 V
    MT1M NM_176870 0.84 0.75 0.79 −1.33 1.16E−11 V
    MT1IP NR_003669 0.76 0.77 0.76 −1.23 6.35E−07 V
    MT1E NM_175617 0.83 0.80 0.77 −1.63 1.99E−10 V
    MT1DP NR_003658 0.83 0.82 0.77 −1.45 4.35E−12 V
    MT1A NM_005946 0.83 0.86 0.77 −1.69 2.22E−12 V
    MIR650 NR_030755 0.71 0.75 0.62 −2.75 0.000039 V
    LRRN3 NM_001099658 0.73 0.73 0.67 −2.53 0.000011 V
    KRT19 NM_002276 0.51 0.63 0.51 1 0.686226 B
    IL1RN NM_173843 0.67 0.75 0.60 −1.55 0.000095 V
    GSN NM_198252 0.70 0.78 0.57 1.23 0.000294 B
    FAM200B NM_001145191 0.78 0.71 0.73 1.42 2.56E−08 B
    CDK5RAP2 NM_001011649 0.55 0.61 0.56 1.06 0.277074 B
    CCL8 NM_005623 0.83 0.80 0.73 −1.45 0.000005 V
    — ENST00000443397 0.71 0.65 0.75 −2.47 0.000045 V
    — n336681 0.72 0.69 0.70 −3.25 0.00003 V
    — n406211 0.76 0.80 0.71 −1.26 1.81E−07 V
    — n384079 0.70 0.59 0.77 −1.08 0.0002 V
    — n332472 0.74 0.67 0.72 −3.15 0.000034 V
    — n346241 0.73 0.75 0.68 −1.81 0.000077 V
  • Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
  • It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
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Claims (13)

What is claimed is:
1. A method of treating a viral infection comprising:
(a) confirming that a subject has a viral infection in a subject comprising measuring the amount of an RNA which is set forth in Table 1 in a sample derived from the subject, wherein when the amount of said RNA set forth in Table 1 is above a predetermined level, it is confirmed that the subject has a viral infection; and
(b) administering to the subject a therapeutically effective amount of an antiviral agent.
2. The method of claim 1, wherein said RNA is CEACAM 1 or PSTPIP2.
3. The method of claim 1, further comprising measuring the amount of at least one additional RNA to confirm said viral infection, wherein said at least one additional RNA is set forth in any one of Tables 1-6A or 15A-B.
4. The method of claim 3, wherein said at least one additional RNA is set forth in Table 15A to confirm said viral infection.
5. The method of claim 1 further comprising analyzing the expression level of CRP polypeptide or TRAIL polypeptide in said sample.
6. The method of claim 1, wherein the sample is whole blood or a fraction thereof.
7. The method of claim 1, wherein no more than 20 RNA determinants are measured.
8. The method of claim 1, wherein no more than 5 RNA determinants are measured.
9. The method of claim 6, wherein said fraction comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.
10. The method of claim 6, wherein said fraction comprises serum or plasma.
11. The method of claim 1, wherein the viral infection is a respiratory infection.
12. The method of claim 1, wherein the subject displays at least one symptom of an infection.
13. The method of claim 12, wherein said at least one symptom comprises a fever.
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