WO2020096796A1 - Procédé de prédiction de la dengue sévère - Google Patents

Procédé de prédiction de la dengue sévère Download PDF

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WO2020096796A1
WO2020096796A1 PCT/US2019/058336 US2019058336W WO2020096796A1 WO 2020096796 A1 WO2020096796 A1 WO 2020096796A1 US 2019058336 W US2019058336 W US 2019058336W WO 2020096796 A1 WO2020096796 A1 WO 2020096796A1
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polynucleotide
dengue
oligonucleotide
hybridizes
patient
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PCT/US2019/058336
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Purvesh Khatri
Shirit Einav
Timothy E. Sweeney
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the method of analyzing a sample may include: obtaining a biological sample from a patient; and detecting the levels of expression of two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2,
  • the method may comprise analyzing three or more, four or more, five or more, or six or more of the biomarkers.
  • the method may comprise analyzing the expression of DEFA4, CACNA2D2, SPON2, CACNA2D3, CHD3, GRAP2, AK5, and PTPRM; GYG1, CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13; or TOR3A, NCR3, ABI3, C3orfl8, and ENPP5.
  • the expression of up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90 or up to 100 biomarkers or more may be measuring in this assay, including at least 2 (at least 3, at least 4 or at least 5, etc.) of the 20 biomarkers listed above.
  • a subject that has a strong likelihood of progressing to severe dengue may be identified.
  • An at least 8-gene set is strongly associated with the progression to severe dengue and represents a predictive signature, generalizable across ages, host genetic factors, vims strains and sample sources.
  • a 20-gene set that is strongly associated with the progression to severe dengue is provided.
  • a panel of biomarkers is used for prognosis of severe dengue.
  • Biomarker panels of any size can be used in the practice of the methods and/or kits described herein.
  • Biomarker panels for prognosis of severe dengue typically includes at least 2 biomarkers and up to 30 biomarkers, including any number of biomarkers in between, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biomarkers.
  • the methods and/or kits described herein includes a biomarker panel with at least 2, at least 3, at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11 or more biomarkers.
  • biomarker panels are usually more economical, larger biomarker panels (i.e., greater than 30 biomarkers) may have the advantage of providing more detailed information and can also be used in some cases.
  • the two or more comprise DEFA4, CACNA2D2, SPON2,
  • the two or more comprise GYG1, CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13.
  • the two or more comprise TOR3A, NCR3, ABI3, C3orfl8, and ENPP5 biomarkers in the biological sample.
  • the method may further include generating a report indicating whether the patient has a high risk of severe dengue, wherein the report is based on the gene expression data obtained by detecting the levels of expression of two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2, CACNA2D2,
  • NCR3, ABI3, C3orfl8, and ENPP5 biomarkers in the biological sample are NCR3, ABI3, C3orfl8, and ENPP5 biomarkers in the biological sample.
  • the method may further include forwarding the report to a third party.
  • the method may include determining whether the patient is at high risk of severe dengue using the data from the detecting the levels of expression of a set of biomarkers in the biological sample.
  • the method may further include monitoring the patient for a condition, wherein the condition includes kidney failure, bleeding, plasma leakage, shock, and organ failure.
  • the method may further include admitting to a hospital only if the patient is at a high risk of severe dengue.
  • a method of diagnosing or prognosing severe dengue in a patient including: obtaining a biological sample from a human patient; detecting levels of expression of two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1,
  • TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 biomarkers in the biological sample diagnosing the patient with severe dengue when increased levels of expression of DEFA4, GYG1 and TOR3A biomarkers, and decreased levels of expression of PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1, TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 biomarkers, compared to the reference value ranges for the biomarkers for a control subject are detected.
  • the methods described herein include administering an effective amount of a pharmaceutical drug to the patient. In some embodiments, the methods described herein include determining a dengue score for each biological sample by subtracting the mean expression of the levels of expression of PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1, TRERF1, GBP2,
  • SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 biomarkers from the mean expression of the levels of expression of DEFA4, GYG1 and TOR3A biomarkers.
  • a method for treating a patient having a high risk of severe dengue comprising: receiving a report indicating whether the patient has a high risk of severe dengue, wherein the report is based on the gene expression data obtained by measuring the amount of RNA transcripts encoded by two or more of DEFA4,
  • the treating includes admitting to a hospital only if the patient is at a high risk of severe dengue
  • Kits for performing the methods described herein are also provided.
  • FIG. 1 Panels A-D depicts a 20-gene set predictive of severe dengue.
  • FIG. 2 depicts publicly available datasets used for the discovery and validation of the
  • FIG. 3 Panels A-T depicts forest plots of the over-expressed and under-expressed genes derived in the forward searches.
  • FIG. 4 depicts the over-expressed and under-expressed genes identified in the discovery cohort via the multi-cohort analysis.
  • FIG. 5 Panels A-B depicts violin plots showing the performance of the 20-gene set to separate Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) from Dengue Fever (DF) in the 7 datasets of the discovery cohort (FIG. 5, Panel A) and 3 datasets of the validation cohort (FIG. 5, Panel B).
  • DHF/DSS Dengue Hemorrhagic Fever/Dengue Shock Syndrome
  • DF Dengue Fever
  • FIG. 6 Panels A-H depicts in silico and prospective validation of the 20-gene set
  • FIG. 7 Panels A-B demonstrates that routine laboratory parameters are ineffective in predicting development of severe dengue and at most part do not correlate with the dengue severity score.
  • dengue virus refers to members of the Flaviviridae family of enveloped viruses with a single-stranded positive-sense RNA genome (see, e.g., Frontiers in Dengue Virus Research, Hanley and Weaver (editors), Caister Academic Press, 2010).
  • the term dengue vims may include any serotype of dengue vims, such as serotypes 1-4, which is capable of causing disease in an animal or human subject.
  • the term encompasses any subtype of dengue virus that causes disease in humans, including strains DEN 1 Hawaii 1944, Den 2 New Guinea C strain, DEN 3 strain H87, and DEN 4 strain H241. A large number of dengue isolates have been partially or completely sequenced. See, e.g., the Broad Institute Dengue Vims Portal (website at
  • viprbrc.org/brc/home.do?decorator flavi_dengue) and the GenBank database, which contain complete sequences for dengue vimses, including serotypes 1-4.
  • the term“severe dengue” refers to a disease or condition classified according to WHO dengue classification methods, including but not limited to the 2009 WHO criteria (Alexander et almen 2011; WHO, 2009) and 1997 WHO criteria (WHO, 1997).
  • Classic warning signs of severe dengue include a decrease in temperature (below 38°C); severe abdominal pain; rapid breathing; persistent vomiting; blood in vomit; fluid accumulation in the body; mucosal (gums and nose) bleeding; liver enlargement; rapid decrease in platelet count; lethargy; and restlessness.
  • DSS severe plasma leakage leading to shock
  • fluid accumulation with respiratory distress severe bleeding as evaluated by a clinician
  • severe organ involvement such as a liver having AST or ALT > 1000, a central nervous system with impaired consciousness or failure of heart and other organs
  • kidney failure shock for 5-7 days
  • severe skin bleeding with spots of blood on the skin petechiae
  • large patches of blood under the skin ecchymoses
  • black stools blood in urine (hematuria); and respiratory distress.
  • a “biomarker” in the context of the methods and/or kits described herein refers to a biological compound, such as a polynucleotide which is differentially expressed in a sample taken from a dengue patient as compared to a comparable sample taken from a patient without dengue or severe dengue.
  • the biomarker can be a nucleic acid, a fragment of a nucleic acid, a polynucleotide, or an oligonucleotide that can be detected and/or quantified.
  • Biomarkers include polynucleotides including nucleotide sequences from genes or RNA transcripts of genes, including but not limited to, DEFA4, GYG1, TOR3A, PTPRM,
  • polypeptide and protein refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, hydroxylation, oxidation, and the like.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • polynucleotide oligonucleotide
  • nucleic acid molecule a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single- stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
  • polyribonucleotides containing D-ribose
  • any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • a biomarker can be a polynucleotide which is present at an elevated level or at a decreased level in samples of patients with severe dengue compared to samples of control subjects.
  • a biomarker can be a polynucleotide which is detected at a higher frequency or at a lower frequency in samples of patients at high risk of severe dengue compared to samples of control subjects.
  • a biomarker can be differentially present in terms of quantity, frequency or both.
  • a polynucleotide is differentially expressed between two samples if the amount of the polynucleotide in one sample is statistically significantly different from the amount of the polynucleotide in the other sample.
  • a polynucleotide is differentially expressed in two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other.
  • a polynucleotide is differentially expressed in two sets of samples if the frequency of detecting the polynucleotide in samples of patients at high risk of severe dengue is statistically significantly higher or lower than in the control samples.
  • a polynucleotide is differentially expressed in two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples.
  • a “similarity value” is a number that represents the degree of similarity between two things being compared.
  • a similarity value may be a number that indicates the overall similarity between a patient's expression profile using specific phenotype-related biomarkers and reference value ranges for the biomarkers in one or more control samples or a reference expression profile.
  • the similarity value may be expressed as a similarity metric, such as a correlation coefficient, or may simply be expressed as the expression level difference, or the aggregate of the expression level differences, between levels of biomarkers in a patient sample and a control sample or reference expression profile.
  • subject refers to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans.
  • Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.
  • the methods and/or kits described herein find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • a“hospital” refers to an institution, observation ward, clinic, hospice, emergency room, or area providing medical and/or surgical treatment and nursing care for sick or injured individuals.
  • a "biological sample” refers to a sample of tissue, cells, or fluid isolated from a subject, including but not limited to, for example, blood, buffy coat, plasma, serum, blood cells (e.g., peripheral blood mononucleated cells (PBMCS), band cells, neutrophils, metamyelocytes, monocytes, or T cells), fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, organs, biopsies and also samples of in vitro cell culture constituents, including, but not limited to, conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
  • PBMCS peripheral blood mononucleated cells
  • band cells neutrophils, metamyelocytes, monocytes, or T cells
  • fecal matter e.g., urine, bone
  • test amount of a biomarker refers to an amount of a biomarker present in a sample being tested.
  • a test amount can be either an absolute amount (e.g., mg/ml) or a relative amount (e.g., relative intensity of signals).
  • a "control amount" of a biomarker can be any amount or a range of amount which is to be compared against a test amount of a biomarker.
  • a control amount of a biomarker can be the amount of a biomarker in a person without a life-threatening condition (e.g., person without dengue or severe dengue) or healthy person.
  • a control amount can be either in absolute amount (e.g., mg/ml) or a relative amount (e.g., relative intensity of signals).
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab') 2 and F(ab) fragments; F v molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al.
  • Detectable moieties or “detectable labels” contemplated for use in the methods and/or kits described herein include, but are not limited to, radioisotopes, fluorescent dyes such as fluorescein, phycoerythrin, Cy-3, Cy-5, allophycoyanin, DAPI, Texas Red, rhodamine, Oregon green, Lucifer yellow, and the like, green fluorescent protein (GFP), red fluorescent protein (DsRed), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), Cerianthus Orange Fluorescent Protein (cOFP), alkaline phosphatase (AP), beta- lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo r , G4l8 r ) dihydrofolate reductase (DHFR), hygromycin-B-phosphotransfera
  • Enzyme tags are used with their cognate substrate.
  • the terms also include color-coded microspheres of known fluorescent light intensities (see e.g., microspheres with xMAP technology produced by Luminex (Austin, TX); microspheres containing quantum dot nanocrystals, for example, containing different ratios and combinations of quantum dot colors (e.g., Qdot nanocrystals produced by Life Technologies (Carlsbad, CA); glass coated metal nanoparticles (see e.g., SERS nanotags produced by Nanoplex Technologies, Inc.
  • Diagnosis generally includes determination as to whether a subject is likely affected by a given disease, disorder or dysfunction. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, i.e., a biomarker, the presence, absence, or amount of which is indicative of the presence or absence of the disease, disorder or dysfunction.
  • diagnostic indicators i.e., a biomarker, the presence, absence, or amount of which is indicative of the presence or absence of the disease, disorder or dysfunction.
  • Prognosis as used herein generally refers to a prediction of the probable course and outcome of a clinical condition or disease.
  • a prognosis of a patient is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease (e.g., dengue fever, dengue hemorrhagic fever and dengue shock syndrome). It is understood that the term “prognosis” does not necessarily refer to the ability to predict the course or outcome of a condition with 100% accuracy.
  • prognosis refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.
  • kits described herein relate to the use of biomarkers either alone or in combination with clinical parameters for aiding diagnosis, prognosis, and treatment of patients.
  • the inventors have discovered biomarkers whose expression profiles can be used for prognosis of severe dengue in patients with severe dengue.
  • Biomarkers that can be used in the practice of the methods and/or kits described herein include polynucleotides including nucleotide sequences from genes or RNA transcripts of genes, including but not limited to, DEFA4, GYG1, TOR3A, PTPRM,
  • biomarkers that can be used in the practice of the methods and/or kits described herein include polynucleotides including nucleotide sequences from genes or RNA transcripts of genes, including but not limited to, DEFA4, CACNA2D2, SPON2,
  • biomarkers that can be used in the practice of the methods and/or kits described herein include
  • biomarkers that can be used in the practice of the methods and/or kits described herein include polynucleotides including nucleotide sequences from genes or RNA transcripts of genes, including but not limited to, TOR3A, NCR3, ABI3, C3orfl8, and ENPP5. Differential expression of these biomarkers is associated with a high risk of severe dengue and therefore expression profiles of these biomarkers are useful for prognosis of severe dengue in patients.
  • a method of determining severe dengue risk of a subject including measuring the level of a plurality of biomarkers in a biological sample derived from a subject suspected of having a life-threatening condition, and analyzing the levels of the biomarkers and comparing with respective reference value ranges for the biomarkers, wherein differential expression of one or more biomarkers in the biological sample compared to one or more biomarkers in a control sample indicates that the subject is at high risk of severe dengue.
  • the reference value ranges can represent the levels of one or more biomarkers found in one or more samples of one or more subjects without an illness (e.g., healthy subject or subject without infection).
  • the reference values can represent the levels of one or more biomarkers found in one or more samples of one or more subjects with a critical illness (e.g., a subject with severe dengue).
  • the levels of the biomarkers are compared to time- matched reference values ranges for non-inf ected and infected/dengue subjects.
  • the biological sample obtained from the subject to be diagnosed is typically whole blood, huffy coat, plasma, serum, or blood cells (e.g., peripheral blood mononucleated cells (PBMCS), band cells, metamyelocytes, neutrophils, monocytes, or T cells), but can be any sample from bodily fluids, tissue or cells that contain the expressed biomarkers.
  • a "control" sample refers to a biological sample, such as a bodily fluid, tissue, or cells that are not diseased. That is, a control sample is obtained from a normal subject (e.g. an individual known to not have a life-threatening condition), a person who does not have severe dengue.
  • a biological sample can be obtained from a subject by conventional techniques. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.
  • a panel of biomarkers is used for prognosis of severe dengue risk.
  • Biomarker panels of any size can be used in the practice of the methods and/or kits described herein.
  • Biomarker panels for prognosis of severe dengue typically include at least 2 biomarkers and up to 30 biomarkers, including any number of biomarkers in between, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 biomarkers.
  • the methods and/or kits described herein include a biomarker panel including at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11 or more biomarkers.
  • the methods and/or kits described herein include a panel of biomarkers for prognosis of severe dengue risk including one or more polynucleotides including a nucleotide sequence from a gene or an RNA transcript of a gene selected from the group consisting of DEFA4, CACNA2D2, SPON2, CACNA2D3, CHD3, GRAP2, AK5, and PTPRM.
  • the panel of biomarkers includes a DEFA4
  • polynucleotide a CACNA2D2 polynucleotide, a SPON2 polynucleotide, an CACNA2D3 polynucleotide, an CHD3 polynucleotide, a GRAP2 polynucleotide, a AK5 polynucleotide, and a PTPRM polynucleotide.
  • the methods and/or kits described herein include a panel of biomarkers for prognosis of severe dengue risk including one or more polynucleotides including a nucleotide sequence from a gene or an RNA transcript of a gene selected from the group consisting of GYG1, CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13.
  • the panel of biomarkers includes a GYG1 polynucleotide, a CX3CR1 polynucleotide, a TRERF1 polynucleotide, an GBP2 polynucleotide, an
  • TMEM63C polynucleotide a SERINC5 polynucleotide, and a SOXl3polynucleotide.
  • the methods and/or kits described herein include a panel of biomarkers for prognosis of severe dengue risk including one or more polynucleotides including a nucleotide sequence from a gene or an RNA transcript of a gene selected from the group consisting of TOR3A, NCR3, ABI3, C3orfl8, and ENPP5.
  • the panel of biomarkers includes a TOR3 A polynucleotide, a NCR3 polynucleotide, a ABI3 polynucleotide, an C3orf 18 polynucleotide, and a ENPP5 polynucleotide.
  • a severe dengue gene score is used for prognosis of severe dengue risk.
  • the severe dengue gene score is calculated by subtracting the geometric mean of the expression levels of all measured biomarkers that are underexpressed compared to control reference values for the biomarkers from the geometric mean of the expression levels of all measured biomarkers that are overexpressed compared to control reference values for the biomarkers, and multiplying the difference by the ratio of the number of biomarkers that are overexpressed to the number of biomarkers that are underexpressed compared to control reference values for the biomarkers.
  • a higher severe dengue gene score for the subject compared to reference value ranges for control subjects indicates that the subject has a high risk of severe dengue.
  • the methods described herein may be used to identify patients at high risk of severe dengue who should be monitored. For example, patients identified as having a high risk of severe dengue by the methods described herein can be sent immediately to a hospital for treatment, whereas patients identified as having a low risk of severe dengue may be further monitored and/or treated in a regular hospital ward, or potentially discharged. Both patients and clinicians can benefit from better estimates of severe dengue risk, which allows timely discussions of patients’ preferences and their choices regarding life-saving measures. Better molecular phenotyping of patients also makes possible improvements in clinical trials, both in 1) patient selection for drugs and interventions and 2) assessment of observed-to-expected ratios of subject severe dengue.
  • a patient diagnosed with a viral infection is further administered a therapeutically effective dose of an antiviral agent, such as a broad- spectrum antiviral agent, an antiviral vaccine, a neuraminidase inhibitor (e.g., zanamivir (Relenza) and oseltamivir (Tamiflu)), a nucleoside analogue (e.g., acyclovir, zidovudine (AZT), and lamivudine), an antisense antiviral agent (e.g., phosphorothioate antisense antiviral agents (e.g., Fomivirsen (Vitravene) for cytomegalovirus retinitis), morpholino antisense antiviral agents), an inhibitor of viral uncoating (e.g., Amantadine and rimantadine for influenza, Pleconaril for rhino viruses), an inhibitor of viral entry (e.g., Fuzeon for HIV), an inhibitor of viral entry (
  • antiviral agents include Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla (fixed dose drug), Balavir, Cidofovir, Combivir (fixed dose drug), Dolutegravir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir, Fixed dose combination (antiretroviral), Fomivirsen, Fos amprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type III, Interferon type II, Interferon type I, Interferon, Lamivudin
  • Podophyllotoxin Protease inhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Synergistic enhancer (antiretroviral), Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir,
  • Antibiotics may include broad spectrum, bactericidal, or bacteriostatic antibiotics.
  • Exemplary antibiotics include aminoglycosides such as Amikacin, Amikin,
  • Spectinomycin(Bs), and Trobicin ansamycins such as Geldanamycin, Herbimycin,
  • carbacephems such as Loracarbef and Lorabid
  • carbapenems such as Ertapenem, Invanz, Doripenem, Doribax, Imipenem/Cilastatin, Primaxin, Meropenem, and Merrem
  • cephalosporins such as Cefadroxil, Duricef, Cefazolin, Ancef, Cefalotin or
  • Cefalothin Keflin, Cefalexin, Keflex, Cefaclor, Distaclor, Cefamandole, Mandol, Cefoxitin,
  • Cefoperazone Cefotaxime
  • Cefpodoxime Ceftazidime
  • Ceftibuten Ceftizoxime
  • Cleocin, Lincomycin, and Lincocin lipopeptides such as Daptomycin and Cubicin;
  • Troleandomycin Tao, Telithromycin, Ketek, Spiramycin, and Rovamycine
  • monobactams such as Aztreonam and Azactam
  • nitrofurans such as Furazolidone, Furoxone
  • Nitrofurantoin, Macrodantin, and Macrobid Nitrofurantoin, Macrodantin, and Macrobid; oxazolidinones such as Linezolid, Zyvox,
  • Vee-K Piperacillin, Pipracil, Penicillin G, Pfizerpen, Temocillin, Negaban, Ticarcillin, and
  • Ticar penicillin combinations such as Amoxicillin/clavulanate, Augmentin,
  • Timentin polypeptides such as Bacitracin, Colistin, Coly-Mycin-S, and Polymyxin B;
  • quinolones/fluoroquinolones such as Ciprofloxacin, Cipro, Ciproxin, Ciprobay, Enoxacin,
  • Penetrex Gatifloxacin, Tequin, Gemifloxacin, Factive, Levofloxacin, Levaquin,
  • Tegopen Dicloxacillin, Dynapen, Flucloxacillin, Floxapen, Mezlocillin, Mezlin, Methicillin,
  • Tetracycline and Sumycin, Achromycin V, and Steclin drugs against mycobacteria such as Clofazimine, Lamprene, Dapsone, Avlosulfon, Capreomycin, Capastat, Cycloserine, Seromycin, Ethambutol, Myambutol, Ethionamide, Trecator, Isoniazid, I.N.H.,
  • Rifapentine, Priftin, and Streptomycin others antibiotics such as Arsphenamine, Salvarsan, Chloramphenicol, Chloromycetin, Fosfomycin, Monurol, Monuril, Fusidic acid, Fucidin, Metronidazole, Flagyl, Mupirocin, Bactroban, Platensimycin, Quinupristin/Dalfopristin, Synercid, Thiamphenicol, Tigecycline, Tigacyl, Tinidazole, Tindamax Fasigyn,
  • the methods described herein further include administering a treatment to the patient, an antimicrobial therapy to the patient, administering an immune- modulating therapy to the patient, or administering an organ- specific treatment to the patient.
  • the organ-specific treatment includes either or both of connecting the patient to any one or more of a mechanical ventilator, a pacemaker, a defibrillator, a dialysis or renal replacement therapy machine, an invasive monitor including a pulmonary artery catheter, arterial blood pressure catheter, or central venous pressure catheter, or
  • treatment includes admitting to a hospital only if the patient is at a high risk of severe dengue.
  • the methods and kits described herein include diagnosing or prognosing severe dengue in a patient, said method including: obtaining a biological sample from a human patient; detecting levels of expression of two or more of DEFA4, GYG1,
  • TOR3A PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3,
  • CX3CR1, TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 biomarkers in the biological sample diagnosing the patient with severe dengue when increased levels of expression of DEFA4, GYG1 and TOR3A biomarkers, and decreased levels of expression of PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, ENPP5 biomarkers, compared to the reference value ranges for the biomarkers for a control subject are detected.
  • the methods further include diagnosing the patient with severe dengue when increased levels of expression of DEFA4 biomarker, and decreased levels of expression of CACNA2D2, SPON2, CACNA2D3, CHD3, GRAP2, AK5, and PTPRM biomarkers, compared to the reference value ranges for the biomarkers for a control subject are detected.
  • the methods further include diagnosing the patient with severe dengue when increased levels of expression of GYG1 biomarker, and decreased levels of expression of CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13 biomarkers, compared to the reference value ranges for the biomarkers for a control subject are detected.
  • the methods further include diagnosing the patient with severe dengue when increased levels of expression of TOR3A biomarker, and decreased levels of expression of NCR3, ABI3, C3orfl8, and ENPP5 biomarkers, compared to the reference value ranges for the biomarkers for a control subject are detected.
  • the method further includes calculating a severe dengue gene score for the patient based on the levels of the biomarkers, wherein a higher severe dengue gene score for the patient compared to a control subject indicates that the patient is at high risk of severe dengue; and administering a treatment to the diagnosed or prognosed patient.
  • Severe dengue treatment may include, for example, monitoring, administering antimicrobial therapy, supportive care, or an immune-modulating therapy, or a combination thereof.
  • Antimicrobial therapy may include administration of one or more drugs against all pathogens the patient is likely to be infected with (e.g., bacterial and/or fungal and/or viral) with preferably broad-spectrum coverage using combinations of antimicrobial agents.
  • Combination antimicrobial therapy may include at least two different classes of antibiotics
  • antibiotics may be administered in combination with antifungal and/or antiviral agents.
  • Supportive therapy for severe dengue may include administration of oxygen, blood transfusions, mechanical ventilation, fluid therapy (e.g., fluid administration with crystalloids and/or albumin continued until the patient shows hemodynamic improvement), nutrition (e.g., oral or enteral feedings), blood glucose management, vasopressor therapy
  • Immune-modulating therapy may include administration of activated protein C, immunoglobulin therapy, anti-platelet therapy, cytokine-blocking therapy, dialysis for pathogenic proteins or with antibiotic cartridges, or any combination thereof.
  • the biomarkers in a sample can be measured by any suitable method known in the art. Measurement of the expression level of a biomarker can be direct or indirect. For example, the abundance levels of RNAs or proteins can be directly quantitated. Alternatively, the amount of a biomarker can be determined indirectly by measuring abundance levels of cDNAs, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, proteins, or other molecules (e.g., metabolites) that are indicative of the expression level of the biomarker.
  • the methods for measuring biomarkers in a sample have many applications.
  • one or more biomarkers can be measured to aid in the prognosis of severe dengue risk, to determine the appropriate treatment for a subject, to monitor responses in a subject to treatment, or to identify therapeutic compounds that modulate expression of the biomarkers in vivo or in vitro.
  • the expression levels of the biomarkers are determined by measuring polynucleotide levels of the biomarkers.
  • the levels of transcripts of specific biomarker genes can be determined from the amount of mRNA, or polynucleotides derived therefrom, present in a biological sample.
  • Polynucleotides can be detected and quantitated by a variety of methods including, but not limited to, microarray analysis, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot, and serial analysis of gene expression (SAGE). See, e.g., Draghici Data Analysis Tools for
  • microarrays are used to measure the levels of biomarkers.
  • An advantage of microarray analysis is that the expression of each of the biomarkers can be measured simultaneously, and microarrays can be specifically designed to provide a diagnostic expression profile for a particular disease or condition (e.g., severe dengue).
  • Microarrays are prepared by selecting probes which include a polynucleotide sequence, and include immobilizing such probes to a solid support or surface.
  • the probes may include DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA.
  • the polynucleotide sequences of the probes may also include DNA and/or RNA analogues, or combinations thereof.
  • the polynucleotide sequences of the probes may be full or partial fragments of genomic DNA.
  • the polynucleotide sequences of the probes may also be synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
  • the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.
  • Probes used in the methods and/or kits described herein are preferably immobilized to a solid support which may be either porous or non-porous.
  • the probes may be polynucleotide sequences which are attached to a nitrocellulose or nylon membrane or filter covalently at either the 3' or the 5' end of the polynucleotide.
  • hybridization probes are well known in the art (see, e.g., Sambrook, et ak, Molecular Cloning: A
  • the solid support or surface may be a glass or plastic surface.
  • hybridization levels are measured to microarrays of probes consisting of a solid phase on the surface of which are immobilized a population of polynucleotides, such as a population of DNA or DNA mimics, or, alternatively, a population of RNA or RNA mimics.
  • the solid phase may be a nonporous or, optionally, a porous material such as a gel.
  • the microarray includes a support or surface with an ordered array of binding (e.g., hybridization) sites or "probes" each representing one of the biomarkers described herein.
  • the microarrays are addressable arrays, and more preferably positionally addressable arrays. More specifically, each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position in the array (i.e., on the support or surface).
  • Each probe is preferably covalently attached to the solid support at a single site.
  • Microarrays can be made in a number of ways, of which several are described below. However they are produced, microarrays share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably, microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. Microarrays are generally small, e.g., between 1 cm 2 and 25 cm 2 ; however, larger arrays may also be used, e.g., in screening arrays.
  • a given binding site or unique set of binding sites in the microarray will specifically bind (e.g., hybridize) to the product of a single gene in a cell (e.g., to a specific mRNA, or to a specific cDNA derived therefrom).
  • a single gene in a cell e.g., to a specific mRNA, or to a specific cDNA derived therefrom.
  • other related or similar sequences will cross hybridize to a given binding site.
  • the "probe" to which a particular polynucleotide molecule specifically hybridizes contains a complementary polynucleotide sequence.
  • the probes of the microarray typically consist of nucleotide sequences of no more than 1,000 nucleotides. In some embodiments, the probes of the array consist of nucleotide sequences of 10 to 1,000 nucleotides. In one embodiment, the nucleotide sequences of the probes are in the range of 10-200 nucleotides in length and are genomic sequences of one species of organism, such that a plurality of different probes is present, with sequences complementary and thus capable of hybridizing to the genome of such a species of organism, sequentially tiled across all or a portion of the genome.
  • the probes are in the range of 10-30 nucleotides in length, in the range of 10-40 nucleotides in length, in the range of 20-50 nucleotides in length, in the range of 40-80 nucleotides in length, in the range of 50-150 nucleotides in length, in the range of 80-120 nucleotides in length, or are 60 nucleotides in length.
  • the probes may include DNA or DNA "mimics” (e.g., derivatives and analogues) corresponding to a portion of an organism's genome.
  • the probes of the microarray are complementary RNA or RNA mimics.
  • DNA mimics are polymers composed of subunits capable of specific, Watson-Crick-like hybridization with DNA, or of specific hybridization with RNA.
  • the nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates).
  • Isothermal amplification methods do not require changing or cycling the reaction temperature and, as such, can be done by incubating a reaction at a constant temperature.
  • Isothermal amplification methods include, but are not limited to, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HD A) and nicking enzyme amplification reaction (NEAR), as well as others.
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HD A helicase-dependent amplification
  • NEAR nicking enzyme amplification reaction
  • DNA can be obtained, e.g., by polymerase chain reaction (PCR) amplification of genomic DNA or cloned sequences.
  • PCR primers are preferably chosen based on a known sequence of the genome that will result in amplification of specific fragments of genomic DNA.
  • Computer programs that are well known in the art are useful in the design of primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences).
  • each probe on the microarray will be between 10 bases and 50,000 bases, usually between 300 bases and 1,000 bases in length.
  • PCR methods are well known in the art, and are described, for example, in Innis et ak, eds., PCR Protocols: A Guide To Methods And Applications, Academic Press Inc., San Diego, Calif. (1990); herein incorporated by reference in its entirety. It will be apparent to one skilled in the art that controlled robotic systems are useful for isolating and amplifying nucleic acids.
  • An alternative, preferred means for generating polynucleotide probes is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or
  • Synthetic sequences are typically between about 10 and about 500 bases in length, more typically between about 20 and about 100 bases, and most preferably between about 40 and about 70 bases in length.
  • synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine.
  • nucleic acid analogues may be used as binding sites for hybridization.
  • nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et ak, Nature 363:566-568 (1993); U.S. Pat. No. 5,539,083).
  • Probes are preferably selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure. See Friend et ak, International Patent Publication WO 01/05935, published Jan. 25, 2001; Hughes et ak, Nat. Biotech. 19:342-7 (2001).
  • positive control probes e.g., probes known to be complementary and hybridizable to sequences in the target polynucleotide molecules
  • negative control probes e.g., probes known to not be complementary and hybridizable to sequences in the target polynucleotide molecules
  • positive controls are synthesized along the perimeter of the array.
  • positive controls are synthesized in diagonal stripes across the array.
  • the reverse complement for each probe is synthesized next to the position of the probe to serve as a negative control.
  • sequences from other species of organism are used as negative controls or as "spike-in" controls.
  • the probes are attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
  • a solid support or surface which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
  • One method for attaching nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al, Science 270:467-470 (1995). This method is especially useful for preparing microarrays of cDNA (See also, DeRisi et al, Nature Genetics 14:457-460 (1996); Shalon et al., Genome Res. 6:639-645 (1996); and Schena et al., Proc. Natl. Acad. Sci. U.S.
  • a second method for making microarrays produces high-density oligonucleotide arrays. Techniques are known for producing arrays containing thousands of
  • oligonucleotides complementary to defined sequences at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Pat. Nos.
  • oligonucleotides e.g., 60-mers
  • the array produced is redundant, with several oligonucleotide molecules per RNA.
  • microarrays may also be used.
  • any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001) could be used.
  • dot blots on a nylon hybridization membrane see Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001.
  • very small arrays will frequently be preferred because hybridization volumes will be smaller.
  • Microarrays can also be manufactured by means of an inkjet printing device for oligonucleotide synthesis, e.g., using the methods and systems described by Blanchard in
  • microdroplets of a high surface tension solvent such as propylene carbonate.
  • the microdroplets have small volumes (e.g., 100 pL or less, more preferably 50 pL or less) and are separated from each other on the microarray (e.g., by hydrophobic domains) to form circular surface tension wells which define the locations of the array elements (i.e., the different probes).
  • Microarrays manufactured by this ink-jet method are typically of high density, preferably having a density of at least about 2,500 different probes per 1 cm 2 .
  • the polynucleotide probes are attached to the support covalently at either the 3' or the 5' end of the polynucleotide.
  • Biomarker polynucleotides which may be measured by microarray analysis can be expressed RNA or a nucleic acid derived therefrom (e.g., cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter), including naturally occurring nucleic acid molecules, as well as synthetic nucleic acid molecules.
  • the target polynucleotide molecules include RNA, including, but by no means limited to, total cellular RNA, poly(A) + messenger RNA (mRNA) or a fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (i.e., cRNA; see, e.g., Linsley & Schelter, U.S. patent application Ser. No.
  • RNA can be extracted from a cell of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et ak, 1979, Biochemistry 18:5294-5299), a silica gel-based column (e.g., RNeasy (Qiagen, Valencia, Calif.) or StrataPrep (Stratagene, La Jolla, Calif.)), or using phenol and chloroform, as described in Ausubel et ak, eds., 1989, Current Protocols In Molecular Biology, Vol. Ill, Green
  • Poly(A) + RNA can be selected, e.g., by selection with oligo-dT cellulose or, alternatively, by oligo-dT primed reverse transcription of total cellular RNA.
  • RNA can be fragmented by methods known in the art, e.g., by incubation with ZnCh, to generate fragments of RNA.
  • total RNA, mRNA, or nucleic acids derived therefrom are isolated from a sample taken from a patient suspected of having a life-threatening condition (e.g., severe dengue).
  • Biomarker polynucleotides that are poorly expressed in particular cells may be enriched using normalization techniques (Bonaldo et al., 1996, Genome Res. 6:791-806).
  • the biomarker polynucleotides can be detectably labeled at one or more nucleotides. Any method known in the art may be used to label the target polynucleotides. Preferably, this labeling incorporates the label uniformly along the length of the RNA, and more preferably, the labeling is carried out at a high degree of efficiency.
  • polynucleotides can be labeled by oligo-dT primed reverse transcription. Random primers (e.g., 9-mers) can be used in reverse transcription to uniformly incorporate labeled nucleotides over the full length of the polynucleotides. Alternatively, random primers may be used in conjunction with PCR methods or T7 promoter-based in vitro transcription methods in order to amplify polynucleotides.
  • the detectable label may be a luminescent label.
  • fluorescent labels include, but are not limited to, fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative.
  • fluorescent labels including, but not limited to, fluorescent phosphoramidites such as FluorePrime (Amersham Pharmacia, Piscataway, N.J.), Fluoredite (Miilipore, Bedford, Mass.), FAM (ABI, Foster City, Calif.), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.) can be used.
  • fluorescent phosphoramidites such as FluorePrime (Amersham Pharmacia, Piscataway, N.J.), Fluoredite (Miilipore, Bedford, Mass.), FAM (ABI, Foster City, Calif.), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.) can be used.
  • the detectable label can be a radiolabeled nucleotide.
  • biomarker polynucleotide molecules from a patient sample are labeled differentially from the corresponding polynucleotide molecules of a reference sample.
  • the reference can include polynucleotide molecules from a normal biological sample (i.e., control sample, e.g., blood from a subject not having severe dengue) or from a reference biological sample, (e.g., blood from a subject having severe dengue).
  • Nucleic acid hybridization and wash conditions are chosen so that the target polynucleotide molecules specifically bind or specifically hybridize to the complementary polynucleotide sequences of the array, preferably to a specific array site, wherein its complementary DNA is located.
  • Arrays containing double-stranded probe DNA situated thereon are preferably subjected to denaturing conditions to render the DNA single- stranded prior to contacting with the target polynucleotide molecules.
  • Arrays containing single- stranded probe DNA may need to be denatured prior to contacting with the target polynucleotide molecules, e.g., to remove hairpins or dimers which form due to self-complementary sequences.
  • Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, or DNA) of probe and target nucleic acids.
  • length e.g., oligomer versus polynucleotide greater than 200 bases
  • type e.g., RNA, or DNA
  • oligonucleotides As the oligonucleotides become shorter, it may become necessary to adjust their length to achieve a relatively uniform melting temperature for satisfactory hybridization results.
  • General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), and in Ausubel et al., Current Protocols In Molecular Biology, vol. 2, Current Protocols Publishing, New York (1994).
  • Typical hybridization conditions for the cDNA microarrays of Schena et al. are hybridization in 5.times.SSC plus 0.2% SDS at 65°C for fthe hours, followed by washes at 25°C in low stringency wash buffer (lxSSC plus 0.2% SDS), followed by 10 minutes at 25°C in higher stringency wash buffer (O.lxSSC plus 0.2% SDS) (Schena et al., Proc. Natl. Acad. Sci. U.S.A. 93:10614 (1993)).
  • hybridization conditions are also provided in, e.g., Tijessen, 1993, Hybridization With Nucleic Acid Probes, Elsevier Science Publishers B.V.; and Kricka, 1992, Nonisotopic Dna Probe Techniques, Academic Press, San Diego, Calif.
  • Particularly preferred hybridization conditions include hybridization at a temperature at or near the mean melting temperature of the probes (e.g., within 5l°C, more preferably within 21 °C) in 1 M NaCl, 50 mM MES buffer (pH 6.5), 0.5% sodium sarcosine and 30% formamide.
  • the fluorescence emissions at each site of a microarray may be, preferably, detected by scanning confocal laser microscopy.
  • a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
  • a laser may be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996, "A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization," Genome Research 6:639-645, which is incorporated by reference in its entirety for all purposes).
  • Arrays can be scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective. Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultiplier tubes. Fluorescence laser scanning devices are described in Schena et al., Genome Res. 6:639-645 (1996), and in other references cited herein. Alternatively, the fiber-optic bundle described by Ferguson et al., Nature Biotech. 14:1681-1684 (1996), may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
  • the methods described herein include detecting the amount of RNA transcripts encoded by two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1, TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 in a sample of RNA obtained from the patient.
  • the methods described herein include detecting the amount of RNA transcripts encoded by two or more of DEFA4, CACNA2D2, SPON2, CACNA2D3, CHD3, GRAP2, AK5, and PTPRM in a sample of RNA obtained from the patient.
  • the methods described herein include detecting the amount of RNA transcripts encoded by two or more of GYG1, CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13 in a sample of RNA obtained from the patient. In one embodiment, the methods described herein include detecting the amount of RNA transcripts encoded by two or more of TOR3A, NCR3, ABI3, C3orfl8, and ENPP5 in a sample of RNA obtained from the patient.
  • the methods and/or kits described herein include two or more of: an oligonucleotide that hybridizes to a DEFA4 polynucleotide, an oligonucleotide that hybridizes to a GYG1 polynucleotide, an oligonucleotide that hybridizes to a TOR3A polynucleotide, an oligonucleotide that hybridizes to a PTPRM polynucleotide, an oligonucleotide that hybridizes to an SPON2 polynucleotide, an oligonucleotide that hybridizes to a GRAP2 polynucleotide, an oligonucleotide that hybridizes to a CACNA2D2 polynucleotide, an oligonucleotide that hybridizes to a CACNA2D3 polynucleotide, an oligonucleotide that hybridizes to a
  • the methods and/or kits described herein include two or more of: an oligonucleotide that hybridizes to a DEFA4 polynucleotide, an oligonucleotide that hybridizes to a CACNA2D2 polynucleotide, an oligonucleotide that hybridizes to a SPON2 polynucleotide, an oligonucleotide that hybridizes to a CACNA2D3 polynucleotide, an oligonucleotide that hybridizes to an CHD3 polynucleotide, an oligonucleotide that hybridizes to a GRAP2 polynucleotide, an oligonucleotide that hybridizes to a AK5 polynucleotide, and an oligonucleotide that hybridizes to a PTPRM polynucleotide.
  • the methods and/or kits described herein include two or more of: an oligonucleotide that hybridizes to a GYG1 polynucleotide, an oligonucleotide that hybridizes to a CX3CR1 polynucleotide, an oligonucleotide that hybridizes to a TRERF1 polynucleotide, an oligonucleotide that hybridizes to a GBP2 polynucleotide, an oligonucleotide that hybridizes to a GYG1 polynucleotide, an oligonucleotide that hybridizes to a CX3CR1 polynucleotide, an oligonucleotide that hybridizes to a TRERF1 polynucleotide, an oligonucleotide that hybridizes to a GBP2 polynucleotide, an oligonucleotide that hybridizes to
  • oligonucleotide that hybridizes to an TMEM63C polynucleotide an oligonucleotide that hybridizes to a SERINC5 polynucleotide, and an oligonucleotide that hybridizes to a SOX13 polynucleotide.
  • the methods and/or kits described herein includetwo or more of: an oligonucleotide that hybridizes to a TOR3A polynucleotide, an oligonucleotide that hybridizes to a NCR3 polynucleotide, an oligonucleotide that hybridizes to a ABI3 polynucleotide, an oligonucleotide that hybridizes to a C3orfl8 polynucleotide, and an oligonucleotide that hybridizes to an ENPP5 polynucleotide.
  • detecting the levels of expression of the two or more biomarkers includes performing microarray analysis, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), a Northern blot, a serial analysis of gene expression (SAGE), isothermal amplification such as LAMP or RPA, or next generation sequencing (NGS).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • SAGE serial analysis of gene expression
  • isothermal amplification such as LAMP or RPA
  • NGS next generation sequencing
  • Various methods are known for sequencing polymers composed of two essential biological building-blocks, amino acids, carbohydrates and nucleotides.
  • existing methods for peptide sequence determination include the N-terminal chemistry of the Edman degradation, N- and C-terminal enzymatic methods, and C-terminal chemical methods.
  • existing methods for sequencing oligonucleotides include the Maxam- Gilbert base-specific chemical cleavage method and the enzymatic ladder synthesis with dideoxy base-specific termination method. Any method known in the art may be used for sequencing.
  • Polynucleotides can also be analyzed by other methods including, but not limited to, northern blotting, nuclease protection assays, RNA fingerprinting, polymerase chain reaction, ligase chain reaction, Qbeta replicase, isothermal amplification method, strand displacement amplification, transcription based amplification systems, nuclease protection (Sl nuclease or RNAse protection assays), SAGE as well as methods disclosed in
  • a standard Northern blot assay can be used to ascertain an RNA transcript size, identify alternatively spliced RNA transcripts, and the relative amounts of mRNA in a sample, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art.
  • Northern blots RNA samples are first separated by size by electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, cross-linked, and hybridized with a labeled probe.
  • Nonisotopic or high specific activity radiolabeled probes can be used, including random-primed, nick- translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and
  • sequences with only partial homology may be used as probes.
  • the labeled probe e.g., a radiolabelled cDNA, either containing the full-length, single stranded DNA or a fragment of that DNA sequence may be at least 20, at least 30, at least 50, or at least 100 consecutive nucleotides in length.
  • the probe can be labeled by any of the many different methods known to those skilled in this art.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • Isotopes that can be used include, but are not limited to, 3 H, 14 C, 32 P, 35 S, 36 Cl, 35 Cr, 57 Co,
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Any enzymes known to one of skill in the art can be utilized. Examples of such enzymes include, but are not limited to, peroxidase, beta-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • Nuclease protection assays can be used to detect and quantitate specific mRNAs.
  • an antisense probe (labeled with, e.g., radiolabeled or nonisotopic) hybridizes in solution to an RNA sample.
  • An acrylamide gel is used to separate the remaining protected fragments.
  • solution hybridization is more efficient than membrane-based hybridization, and it can accommodate up to 100 qg of sample RNA, compared with the 20-30 qg maximum of blot hybridizations.
  • RNA probes Oligonucleotides and other single- stranded DNA probes can only be used in assays containing S 1 nuclease.
  • the single- stranded, antisense probe must typically be completely homologous to target RNA to prevent cleavage of the probe: target hybrid by nuclease.
  • Serial Analysis Gene Expression can also be used to determine RNA abundances in a cell sample. See, e.g., Velculescu et al., 1995, Science 270:484-7; Carulli, et al., 1998, Journal of Cellular Biochemistry Supplements 30/31:286-96; herein
  • RNA is extracted from cells.
  • RNA is converted into cDNA using a biotinylated oligo (dT) primer, and treated with a four-base recognizing restriction enzyme (Anchoring Enzyme:
  • AE oligonucleotide adapter
  • linkers are composed of: (1) a protruding single strand portion having a sequence complementary to the sequence of the protruding portion formed by the action of the anchoring enzyme, (2) a 5' nucleotide recognizing sequence of the IIS-type restriction enzyme (cleaves at a predetermined location no more than 20 bp away from the recognition site) serving as a tagging enzyme (TE), and (3) an additional sequence of sufficient length for constructing a PCR-specific primer.
  • the linker- linked cDNA is cleaved using the tagging enzyme, and only the linker- linked cDNA sequence portion remains, which is present in the form of a short-strand sequence tag.
  • amplification product is obtained as a mixture including myriad sequences of two adjacent sequence tags (ditags) bound to linkers A and B.
  • the amplification product is treated with the anchoring enzyme, and the free ditag portions are linked into strands in a standard linkage reaction.
  • the amplification product is then cloned. Determination of the clone's nucleotide sequence can be used to obtain a read-out of consecutive ditags of constant length. The presence of mRNA corresponding to each tag can then be identified from the nucleotide sequence of the clone and information on the sequence tags.
  • Quantitative reverse transcriptase PCR can also be used to determine the expression profiles of biomarkers (see, e.g., U.S. Patent Application Publication No.
  • the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
  • the two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia vims reverse transcriptase (MLV-RT).
  • AMV-RT avilo myeloblastosis virus reverse transcriptase
  • MMV-RT Moloney murine leukemia vims reverse transcriptase
  • the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling.
  • extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions.
  • the derived cDNA can then be used as a template in the subsequent PCR reaction.
  • the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity.
  • TAQMAN PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
  • Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction.
  • a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers.
  • the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe.
  • the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner.
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
  • TAQMAN RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700 sequence detection system. (Perkin-Elmer- Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
  • the 5' nuclease procedure is ran on a real-time quantitative PCR device such as the ABI PRISM 7700 sequence detection system.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer.
  • the system includes software for running the instrument and for analyzing the data. 5 '-Nuclease assay data are initially expressed as Ct, or the threshold cycle.
  • Fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction.
  • the point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).
  • RT-PCR is usually performed using an internal standard.
  • the ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
  • RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and beta-actin.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • beta-actin beta-actin
  • RT-PCR measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TAQMAN probe).
  • Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • quantitative competitive PCR where internal competitor for each target sequence is used for normalization
  • quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • Biomarker data may be analyzed by a variety of methods to identify biomarkers and determine the statistical significance of differences in observed levels of biomarkers between test and reference expression profiles in order to evaluate whether a patient is at risk of severe dengue.
  • patient data is analyzed by one or more methods including, but not limited to, multivariate linear discriminant analysis (LDA), receiver operating characteristic (ROC) analysis, principal component analysis (PCA), ensemble data mining methods, significance analysis of microarrays (SAM), cell specific significance analysis of microarrays (csSAM), spanning-tree progression analysis of density-normalized events (SPADE), and multi-dimensional protein identification technology (MUDPIT) analysis.
  • LDA multivariate linear discriminant analysis
  • ROC receiver operating characteristic
  • PCA principal component analysis
  • SAM significance analysis of microarrays
  • csSAM cell specific significance analysis of microarrays
  • SPADE spanning-tree progression analysis of density-normalized events
  • MUDPIT multi-dimensional protein identification technology
  • kits for prognosis of severe dengue in a subject wherein the kits can be used to detect the biomarkers described herein.
  • the kits can be used to detect any one or more of the biomarkers described herein, which are differentially expressed in samples from patients.
  • the kit may include one or more agents for detection of biomarkers, a container for holding a biological sample isolated from a human subject suspected of having a life-threatening condition; and printed instructions for reacting agents with the biological sample or a portion of the biological sample to detect the presence or amount of at least one biomarker in the biological sample.
  • the agents may be packaged in separate containers.
  • the kit may further include one or more control reference samples and reagents for performing an immunoassay or microarray analysis.
  • the kit includes agents for measuring the levels of two or more of two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1, TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5.
  • the two or more include DEFA4, CACNA2D2, SPON2, CACNA2D3, CHD3, GRAP2, AK5, and PTPRM.
  • the two or more include GYG1, CX3CR1, TRERF1, GBP2, TMEM63C, SERINC5, and SOX13.
  • the kit may comprise reagents for measuring up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90 or up to 100 biomarkers, including the reagents required for the analysis of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2,
  • the two or more include TOR3A, NCR3, ABI3, C3orfl8, and ENPP5.
  • the kit may include agents for detecting biomarkers of a panel including a DEFA4 polynucleotide, a CACNA2D2 polynucleotide, a SPON2 polynucleotide, a CACNA2D3 polynucleotide, an CHD3 polynucleotide, a GRAP2 polynucleotide, a AK5 polynucleotide, and a PTPRM polynucleotide.
  • the kit may include agents for detecting biomarkers of a panel including two or more of a DEFA4 polynucleotide, a GYG1 polynucleotide, a TOR3A polynucleotide, a PTPRM polynucleotide, a SPON2
  • polynucleotide a GRAP2 polynucleotide, a CACNA2D2 polynucleotide, a CACNA2D3 polynucleotide, a TMEM63C polynucleotide, a AK5 polynucleotide, a CHD3
  • polynucleotide a CX3CR1 polynucleotide, a TRERF1 polynucleotide, a GBP2
  • polynucleotide a SERINC5 polynucleotide, a SOX13 polynucleotide, a NCR3
  • kits for practicing the subject methods as described above.
  • the kit may reagents for measuring the amount of
  • PTPRM SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1,
  • the kit may include, for each RNA transcript, a sequence-specific oligonucleotide that hybridizes to the transcript.
  • the sequence- specific oligonucleotide may be biotinylated and/or labeled with an optically-detectable moiety.
  • the kit may include, for each RNA transcript, a pair of PCR primers that amplify a sequence from the RNA transcript, or cDNA made from the same.
  • the kit may include an array of oligonucleotide probes, wherein the array includes, for each RNA transcript, at least one sequence-specific oligonucleotide that hybridizes to the transcript.
  • the oligonucleotide probes may be spatially addressable on the surface of a planar support, or tethered to optically addressable beads, for example.
  • kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.
  • the subject kit may further include instructions for using the components of the kit to practice the subject method.
  • the methods and/or kits described herein include a computer implemented method for determining severe dengue risk of a patient suspected of having a life-threatening condition.
  • the computer performs steps including: receiving inputted patient data including values for the levels of one or more biomarkers in a biological sample from the patient; analyzing the levels of one or more biomarkers and comparing with respective reference value ranges for the biomarkers; calculating a severe dengue gene score for the patient based on the levels of the biomarkers, wherein a higher severe dengue gene score for the patient compared to a control subject indicates that the patient is at high risk of severe dengue; and displaying information regarding the severe dengue risk of the patient.
  • the inputted patient data includes values for the levels of a plurality of biomarkers in a biological sample from the patient.
  • the inputted patient data includes values for the levels of two or more of DEFA4, GYG1, TOR3A, PTPRM, SPON2, GRAP2, CACNA2D2, CACNA2D3, TMEM63C, AK5, CHD3, CX3CR1, TRERF1, GBP2, SERINC5, SOX13, NCR3, ABI3, C3orfl8, and ENPP5 polynucleotides.
  • the methods described herein further include information, in electronic or paper form, comprising instructions to correlate the detected levels of each biomarker with severe dengue.
  • the methods described herein may involve creating a report that shows the severe dengue gene score of the subject, e.g., in an electronic form, and forwarding the report to a doctor or other medical professional to help identify a suitable course of action, e.g., to identify a suitable therapy for the subject.
  • the report may be used along with other metrics as a diagnostic to determine whether the subject has a high risk of a disease or condition (e.g. severe dengue).
  • a report can be forwarded to a“remote location”, where“remote location,” means a location other than the location at which the image is examined.
  • a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc.
  • office, lab, etc. another location in the same city
  • another location in a different city e.g., another location in a different city
  • another location in a different state e.g., another location in a different state
  • another location in a different country etc.
  • the two items can be in the same room but separated, or at least in different rooms or different buildings, and can be at least one mile, ten miles, or at least one hundred miles apart.
  • “Communicating” information references transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network).
  • “Forwarding" an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. Examples of communicating media include radio or infra-red transmission channels as well as a network connection to another computer or networked device, and the internet or including email transmissions and information recorded on websites and the like.
  • the report may be analyzed by an MD or other qualified medical professional, and a report based on the results of the analysis of the image may be forwarded to the subject from which the sample was obtained.
  • a diagnostic system for performing the computer implemented methods described herein.
  • a diagnostic system may include a computer containing a processor, a storage component (i.e., memory), a display component, and other components typically present in general purpose computers.
  • the storage component stores information accessible by the processor, including instructions that may be executed by the processor and data that may be retrieved, manipulated or stored by the processor.
  • the storage component includes instructions for determining the severe dengue risk of the subject.
  • the storage component includes instructions for calculating the severe dengue gene score for the subject based on biomarker expression levels, as described herein.
  • the storage component may further include instructions for performing multivariate linear discriminant analysis (LDA), receiver operating characteristic (ROC) analysis, principal component analysis (PCA), ensemble data mining methods, cell specific significance analysis of microarrays (csSAM), or multi-dimensional protein identification technology (MUDPIT) analysis.
  • the computer processor is coupled to the storage component and configured to execute the instructions stored in the storage component in order to receive patient data and analyze patient data according to one or more algorithms.
  • the display component displays information regarding the diagnosis and/or prognosis (e.g., severe dengue risk) of the patient.
  • the storage component may be of any type capable of storing information accessible by the processor, such as a hard-drive, memory card, ROM, RAM, DVD, CD-ROM, USB Flash drive, write-capable, and read-only memories.
  • the processor may be any well-known processor, such as processors from Intel Corporation. Alternatively, the processor may be a dedicated controller such as an ASIC.
  • the instructions may be any set of instructions to be executed directly (such as machine code) or indirectly (such as scripts) by the processor.
  • instructions such as machine code
  • steps such as scripts
  • programs may be used interchangeably herein.
  • the instructions may be stored in object code form for direct processing by the processor, or in any other computer language including scripts or collections of independent source code modules that are interpreted on demand or compiled in advance.
  • Data may be retrieved, stored or modified by the processor in accordance with the instructions.
  • the data may be stored in computer registers, in a relational database as a table having a plurality of different fields and records, XML documents, or flat files.
  • the data may also be formatted in any computer-readable format such as, but not limited to, binary values, ASCII or Unicode.
  • the data may include any information sufficient to identify the relevant information, such as numbers, descriptive text, proprietary codes, pointers, references to data stored in other memories (including other network locations) or information which is used by a function to calculate the relevant data.
  • the processor and storage component may include multiple processors and storage components that may or may not be stored within the same physical housing. For example, some of the instructions and data may be stored on removable CD- ROM and others within a read-only computer chip. Some or all of the instructions and data may be stored in a location physically remote from, yet still accessible by, the processor. Similarly, the processor may actually include a collection of processors which may or may not operate in parallel.
  • computer is a server communicating with one or more client computers.
  • Each client computer may be configured similarly to the server, with a processor, storage component and instructions.
  • Each client computer may be a personal computer, intended for use by a person, having all the internal components normally found in a personal computer such as a central processing unit (CPU), display (for example, a monitor displaying information processed by the processor), CD-ROM, hard-drive, user input device (for example, a mouse, keyboard, touch-screen or microphone), speakers, modem and/or network interface device (telephone, cable or otherwise) and all of the components used for connecting these elements to one another and permitting them to communicate (directly or indirectly) with one another.
  • CPU central processing unit
  • display for example, a monitor displaying information processed by the processor
  • CD-ROM compact disc read-only memory
  • hard-drive for example, hard-drive
  • user input device for example, a mouse, keyboard, touch-screen or microphone
  • speakers modem and/or network interface device
  • modem and/or network interface device telephone,
  • client computers may include a full-sized personal computer, many aspects of the system and method are particularly advantageous when used in connection with mobile devices capable of wirelessly exchanging data with a server over a network such as the Internet.
  • client computer may be a wireless-enabled PDA such as a Blackberry phone, Apple iPhone, Android phone, or other Internet-capable cellular phone.
  • the user may input information using a small keyboard, a keypad, a touch screen, or any other means of user input.
  • the computer may have an antenna for receiving a wireless signal.
  • the server and client computers are capable of direct and indirect communication, such as over a network. Although only a few computers may be used, it should be appreciated that a typical system can include a large number of connected computers, with each different computer being at a different node of the network.
  • the network, and intervening nodes may include various combinations of devices and communication protocols including the Internet, World Wide Web, intranets, virtual private networks, wide area networks, local networks, cell phone networks, private networks using communication protocols proprietary to one or more companies, Ethernet, WiFi and HTTP. Such communication may be facilitated by any device capable of transmitting data to and from other computers, such as modems (e.g., dial-up or cable), networks and wireless interfaces.
  • the server may be a web server.
  • information may be sent via a medium such as a disk, tape, flash drive, DVD, or CD-ROM.
  • the information may be transmitted in a non-electronic format and manually entered into the system.
  • some functions are indicated as taking place on a server and others on a client, various aspects of the system and method may be implemented by a single computer having a single processor.
  • Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass); kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa);
  • kilobases kb
  • base pairs bp
  • nucleotides nt
  • intramuscular i.m.
  • intraperitoneal i.p.
  • subcutaneous s.c.
  • the 20-gene gene set was validated both in existing cohorts and in a prospective cohort from Colombia and demonstrated that it performed well under both the current and former WHO dengue classification methods.
  • the discovery and validation cohorts were from 8 countries on 3 continents, providing strong evidence that the 20-gene set is not modulated by the underlying genetic background of the patients or the vims strains.
  • the 20-gene dengue severity scores declined during the disease course, suggesting that infection triggered this host response.
  • the gene set was strongly associated with the progression to severe dengue and represents a predictive signature, generalizable across ages, host genetic factors, vims strains and sample sources, with potential implications for the development of a host response-based dengue prognostic assay.
  • NIH GEO and ArrayExpress Two public gene expression microarray repositories (NIH GEO and ArrayExpress) were searched for all human gene expression dengue datasets. Datasets that examined clinical cohorts of dengue infection in whole blood or PBMCs were retained for further study, and datasets that examined only dengue with no severe dengue, were done in patients on steroid treatment, were non-clinical (e.g. cell culture studies), or used on-chip two-sample arrays were excluded. The remaining 10 datasets contained 530 samples from 7 countries from both adult and pediatric patients (FIG. 2).
  • DerSimonian- Laird was used because of the previously published analysis of various random effects inverse variance models across a range of diseases (Sweeney et al., 2017) that showed DerSimonian- Laird provided compromise to identify differentially expressed genes while reducing false positives.
  • a forward search was first ran, using the Metaintegrator R package, as previously described (Haynes et al., 2017; Sweeney et al., 20l6a). Briefly, the algorithm started with the single gene with the best discriminatory power, and then at each subsequent step added the gene with the best possible increase in weighted AUC (area under the curve; the sum of the AUC for each dataset times the number of samples in that dataset) to the set of genes, until no further additions can increase the weighted AUC more than some threshold amount (here 0.005 x the total number of samples).
  • a dengue score was defined as follows: for each sample, the mean expression of the down-regulated genes is subtracted from the mean expression of the up-regulated genes to yield a dengue score.
  • the forward search was ran exhaustively, such that once a gene set had been identified, those genes were removed from the remaining pool and the forward search was ran again.
  • An arbitrary minimum threshold was set for performance of a mean AUC of 0.75 in the discovery data, which yielded three gene sets with a total of 20 genes. The entire list of 20 genes was then pooled to make a single dengue score.
  • the 20-gene set was validated in three independent clinical dengue gene expression datasets, comparing its ability to differentiate DF from DHF/DSS. Between-groups dengue score comparisons were done with the Wilcoxon rank sum test. Significance levels were set at two-tailed p ⁇ 0.05, unless specified otherwise. All computation and calculations were done in the R language for statistical computing (version 3.0.2).
  • Discharge diagnoses were also blindly classified by infectious diseases specialists according to the 1997 WHO criteria into DF, DHF, and/or DSS criteria.
  • the first venous blood sample was collected upon enrollment on the first day of presentation (FIG. 6, Panel B). Patients presenting with dengue with warning signs provided additional blood samples every 48 to 72 hours during their hospital admission. When possible, an additional sample was obtained from all patients following defervescence (1-17 weeks after the initial presentation) during a routine visit to the infectious diseases clinic (FIG. 6, Panel B). 2.5 ml of whole blood were collected in Paxgene tubes (PreAnalytiX) and stored at -80 °C. Serum samples were obtained for additional assays. Samples transport, reception, and processing were strictly controlled using personal data assistants (PDAs) with barcode scanners.
  • PDAs personal data assistants
  • rRT-PCR real-time reverse transcriptase PCR
  • Multiplexed antigen microarrays including DENV-2 vims-like particles spotted in triplicate were fabricated on pGOLD slides (Nirmidas Biotech, California) and serologic testing performed, as described (Zhang et ak, 2017). Briefly, for DENV IgG and IgM testing, each well was incubated with human sera (400 times dilution) for 40 min, followed by incubation of a mixture of anti-human IgG-IRDye680 conjugate and anti-human IgM- IRDye800 conjugate for 15 min (Vector-Laboratories, Burlingame, CA). Each well was washed between incubation procedures.
  • the biochip was then scanned with a MidaScan-IR near-infrared scanner.
  • IRDye680 and IRDye800 fluorescence images were generated, and the median fluorescence signal for each channel on each microarray spot was quantified by MidaScan software.
  • the average of the three median fluorescence signals for three spots was calculated and normalized by positive and negative reference samples through a two-point calibration.
  • Previously defined cutoffs based on mean levels +3 S.D. were used (Zhang et al., 2017).
  • DENV IgG avidity was performed as above in duplicate wells, except that following primary incubation, one well was incubated with 10 M urea for 10 min. Then, anti-human IgG-IRDye680 conjugate was applied to each well and incubated for 15 min.
  • DENV IgG avidity was calculated by dividing the normalized DENV IgG result of the sample tested with urea treatment by the normalized DENV IgG result of the sample without urea treatment. High avidity (>0.6) is indicative of a past infection, whereas low avidity ( ⁇ 0.6) is consistent with a recent infection.
  • the Biomark Microfluidic qPCR Array was used to quantify the individual transcripts of the signature at the Stanford Human Immune Monitoring Center, as previously described(Cheow et ak, 2015). 50 ng of total RNA was reverse transcribed at 50°C for 15 minutes using the High Capacity Reverse Transcription kit (ABI). Preamplification was performed on a thermocycler following addition of the TaqMan PreAmp Master Mix Kit
  • Taq polymerase reaction was initiated by bringing the sample to 95 °C for 2 minutes.
  • the cDNA was preamplified by denaturing for 10 cycles at 95 °C for 15 seconds and annealing at
  • Reagent, and preamplified cDNA were mixed and loaded into the 48.48 Dynamic Array (Fluidigm) sample inlets, followed by loading 10X Taqman gene expression assays into the assay inlets. Manufacturer’s instructions for chip priming, pipetting, mixing, and loading onto the BioMark system were followed. RT-PCR was carried out at the following conditions: 10 min at 95 °C followed by 50 cycles of 15 sec at 95°C and 1 min at 60°C. Data were analyzed using software. All reactions were performed in duplicate and Ct values were normalized to 18S RNA and beta-actin. TaqMan reagents are listed below.
  • Example 1 In silico discovery and validation of a 20-gene set predictive of severe dengue in existing cohorts
  • Different combinations of at least 2, at least 3, at least 4, at least 5, etc. of the above genes may be used in certain cases.
  • FIG. 1 Panel A depicts a schematic of the multi-cohort analysis workflow for the discovery and validation of the 20-gene set.
  • FIG. 1 Panel B depicts representative forest plots of an over-expressed (DEFA4, left) and under-expressed (PTPRM, right) genes derived in the forward searches.
  • the x-axis represents standardized mean difference between DHF/DSS and DF.
  • the size of the blue rectangles is inversely proportional to the standard error of mean in the study.
  • Whiskers represent the 95% Cl.
  • the orange diamonds represent overall, combined mean difference for a given gene. Width of the diamonds represents the 95% Cl of overall combined mean difference.
  • FIG. 1 Panel B depicts representative forest plots of an over-expressed (DEFA4, left) and under-expressed (PTPRM, right) genes derived in the forward searches.
  • the x-axis represents standardized mean difference between DHF/DSS and DF.
  • the size of the blue rectangles is inversely proportional to
  • FIG. 1 Panel C depicts ROC curves comparing patients with DF with patients with DHF and/or DSS in the 7 discovery data sets.
  • FIG. 1 Panel D depicts a representative violin plot showing the performance of the 20-gene set for separating DHF and/or DSS from DF in one of the discovery cohorts (GSE13052- GPL2700). Wilcoxon P value is shown.
  • ROC receiver operating characteristic.
  • FIG. 2 depicts publicly available datasets used for the discovery and validation of the 20-gene set.
  • FIG. 3 Panels A-T depicts forest plots of the over-expressed and under-expressed genes derived in the forward searches.
  • the x axis represents standardized mean difference between DHF/DSS and DF.
  • the size of the blue rectangles is inversely proportional to the standard error of mean in the study.
  • Whiskers represent the 95% Cl.
  • the orange diamonds represent overall, combined mean difference for a given gene. Width of the diamonds represents the 95% Cl of overall combined mean difference.
  • FIG. 4 depicts over-expressed and under-expressed genes identified in the discovery cohort via the multi-cohort analysis.
  • FIG. 5, Panels A-B depict violin plots showing the performance of the 20-gene set to separate DHF/DSS from DF in the 7 datasets of the discovery cohort (FIG. 5, Panel A) and 3 datasets of the validation cohort (FIG. 5, Panel B).
  • Example 2 Validation of the 20-gene set in a prospective new cohort of dengue patients
  • the transcripts for individual genes were quantified by high-throughput microfluidic qRT-PCR assays (Cheow et al., 2015) in samples of confirmed dengue patients.
  • Colombia cohort To account for this difference in diagnosis, the Colombia cohort data was re-analyzed after blindly classifying patients based on the 1997 WHO criteria.
  • the 20-gene set performed equally in DENV infected children and adults, suggesting that it is not affected by age-dependent variations in immune responses. It also performed well in several immunosuppressed patients and a pregnant patient with severe dengue, yet it unnecessarily predicted severe dengue in two early postpartum patients (Table S5). Larger cohort studies are required to determine its utility in these special populations.
  • FIG. 6, Panel A depicts ROC curves comparing patients with DHF and/or DSS with DF patients in the 3 existing validation data sets.
  • FIG. 6, Panel B depicts a schematic of patient enrollment and sample collection in the prospective Colombia cohort. In brackets are the number of samples available for each disease category/the number of patients in each disease category.
  • FIG. 6, Panel C depicts a ROC curve comparing patients with severe dengue (SD) with patients with dengue with (DWS) or without (D) warning signs in the Colombia cohort.
  • FIG. 6, Panel D depicts a ROC curve comparing patients with SD with patients with DWS in the Colombia cohort.
  • FIG. 6, Panel E depicts violin plots showing the performance of the 20-gene set to separate SD from D and DWS in the Colombia cohort.
  • FIG. 6, Panel F depicts a ROC curve comparing patients with DHF and/or DSS with DF (1997 WHO criteria).
  • FIG. 6, Panel G depicts violin plots showing the performance of the
  • FIG. 6 Panel H depicts dengue severity scores in longitudinal samples from individuals in the Colombia cohort over time.
  • FIG. 7, Panel A depicts ROC curves comparing patients with SD with patients with D and DWS based on hematocrit level (FIG. 7, Panel A, top left), platelet count (FIG. 7, Panel A, top right), hematocrit level and platelet count (FIG. 7, Panel A, bottom left) upon presentation in the Colombia cohort.
  • FIG. 7, Panel A, bottom right depicts a ROC curve comparing patients with SD with patients with D and DWS based on the combination of the 20-gene set with hematocrit level and platelet count upon presentation in the Colombia cohort.
  • Panel B depicts a correlation of the dengue severity score with nadir platelet count, peak hematocrit, nadir of total leukocytes, neutrophils, lymphocytes, monocytes, viral load in serum, prior exposure to dengue, and dengue serotype via linear regression analysis. Mean+SD are shown in the lower mid and right panels.
  • Table 1 Colombia cohort. Demographic, clinical, and laboratory characteristics of study population.
  • the 20-gene set predicted the progression to severe dengue early in the course of dengue infection with high sensitivity and specificity (100% and 76-79% in the Colombia cohort, respectively) (Table 3) and was robust to clinical heterogeneity of DENY infection.
  • Example 3 The 20-gene set is enriched in NK and NKT cells
  • the following gene sets (which are subsets of the 20 genes) can be used to predict severe dengue.
  • the 20 gene set is made up of the combination of these gene sets:
  • 77gpl2 mean pl2700 pl6104 pi 15615 gpl570 pll3158 2gpl570
  • Flavivirus NS3 and NS5 proteins interaction network a high-throughput yeast two-hybrid screen. BMC Microbiology 11, 234.
  • Chymase Level Is a Predictive Biomarker of Dengue Hemorrhagic Fever in Pediatric and Adult Patients. The Journal of infectious diseases 216, 1112-1121.

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Abstract

L'invention concerne un procédé basé sur l'expression génique pour le diagnostic ou le pronostic d'un patient souffrant d'une dengue sévère. L'invention concerne également un kit pour exécuter le procédé.
PCT/US2019/058336 2018-11-06 2019-10-28 Procédé de prédiction de la dengue sévère WO2020096796A1 (fr)

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CN112226525A (zh) * 2020-09-15 2021-01-15 石家庄市人民医院(石家庄市第一医院、石家庄市肿瘤医院、河北省重症肌无力医院、石家庄市心血管病医院) 重症肌无力诊断用试剂

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