WO2017066538A1

WO2017066538A1 - Combination of serotonin and a cytokine as an early, prognostic predictor of severe dengue

Info

Publication number: WO2017066538A1
Application number: PCT/US2016/056999
Authority: WO
Inventors: Liang CUI; Yie Hou LEE; Steven R. Tannenbaum
Original assignee: Massachusetts Institute Of Technology
Priority date: 2015-10-14
Filing date: 2016-10-14
Publication date: 2017-04-20

Abstract

The invention relates to a method of determining if a subject has developed or is at risk of developing dengue hemorrhagic fever, wherein the method includes determining a serotonin concentration and a cytokine concentration, e.g. an interferon-gamma concentration, in a biological sample obtained from the subject, and comparing the serotonin concentration and the cytokine, e.g. interferon-gamma, concentration in the biological sample with a control serotonin concentration and a control cytokine, e.g. interferon-gamma, concentration, wherein the presence of a difference of the serotonin and cytokine, e.g. interferon-gamma, concentrations in the biological sample as compared to the control serotonin and control cytokine, e.g. interferon- gamma, concentrations indicates the subject has developed or is at risk of developing dengue hemorrhagic fever.

Description

COMBINATION OF SEROTONIN AND A CYTOKINE AS AN EARLY, PROGNOSTIC

PREDICTOR OF SEVERE DENGUE

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 62/241,252, filed October 14, 2015. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Due to a lack of anti-dengue therapeutics and vaccines, and that patient care is purely supportive, early triage (<96 h upon onset of fever) of dengue patients in the acute, febrile phase is important for the appropriate patient management and monitoring. However, the prediction of severe dengue proven challenging based on clinical symptoms alone - plasma leakage is varied and difficult to evaluate (pleural effusion, ascites or increased gall bladder wall thickness) and daily or even more frequent determining of hematocrit levels requires hospitalization for at least 2-3 days around the defervescence period. This becomes a major hurdle during the time of a dengue outbreak within an endemic region. In addition, effective triage can only be made at the time of symptom presentation (onset of defervescence that is >96 h upon onset of fever).

Accordingly, there is a significant unmet need for a rapid and accurate assay that allows for early prediction of severe dengue.

SUMMARY OF THE INVENTION

[0003] Provided herein is a rapid, quantitative assay for the combination of serotonin and a cytokine, e.g., interferon-gamma, for the purpose of early prognosis of patients with propensity to develop severe dengue. Quantitative information of serotonin and cytokine, e.g., interferon- gamma, concentrations can be used to determine specific concentration cutoffs to derive the best sensitivity (true positive) and specificity (true negative) to predict patients likely to develop severe dengue within the first 96 hours upon onset of fever.

[0004] We implemented a high-throughput, quantitative assay through stable-isotope dilution mass spectrometry to sensitively and accurately quantify serotonin levels in sera and urine, and of which correlated significantly with the drop in platelet numbers in dengue cases. We captured interferon-gamma (IFN-γ), basic fibroblast growth factor (FGF basic), granulocyte-colony stimulating factor (G-CSF) and interleukin-8 (IL-8) as highly ranked cytokines, and interferon- gamma as a top-ranked cytokine in dengue hemorrhagic fever (DHF), reflecting the proinflammatory state of dengue infections. Using Receiver Operating Curve (ROC) analysis, the combination of serotonin and interferon-gamma produced an Area Under Curve of 0.92, with sensitivity = 77.8% and specificity = 95.8%. Cutoffs on the concentrations of serotonin and cytokine, e.g., interferon-gamma, can be changed and pre-determined to obtain optimal sensitivity and specificity of predicting risk of severe dengue based on the prevalence of disease or nature of prognosis. Taken together, the combination of serotonin and cytokine, e.g., interferon-gamma, provides quantitative, accurate metrics for the early prognosis of severe dengue.

[0005] In some embodiments, the biological sample is a serum or urine sample. In some embodiments, the biological sample is obtained from the subject within 96 hours of onset of fever (>38.0°C). In some embodiments, the method further comprises managing treatment of the subject based on the statistically significant difference. In some embodiments, the managing treatment of a subject comprises hospitalization of the subject based on the presence of the statistically significant difference. In another aspect, the invention provides a kit or device for evaluating Dengue Fever infection. In some embodiments, the kit or device comprises one or more binding partners for serotonin and/or interferon-gamma, wherein the one or more binding partners are attached to a surface. In some embodiments, the device generates an output that indicates that a subject has or is at risk for DHF. In some embodiments, the kit or device further comprises at least one control binding agent. In some embodiments, the device is a dipstick.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 depicts the positive correlation between serotonin and platelet numbers.

[0007] FIG. 2 depicts the scatter plot of serotonin in serum samples of DF/DHF patients at different stages of the infection.

[0008] FIG. 3 depicts the prognostic performance of combining serotonin and interferon- gamma with receiver operator characteristics (ROC) analysis. [0009] FIGs. 4A-4C depict the prognostic performance of (FIG. 4A) platelets alone, (FIG. 4B) serotonin only and (FIG. 4C) interferon-gamma only with receiver operator characteristic (ROC).

[0010] FIGs. 5A-5B summarize the concentration cutoffs of (FIG. 5A) interferon-gamma and (FIG. 5B) serotonin and their corresponding sensitivity and specificity in predicting severe dengue.

[0011] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Aspects of the invention relate to methods and devices for evaluating Dengue Virus infection, early prediction of DHF and DHF progression. Previous studies have attempted to develop methods for early prediction of DHF by statistical analysis of a variety of clinical laboratory indicators. For example, Potts et al. reported a classification and regression tree (CART) analysis of clinical indicators from patient cohort of 1384 dengue-infected children in Thailand (Potts JA, Gibbons RV, Rothman AL, Srikiatkhachorn A, Thomas SJ, et al. (2010) Prediction of dengue disease severity among pediatric Thai patients using early clinical laboratory indicators. PLoS Negl Trop Dis 4: e769; Potts JA, Thomas SJ, Srikiatkhachorn A, Supradish PO, Li W, et al. (2010) Classification of dengue illness based on readily available laboratory data. Am J Trop Med Hyg 83 : 781-788), while Tanner et al employed a decision tree algorithm in a 1200 patient cohort from Singapore and Vietnam (Tanner L, Schreiber M, Low JG, Ong A, Tolfvenstam T, et al. (2008) Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness. PLoS Negl Trop Dis 2: el96). Both these studies reported a subset of clinical variables that, when applied in a regression tree format, achieved high sensitivity but poor specificity. However, their usefulness in clinical applications may be limited for a number of reasons - 1) exhaustive clinical evaluation is expensive, 2) relies on daily monitoring of admitted patients, therefore not contributing to a significant reduction of hospitalization and 3) heterogeneous presentation of clinical symptoms. Importantly, because clinical variables accompany rather than precede DHF symptoms (e.g., plasma leakage), and for the reasons stated above such clinical variables are better as 'indicators' rather than 'predictors' of DHF.

[0013] Methods that rely on molecular measurements are likely to have a significant impact on prognostic capabilities of severe dengue due to greater sensitivity of molecular detection assays, the appearance of early molecular events preceding clinical manifestations, and the possibility of development of low cost measurement devices of target compounds. Brasier et al. used a logistic regression approach and reported a 3 component biomarker panel consisting of platelet count, lymphocyte count and IL-10 levels that, when measured during the first week following onset of fever, classified DF from DHF patients with an accuracy of >85% in a clinical cohort from Venezuela (Brasier AR, Ju H, Garcia J, Spratt HM, Victor SS, et al. (2012) A Three- Component Biomarker Panel for Prediction of Dengue Hemorrhagic Fever. Am J Trop Med Hyg 86: 10 341-348). However, this broad and late window of measurement makes the clinical application of these markers less than ideal, despite the seemingly high predictive power, and is likely a consequence of the sample grouping strategy employed.

[0014] Here, we demonstrate the utility of the combination of serotonin and interferon- gamma levels as accurate predictors of DHF. The biology of serotonin in dengue is specific, as shown by the significant correlation of serotonin and platelet numbers in the febrile phase, reinforcing the use of this compound in reflecting platelet drop and its attending dysfunction (FIG. 1). We implemented a high-throughput, quantitative assay through stable-isotope dilution mass spectrometry to sensitively and accurately quantify serotonin levels in sera (FIG. 2).

Meanwhile, through multiplex immunoassay and multivariate statistical analysis, we captured interferon-gamma (IFN-γ), basic fibroblast growth factor (FGF basic), granulocyte-colony stimulating factor (G-CSF) and interleukin-8 (IL-8) as highly-ranked cytokines, and interferon- gamma as a top-ranked cytokine in DHF, reflecting the pro-inflammatory state of dengue infections and the overall immune response of the patient. The combination of serotonin and interferon-gamma produced an Area Under Curve of 0.92, with sensitivity = 77.8% and specificity = 95.8% (FIG. 3) and were superior to platelets alone (FIG. 4A), serotonin alone (FIG. 4B) or interferon-gamma alone (FIG. 4C). However, interferon-gamma can be used as a first screen to capture as many patients likely to develop severe dengue (a test with high sensitivity /low specificity) and serotonin as a second screen to eliminate false positives (test with low sensitivity /high specificity) thereby keeping hospitalizations low. Taken together, the combination of serotonin with interferon-gamma not only reflects the biology of dengue, but also provides quantitative, accurate metrics for the early (<96 h from onset of fever) prognosis of DHF.

Dengue Virus

[0015] Dengue Virus causes a collection of human illnesses ranging from mild Dengue Fever (DF) to potentially lethal Dengue Hemorrhagic Fever (DFIF) and Dengue Shock Syndrome (DSS). Viral transmission to humans occurs via the mosquito species of Aedes aegypti and Aedes albopictus. Dengue is endemic to tropical and sub-tropical regions of the world and with over 50-100 million annual cases of dengue infection. Dengue virus occurs in four distinct serotypes (DENV1-4) all of which can cause severe illnesses. A patient presenting symptoms such as fever, headache, muscle and joint pain, nausea and rashes may be exhibiting signs of DF. However accurate diagnostic tests including viral culture, RT-PCR and serological tests are essential to both identify the serotype and to rule out other febrile illnesses such as malaria, typhoid, SARS, yellow fever also common in dengue-endemic regions. Although most DF patients recover after 5-7 days, in a small proportion of patients the initial febrile period is followed by a rapid onset of vascular leakage, thrombocytopenia, and hemorrhage indicating DHF. Continual loss of fluids from internal bleeding can very rapidly result in shock-like symptoms (DSS) resulting in hypotension and cardiovascular collapse, which if uncontrolled can result in death. The WHO has released guidelines for classification of dengue illnesses (Potts JA, Thomas SJ, Srikiatkhachorn A, Supradish PO, Li W, et al. (2010) Classification of dengue illness based on readily available laboratory data. Am J Trop Med Hyg 83 : 781-788), which are often seen as a spectrum of conditions varying in severity from mild DF to life-threatening DHF and DSS. In clinical practice DF patients who display thrombocytopenia (<100,000/dl), bleeding and plasma leakage are generally classified as DHF (Potts JA, Gibbons RV, Rothman AL, Srikiatkhachorn A, Thomas SJ, et al. (2010) Prediction of dengue disease severity among pediatric Thai patients using early clinical laboratory indicators. PLoS Negl Trop Dis 4: e769). However these symptoms become evident only in the critical phase of infection and currently it is not possible to distinguish DF and DHF during the early febrile stages.

[0016] The inability to reliably identify DF cases that will develop into severe DHF frequently results in over-hospitalization of Dengue patients even though none or only a small proportion of them may develop DHF, contributing significantly to the financial burden of Dengue. Due to lack of vaccines and anti-viral therapeutic strategies, optimal disease management and patient care are the most effective means of treating Dengue patients currently. The biggest challenge in Dengue management is the inability to distinguish patients with severe disease potential from those with self-limiting disease. Current methods require continual monitoring of hospitalized patients for laboratory indicators of severity including platelet count, WBC count, Hematocrit and Hemorrhagic indications. In developed countries, this results in over-hospitalization of patients with a huge economic burden while in underdeveloped nations it results in inadequate care of critically ill patients with subsequent high mortality.

[0017] In some embodiments, methods described herein are useful to determine whether a subject having symptoms of a Dengue virus infection is likely to develop severe DHF as opposed to having a self-limiting disease. In some embodiments, if the levels of serotonin and interferon- gamma described herein are indicative that a subject is more likely than not to develop severe DHF, then the subject is subsequently or immediately hospitalized. In some embodiments, a subject that is not hospitalized is monitored regularly (e.g., weekly, every 2-3 days, daily, more than once a day) to determine whether there are any indication that the infection is changing to a severe DHF. For example, the levels of serotonin and interferon-gamma described herein may be evaluated several times (e.g., weekly, every 2-3 days, daily, more than once a day) while a patient has other symptoms (e.g., a fever) associated with a Dengue virus infection. In some embodiments, serotonin and interferon-gamma described herein can be used to classify DHF and DF in subjects within about 96 hours of fever onset. This classification can be used to triage patients, e.g., by sending subjects suspected of having DHF to the hospital.

[0018] The Prospective Adult Dengue Study (PADS) is a cohort study of acutely febrile adults at a tertiary care center, Communicable Diseases Center, Tan Tock Seng Hospital. Adult patients (> 18 years) presenting with acute onset of fever (> 37.5°C) without rhinitis or other clinical alternatives were included in the study (Febrile stage, < 96 hours post onset of fever; Defervescence, Day 5-7, Convalescence, Day 21-28). Venous blood samples were collected, aliquoted and frozen at -80°C for hematological, virological and serological analysis. In the present application, by combining robust sample cohort and analytical methods, a novel set of biomarkers has been identified and is described herein that can be used to classify DF and DHF patients with a sensitivity and specificity of >77%. Because this classification can be obtained from blood measurements within the first 96 hours of onset of febrile symptoms, they are likely to be clinically useful as 'predictors' . The ability to classify patients as having DF or DHF can be used to identify subjects in need of further need of treatment, e.g., hospitalization.

[0019] Accordingly, in one aspect the invention provides novel biomarkers useful for evaluating (e.g., identifying, assisting in identifying, diagnosing, assisting in diagnosing, triaging, or assisting in triaging) a subject with a disease caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. In another aspect, the invention provides kits and devices useful for evaluating (e.g., identifying, assisting in identifying, diagnosing, assisting in diagnosing, triaging, or assisting in triaging) a subject with a disease caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. In yet another aspect, the invention provides biomarkers, kits, and devices for use in evaluation of drug efficacy during clinical trials of potential drugs and vaccines for Dengue Virus. In yet another aspect, the invention provides biomarkers, kits, and devices for use in epidemiological surveys of disease pathology of Dengue Virus. In some aspects, the invention provides methods for early identification or determination of subjects at risk for developing or having developed DHF. In some embodiments, early identification occurs at the early febrile phase (e.g., less than about 96 hours after onset of fever (>38.0°C)).

Biomarkers

[0020] In some aspects, the invention provides biomarkers for use in methods of evaluating (e.g., identifying, assisting in identifying, diagnosing, assisting in diagnosing, triaging, or assisting in triaging) a subject with a disease caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. The methods described herein can be used in combination with other risk factors in evaluating a subject. As defined herein, a biomarker is any organic molecule, e.g., metabolite, protein, DNA, RNA, adducts or any derivative thereof, or a biological entity, e.g., a cell or a virus, that can be used as an indicator of a disease state, e.g., Dengue Fever, Dengue Hemorrhagic Fever, or Dengue Shock Syndrome. The biomarkers in this invention are serotonin (serum/urine metabolite) and cytokine (e.g., interferon-gamma) .

[0021] In some embodiments, the combination of serotonin and cytokine, e.g., interferon- gamma, is used for evaluating a disease in a subject caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. In some embodiments, provided herein is a method of detecting a serotonin concentration and one or more cytokine concentration selected from interferon-gamma, basic fibroblast growth factor, granulocyte- colony stimulating factor, interleukin-8, IL10, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor-A (VEGF-A), VEGF-D, interferon alpha (IFN-alpha), tumor necrosis factor alpha (TNF alpha), beta-Nerve Growth Factor (bNGF), hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF-BB) in a subject who has developed or is at risk of developing dengue hemorrhagic fever, comprising obtaining a sample from the subject; determining the serotonin concentration in the sample by contacting the sample with a detection agent; and determining the one or more cytokine concentration in the sample by contacting the sample with a detection agent against interferon-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, interleukin-8, ILIO, BDNF, VEGF-A, VEGF-D, IFN- alpha, TNF alpha, bNGF, HGF or PDGF-BB. In some embodiments, the method further comprises comparing the serotonin concentration in the sample with a control serotonin concentration. In some embodiments, the method further comprises comparing the one or more cytokine concentration selected from interferon-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, interleukin-8, IL10, BDNF, VEGF-A, VEGF-D, IFN- alpha, TNF alpha, bNGF, HGF or PDGF-BB with one or more corresponding control cytokine concentration.

[0022] In some embodiments, the levels of serotonin and cytokine, e.g., interferon-gamma, are measured from a biological sample collected at one time point. In some embodiments, the time point is less than about 96 hours after onset of a fever in the subject (>38.0°C). In some embodiments, the expression levels of serotonin and cytokine, e.g., interferon-gamma, are measured from a biological sample collected at more than one time points (e.g., at less than about 96 hours after fever onset, at about 5-7 days after fever onset, and at about 3-4 weeks after fever onset).

[0023] The biomarkers provided herein can be detected using any of a variety of detection agents (also referred to herein as "binding partners") known in the art, including antibody or antigen-binding fragment thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, scFv, dAb fragments. The antibody or antigen-binding fragment thereof can be monoclonal, chimeric, or humanized. Detection agents also include other peptide molecules and aptamers that bind specifically to a biomarker. Methods for producing antibodies, peptide molecules, and aptamers are well known in the art.

Serotonin

[0024] In some aspects, the invention provides serotonin and uses thereof evaluating diseases caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever and Dengue Shock Syndrome. Serotonin is derived from tryptophan and primarily found in the gastrointestinal tract (GI tract), blood platelets and the central nervous system. Approximately 90% of the serotonin in human body is located in the enterochromaffin cells in the GI tract, and secreted serotonin from the enterochromaffin cells will eventually get into the blood. Blood platelets actively take up serotonin and store it. For platelet to function or to form a plug, three steps are involved: platelet adhesion, inducers release and platelet aggregation. Serotonin is released when the platelets bind to a clot, and serves as a vasoconstrictor to help regulate homeostasis and blood clotting. Serotonin is also a platelet agonist, and has effects on all three steps mentioned above. Low level of serotonin can reduce platelet adhesion by decreasing the secretion of adhesive proteins, and reduce platelet responses to inducers like ADP and thromboxane A2. Thus, decrease of serotonin can adversely affect platelet function and may contribute to the symptoms of DHF like plasma leakage.

[0025] In some embodiments, decreased level of serotonin at the early febrile phase (e.g., less than about 96 hours after onset of fever) compared to control expression levels indicate a subject at risk of developing or having developed DHF. [0026] Cytokines: Interferon-gamma (IFN-γ), Basic Fibroblast Growth Factor (FGF basic), Granulocyte-Colony Stimulating Factor (G-CSF), Interleukin-8 (IL-8), IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF and PDGF-BB

[0027] In some aspects, the invention provides cytokines, e.g., interferon-gamma, and uses thereof evaluating diseases caused by Dengue Virus, e.g., Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. As used herein, cytokines refer to cell-signaling molecules, such as proteins, peptides or glycoproteins. In some aspects, cytokines refer to subclasses of cell-signaling molecules, such as interleukins, lymphokines, chemokines and interferons. In some embodiments, increased level of interferon-gamma at the early febrile phase (e.g., less than about 96 hours after onset of fever) compared to control expression levels indicate a subject at risk of developing or having developed DHF.

Controls and Control levels

[0028] In some embodiments, methods provided herein comprise measuring the levels of serotonin and cytokine, e.g., interferon-gamma, in a biological sample and then comparing that level to one or more control levels. In some embodiments, the comparing identifies a subject at risk of developing or having developed DHF. As defined herein, a control level is a level of the same biomarker in a biological sample from a control subject or subjects. A control subject may be a normal, healthy subject that shows no Dengue virus-associated symptoms. Dengue virus- associated symptoms include fever, headache, muscle and joint pain, nausea, rashes, vascular leakage, thrombocytopenia, hemorrhage, hypotension and cardiovascular collapse. A control subject may be a subject that presents with one or more Dengue virus-associated symptoms. The control subject may be a subject diagnosed with or suspected of having Dengue Fever. The control subject may be a subject with Dengue Fever that does not develop or is not at risk for developing Dengue Hemorrhagic Fever. In some embodiments, a control level is a predetermined level from a control subject or subjects, such that the control level need not be measured every time the methods described herein are performed.

Samples [0029] Biological samples, as used herein, refer to samples taken or derived from a subject (e.g., a subject who has developed or is at risk of developing dengue hemorrhagic fever).

Examples of biological samples include tissue samples or fluid samples. Examples of biological fluid samples are blood, plasma, serum, urine, saliva, tears, and other bodily fluids. In some embodiments, the methods described herein comprise obtaining or providing a biological sample. In some embodiments the biological sample is blood or serum. In some embodiments, the biological sample is blood. In some embodiments, the biological sample is serum. In some embodiments, the biological sample is collected at one time point. In some embodiments, the biological sample is collected at less than about 96 hours after fever onset in the subject (>38.0°). In some embodiments, the biological sample is collected at more than one time point (e.g., at less than about 96 hours after fever onset, at about 5-7 days after fever onset, and at about >7 days after fever onset).

Subjects

[0030] A subject is preferably a human. A subject may be an adult or a child. A subject may be a patient. A subject may present with one or more Dengue virus-associated symptoms.

Dengue virus-associated symptoms include, but are not limited to, headache, muscle and joint pain, nausea and rashes. A subject may already be known or suspected of having a Dengue virus infection. Determining if a subject has Dengue virus infection can be accomplished by methods well-known in the art, e.g., viral titer or serology (see, e.g., Dengue hemorrhagic fever:

diagnosis, treatment, prevention and control. Geneva: World Health Organization, 1997). In some embodiments, a subject has or is suspected of having a Dengue virus infection. In some embodiments, a subject is suspected of having a Dengue virus infection. In some embodiments, a subject has a Dengue virus infection.

[0031] It may be desirable to distinguish between subjects with primary infection or secondary infection. Accordingly, in some embodiments, a subject has or is suspected of having a primary Dengue virus infection. In some embodiments, a subject has or is suspected of having a secondary Dengue virus infection (e.g., a subject who has been previously infected with one Dengue virus serotype and now has or is suspected of having another infection with a different Dengue virus serotype). In some embodiments a subject has or is suspected of having a primary or secondary infection. Primary and secondary Dengue infections can be distinguished from each other using assays known in the art, e.g., a haemagglutination inhibition (HI) assay, an IgM antibody capture ELISA, or an IgG avidity assay (see, e.g., De Souza VA, Fernandes S, Araujo ES, Tateno AF, Olivera OM. Use of an immunoglobulin G avidity test to discriminate between primary and secondary dengue virus infections. J Clin Microbiol . 2004 Apr ; 42: 1782-1784; Matheus S, Deparis X, Labeau B, Lelarge J, Movran J, Dussart P. Discrimination of primary and secondary dengue virus infection by an immunoglobulin G avidity test using a single acute phase serum sample. J Clin Microbiol. 2005; 43 : 2793-2797; Dengue hemorrhagic fever: diagnosis, treatment, prevention and control. Geneva: World Health Organization, 1997; Chakravarti A, Parthsarathy P, Chakravarti A Evaluation of a rapid immunochromatographic test in the diagnosis of dengue fever. Indian J Pathol Microbiol . 2003; 46(1): 127-8; and Chakravarti A, Matlani M, and Kumar A. Discrimination between primary and secondary dengue infection by using an immunoglobulin G avidity test. Dengue Bulletin. 2008; 32: 67-72). In some embodiments, a subject has or is at risk of developing DHF. In some embodiments, a subject is at risk of developing DHF. In some embodiments, a subject has or is suspected of having DHF. Symptoms of DHF include, but are not limited to onset of vascular leakage, thrombocytopenia, and hemorrhage.

Statistical Significance

[0032] In some embodiments, the methods described herein comprise determining whether a statistically significant difference exists between the levels of serotonin and interferon-gamma as described herein and one or more control levels. In some embodiments, the statistically significant difference indicates that a subject is at risk of developing or has developed DHF. In some embodiments, determining a statistically significant difference comprises performing a statistical test. The statistical test can be one known in the art or described herein, e.g., student's T-test, Chi-squared test, analysis of variance (ANOVA) test, Willcoxon rank sum test, Kruskal- Wallis test, or Pearson product-moment correlation coefficient test. It is to be appreciated that the statistical test can be calculated using a processor, computer, or other calculating device.

[0033] In some embodiments, the methods described herein comprise generating a statistical report or statistically significant profile. In some embodiments, the method comprises determining whether a statistically significant difference exists between the levels of serotonin and interferon-gamma and control levels, wherein the presence of a statistically significant difference is indicative of a subject at risk of developing or having developed DHF. In some embodiments, the method comprises: a) determining levels of serotonin and interferon-gamma and b) determining whether a statistically significant difference exists between the levels of serotonin and interferon-gamma and control levels, wherein the presence of statistically significant difference is indicative of a subject at risk of developing or having developed DHF.

[0034] In some embodiments, the one or more biomarkers further comprise white blood cell count (WBC), red blood cell count (RBC), blood hemoglobin (HGB), hematocrit (HCT), macrophage cell volume (MCV), mean corpuscular haemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), lymphocyte percentage (LYMPH%), lymphocyte count (LYMPH), mixed cell distribution (MXD), neutrophil percentage ( EUT%), neutrophil count (NEUT), red blood cell distribution width-coefficient of variation (RDW-CV), and viral titers. In some embodiments, the method further comprises measuring the levels of one or more biomarkers from a biological sample. In some embodiments, the method further comprises performing a statistical test. In some embodiments, the method further comprises obtaining a biological sample. In some embodiments, the method further comprises identifying a subject at risk of developing or having developed DHF. In some embodiments, the method further comprises managing treatment of a subject based on the statistically significant difference. In some embodiments, the managing treatment of a subject comprises hospitalization of the subject based on the statistically significant difference.

Kits and Devices

[0035] In some embodiments, the invention provides kits and devices for evaluating (e.g., identifying, assisting in identifying, diagnosing, assisting in diagnosing, triaging, detecting or assisting in triaging) a subject as described herein (e.g., a subject having developed DHF or at risk of developing DHF). In some embodiments, the kit or device comprises a first group of binding partners for detecting the biomarkers discussed herein. In some embodiments, a binding partner is any molecule that binds specifically to a biomarker. In some embodiments, the binding partner is an antibody or antigen-binding fragment thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, scFv, or dAb fragments. Methods for producing antibodies and antigen-binding fragments thereof are well known in the art (see, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), WO2006/040153, WO2006/122786, and WO2003/002609). Binding partners also include other peptide molecules and aptamers that bind specifically to a biomarker. Methods for producing peptide molecules and aptamers are well known in the art (see, e.g., published US Patent Application No. 2009/0075834, US Patent Nos. 7435542, 30 7807351 and 7239742).

[0036] In some embodiments, the binding partner(s) are bound to a surface. The binding partners may be bound to the surface covalently or non-covalently. The binding partners may be bound directly to the surface, or may be bound indirectly, e.g., through a linker. Examples of linkers, include, but are not limited to, carbon-containing chains, polyethylene glycol (PEG), nucleic acids, monosaccharide units, and peptides. The surface can be made of any material, e.g., metal, plastic, paper, or any other polymer, or any combination thereof. In some embodiments, the device is a dipstick. In some embodiments, the surface comprises microfluidic channels. In some embodiments, the kit or device comprises binding partners for serotonin and interferon-gamma. In some embodiments, the kit or device comprises one or more control binding agents. Examples of suitable control binding agents include, but are not limited to, a serum albumin binding partner.

[0037] In some embodiments, the kit or device comprises a second group of binding partners. In some embodiments, the second group of binding partners is washed over the biomarkers bound to the first group of binding partners. In some embodiments, the second group of binding partners comprises a detectable label (e.g., a flurophor, a biotin, or an enzyme). In some embodiments, the second group of binding partners are antibodies or antigen binding fragments thereof. In some embodiments, the kit or device comprises a secondary antibody or antibodies comprising a detectable label, wherein the secondary antibody or antibodies that binds to the second group of binding partners.

[0038] In some embodiments, the kit or device generates an output that indicates that a subject has or is at risk for developing DHF. Examples of outputs include, but are not limited to, appearance of one or more indicators, a change in one or more indicators, or disappearance of one or more indicators. Suitable indicators include, but are not limited to, a detectable chemical, e.g., fluorescent dyes or colorimetric dyes. The indicators may be measured, e.g., by eye or using a machine such as a spectrometer, a spectroradiometer, or a spectrocolorimeter. Indicators may be positive, e.g., indicating an increased or elevated level of a biomarker compared to a control, or negative, e.g., indicating a decreased or low level of a biomarker compared to a control. In some embodiments, the kit or device contains at least one positive indicator. In some embodiments, the kit or device contains at least negative indicator. In some embodiments, the kit or device contains at least one positive indicator and at least one negative indicator. By way of a non-limiting example, the device could be a dipstick that can detect serotonin and interferon- gamma.

[0039] As described herein, serotonin is expected to be negative or low in subjects that have or are at risk of developing DHF compared to a subject that has DF. Conversely, interferon- gamma is expected to be high or positive in subjects that have or are at risk of developing DHF compared to a subject that has DF. Thus, serotonin would serve as a negative indicator, while interferon-gamma would serve as a positive indicator of a subject having or being at risk of developing DHF.

[0040] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0041] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of determining if a subject has developed or is at risk of developing dengue hemorrhagic fever, the method comprising:

determining a serotonin concentration and an interferon-gamma (IFN-gamma), basic fibroblast growth factor, granulocyte-colony stimulating factor, interleukin-8 (IL- 8), ILIO, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor-A (VEGF-A), VEGF-D, IFN-alpha, tumor necrosis factor alpha (TNF alpha), beta-Nerve Growth Factor (bNGF), hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF-BB) concentration in a biological sample obtained from the subject; and

comparing the serotonin concentration in the biological sample with a control serotonin concentration and comparing the IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, ILIO, BDNF, VEGF-A, VEGF-D, IFN- alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration in the biological sample with a control IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, ILIO, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration,

wherein the presence of a decrease of the serotonin concentration in the biological sample as compared to the control serotonin concentration and the presence of a difference of the IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, ILIO, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration in the biological sample as compared to the control IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, ILIO, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration indicates the subject has developed or is at risk of developing dengue hemorrhagic fever.

2. The method of claim 1, wherein the biological sample is obtained from the subject at less than about 96 hours after onset of a fever of 38.0°C or higher.

3. The method of claim 1, wherein the biological sample is obtained from the subject at about 5 to 7 days after onset of a fever of 38.0°C or higher.

4. The method of claim 1, wherein the biological sample is obtained from the subject at about greater than 7 days after onset of a fever of 38.0°C or higher.

5. The method of claim 1, wherein the serotonin concentration in the biological sample is lower than that of the control serotonin concentration and the interferon-gamma concentration in the biological sample is higher than that of the control interferon-gamma concentration.

6. The method of claim 1, wherein the biological sample is a tissue sample or a fluid sample obtained from the subject.

7. The method of claim 1, wherein the biological sample is blood or serum obtained from the subject.

8. The method of claim 1, wherein the subject is a human that has or is suspected of having a dengue virus infection.

9. The method of claim 8, wherein the dengue virus infection is a primary dengue virus infection or a secondary dengue virus infection.

10. The method of claim 1, wherein the control serotonin concentration and the control IFN- gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, ILIO, BD F, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB

concentration are obtained from at least one control subject wherein the control subject is a healthy subject that shows no dengue virus associated symptoms, a subject that presents with one or more dengue virus associated symptoms, a subject that is diagnosed with or suspected of having dengue fever, or a subject with dengue fever that does not develop into dengue hemorrhagic fever or is not at risk for developing dengue hemorrhagic fever.

11. The method of claim 10, wherein the dengue virus associated symptoms are at least one of fever, headache, muscle pain, joint pain, nausea, rash, vascular leakage,

thrombocytopenia, hemorrhage, hypotension and cardiovascular collapse.

12. The method of claim 1, wherein the serotonin and interferon-gamma concentrations of the biological sample produce an area under curve of 0.92, using receiver operator curve analysis, with a sensitivity of greater than 77% and a specificity of greater than 95%.

13. The method of claim 1, wherein the method further comprises measuring the level of at least one biomarker from the biological sample, wherein the biological biomarker is at least one of white blood cell count, red blood cell count, blood hemoglobin, hematocrit, macrophage cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, lymphocyte percentage, lymphocyte count, mixed cell distribution, neutrophil percentage, neutrophil count, red blood cell distribution width- coefficient of variation, and viral titers.

14. The method of claim 1, wherein the method further comprises:

managing treatment of the subject based on the decrease of the serotonin concentration of the biological sample as compared to the control serotonin concentration and the difference of IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration of the biological sample as compared to the control IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB concentration.

15. The method of claim 14, wherein the managing treatment comprises hospitalization of the subject.

16. A device, comprising:

i) a surface;

ϋ) a first binding partner; iii) a second binding partner; and

iv) optionally, at least one of a control binding agent and a third or subsequent binding partner;

v) wherein the first and second binding partners are bound to the surface covalently or non-covalently,

vi) wherein the first and second binding partners are bound directly to the surface or indirectly to the surface via a linker, and

vii) wherein the first binding partner is a binding partner for serotonin and the second binding partner is a binding partner for interferon-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, IL-8, IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB.

17. The device of claim 16, wherein the control binding agent is a serum albumin binding partner and the third or subsequent binding partner is a detectable label.

18. The device of claim 16, wherein the device generates an output that indicates that a

subject has or is at risk for developing dengue hemorrhagic fever.

19. The device of claim 16, wherein the device is a dipstick and the dipstick comprises a positive indicator for interferon-gamma and a negative indicator for serotonin.

20. A method of detecting a serotonin concentration and one or more cytokine concentration selected from interferon-gamma (IFN-gamma), basic fibroblast growth factor, granulocyte-colony stimulating factor, interleukin-8, ILIO, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor-A (VEGF-A), VEGF-D, IFN-alpha, tumor necrosis factor alpha (TNF alpha), beta-Nerve Growth Factor (bNGF), hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF-BB) in a subject who has developed or is at risk of developing dengue hemorrhagic fever, comprising:

i) obtaining a sample from the subject who has developed or is at risk of developing dengue hemorrhagic fever; ii) determining the serotonin concentration in the sample by contacting the sample with a detection agent; and

iii) determining the one or more cytokine concentration in the sample by

contacting the sample with a detection agent against IFN-gamma, basic fibroblast growth factor, granulocyte-colony stimulating factor, interleukin-8, IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB.

21. The method of claim 20, further comprising comparing the serotonin concentration in the sample with a control serotonin concentration.

22. The method of claim 20, further comprising comparing the one or more cytokine

concentration selected from IFN-gamma, basic fibroblast growth factor, granulocyte- colony stimulating factor, IL-8, IL10, BDNF, VEGF-A, VEGF-D, IFN-alpha, TNF alpha, bNGF, HGF or PDGF-BB with one or more corresponding control cytokine

concentration.