Viral biomarker assay

Abstract

The invention is in the field of diagnosis of acute infections, and discrimination between infections by different types of pathogen. More specifically, this invention relates to a quantitative assay for their detection by Multiplexed Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) methods.

Description

[0001] VIRAL BIOMARKER ASSAY

[0002] FIELD OF THE INVENTION

[0003] The invention is in the field of diagnosis of acute infections, and discrimination between infections by different types of pathogen. More specifically, this invention relates to a quantitative assay for their detection by Multiplexed Liquid Chromatography Tandem Mass Spectrometry (LC-MS / MS) methods

[0004] BACKGROUND

[0005] Conventional methods for differentiation of acute infections relying on pathogen identification through culture, polymerase chain reaction or antigen detection are time-consuming and / or insensitive, leading to diagnostic delays that result in inappropriate antimicrobial prescription and infection transmission. There is therefore need for in novel biomarkers of infection classes and assays for the biomarkers that can better guide therapeutic and infection control decisions in realtime. Metabolomics technologies for large-scale characterisation of low-molecular-weight metabolites have the potential to aid discovery of novel biomarkers of infectious diseases, metabolic profiling of biofluids has produced candidate biomarkers in only a small number of infectious states. One study identified a two-metabolite serum signature differentiating infected from non-infected patients within a systemic inflammatory response syndrome cohort (Neugebauer et al. (2016) Grit Care Med 44, 1649-1662.). Metabolomic interrogation of cerebrospinal fluid from patients with meningitis was able to differentiate between M. tuberculosis and other infectious causes (Zhang et al. (2019) Infect Genet Evol 68, 253-264). Wang et al. examined the lipidome of 40 patients in a paediatric cohort prior to the COVID-19 pandemic and identified a 3-lipid signature that discriminated bacterial from viral infection (Wang et al. (2019) Sci Rep 9, 17714). A number of more recent studies report metabolic differences between patients with and without SARS-CoV-2 infection, but comparator groups did not include bacterial infections.

[0006] Currently, most biomarkers in the infection diagnosis field aim to detect bacterial infection. The available viral infection diagnosis tests can take up to 48 hours to return a result and are limited in terms of accuracy. At present, all diagnostic tests for viruses are targeted at specific, known pathogens. Traditional viral diagnostics that rely on PCR take several hours to days to return a result. Further limitations of PCR include: one needs to know which pathogen is being targeted, and a PCR test cannot distinguish between an active viral infection and transient virus carriage and false positives may be obtained from detection of remnants from old infections. SUMMARY

[0007] The invention provides a method for quantifying the amount of 3'-deoxy-3',4'-didehydro-cytidine (ddhC) in a sample from a subject, which can be used e.g. in diagnosing active viral infections using 3'-deoxy-3',4'-didehydro-cytidine (ddhC) as a biomarker. ddhC is derived from ddhC- triphosphate (ddhCTP), a product of the enzyme Viperin that is involved in antiviral response. ddhC is present in elevated levels in the serum of patients with viral infections. By employing targeted tandem mass spectrometry analysis, the levels of ddhC in patient serum can be absolutely quantified. The presence of ddhC indicates an active viral infection, enabling its use in viral infection diagnosis. The assay described herein for quantitative measurement of this analyte which is specifically associated viral infections in a single sample using mass spectrometry has not been previously disclosed or demonstrated.

[0008] For the first time, the invention utilises mass spectrometry-mediated quantification of ddhC to enable the rapid and accurate detection of active viral infections with high accuracy. The assay can return a result in 12 minutes or less. The invention is suitable for high-throughput screening of large numbers of samples without requiring time-consuming and expensive preparation (e.g. a separate filtration step to remove lipids) before being subjected to the method of the invention. This invention is the first of its kind to provide an absolute quantification of ddhC levels in patients, surpassing the limitations of existing diagnostic methods. Moreover, it has the unique ability to distinguish between viral and non-viral infections (such as bacterial infections), making it an invaluable tool for improving diagnostic efficiency in clinical settings. Of particular importance is the ability of the invention to detect ddhC as a pan-viral marker. Since ddhC is a non-specific biomarker of active viral infection, the method does not need to be adjusted to be able to detect new types or strains of viral infections - unlike existing techniques. This makes the method an invaluable tool in applications ranging from point of care diagnostics and pandemic preparedness to agricultural applications. By combining precision and distinction, this invention holds great promise for revolutionising viral infection diagnostics.

[0009] The inventors have found that 3'-Deoxy-3',4'-didehydro-cytidine (ddhC), a free base of the antiviral molecule ddhC-triphosphate (ddhCTP), is detectable in serum of patients with an active viral infection. An assay has been developed which uses liquid chromatography combined with mass spectrometry to identify viral infections. The assay method provides accurate and precise ddhC quantification by comparing the results obtained in native sample matrix to surrogate and zero matrices.

[0010] The invention provides a method for determining by mass spectrometry the quantity of ddhC present in a biological sample from a subject comprising (i) preparing the sample for mass spectrometry analysis; (ii) adding internal standard comprising isotope labelled ddhC to the sample; (iii) obtaining a one or more reference samples; and (iv) assaying the sample and the one or more reference samples for the presence of ddhC using mass spectrometry and thereby determining the quantity of ddhC present in the biological sample. The mass spectrometry is preferably liquid chromatography - tandem mass spectrometry (LC-MS-MS).

[0011] The method for determining the quantity of ddhC present in a biological sample can be used in a method of diagnosis. A method for diagnosing a viral infection in a subject is provided comprising: i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the methods described herein, and ii) diagnosing a viral infection if ddhC is greater than a particular threshold. A method for diagnosing a viral infection in a subject is provided comprising: i) determining by mass spectrometry the absolute quantity and / or concentration of ddhC present in a biological sample from a subject according to the methods described herein, and ii) diagnosing a viral infection if ddhC is greater than a particular threshold.

[0012] The method for determining the quantity of ddhC present in a biological sample can be used in a method of treatment. A method for treating a viral infection in a subject is provided, comprising i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the methods described herein, ii) diagnosing a viral infection if absolute quantity of ddhC is greater than a particular threshold; and (iii) administering an antiviral agent to the subject and / or withholding antibacterial agents from the subject. The method for determining the quantity of ddhC present in a biological sample can be used in a method for determining the severity of a viral infection. A method for determining the severity of a viral infection is provided, comprising i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the methods described herein, and ii) the severity of the viral infection is determined using the quantity of ddhC. The method for determining the quantity of ddhC present in a biological sample can be used in a method for assessing the efficacy of a treatment for a viral infection in a subject. Also provided are a kit for determining the concentration of ddhC present in a biological sample, a kit for use in the method of diagnosis, a kit for use in the method of treatment, a kit for use in the method of determination of the severity of a viral infection and a kit for assessing the efficacy of a treatment for a viral infection in a subject. Kits are also provided.

[0013] Brief Description of the Drawings

[0014] FIGURE 1 shows extracted ion chromatograms of the quantifier transitions of13Cs-ddhC (panel A) and ddhC (panel B) from a selected reaction monitoring (SRM) experiment.13Cs-ddhC is a heavy analogue of ddhC where the carbon atoms in the ribose ring are13C carbon.13Cs-ddhC is a standard that is synthesised to account for matrix effect in the absolute quantification of ddhC in biological samples. The X-axes represent time in minutes (min), ranging from 1 min to 1.3 min, with intervals of 0.05 min. The Y-axes represent the relative intensity of ion signals, ranging from 0% to 100%. The relative intensity reflects the abundance or strength of the detected ions at different time points during the chromatographic analysis.13Cs-ddhC was analysed with a parent ion m / z of 231.07 and a quantifier ion m / z of 94.97, and ddhC was analysed with a parent ion m / z of 225.97 and a quantifier ion m / z of 112.04. Both compounds displayed narrow elution peaks observed at 1.14min.

[0015] FIGURE 2 shows the extracted ion chromatogram of ddhC ([M+Na]+m / z 248.065) in the hydrophilic interaction liquid chromatography (HILIC) profiling in positive ionisation mode of different solid phase extraction (SPE) solutions. SPE is used to selectively retain ddhC from healthy controls biofluid samples to be used as analyte-free matrices for use as calibration standards and quality control standards for clinical analysis. In order, the panels represent time- of-flight chromatography - mass spectrometry profiles of different SPE fractions, namely the ACN elution (A), WaterACN elution (B), Water wash (C), HLB load (D), and long term reference (LTR) serum (E) before SPE. The X-axes represent time in minutes (min), ranging from 1 .36 min to 3.06 min. The Y-axis represent the relative intensity of ion signals, ranging from 0% to 100%. Numerical labels represent peak elution time. E shows peak ddhC elution at 1.87 min and 1.89 min. In D, a diminished signal is observed at 1.87 min which indicates that ddhC was selectively retained from LTR serum during the loading step of the sample onto the cartridge. Importantly, apart from ddhC depletion, metabolic profile is preserved between E and D. C shows a peak signal at 1.88 min upon washing with water. This signal is not observed in B and A. This indicates that ddhC is completely eluted upon washing with water. Thus, the serum eluate collected during the loading step on the SPE cartridge represents the closest composition to the original serum deprived of ddhC.

[0016] FIGURE 3 shows a comparison of total ion current chromatograms (TIC) obtained from global HILIC profiling in positive ionisation mode (HPOS). Panel A displays the TIC of the LTR serum load fraction collected from the HLB oasis cartridge. Panel B displays the TIC of the test LTR serum. The X-axes represent time (min), ranging from 0.4min to 7.2min. The Y-axis represent the relative intensity of ion signals, ranging from 0% to 100%. Numerical labels represent peak elution time (top) and m / z ratio (bottom). The data is comparable, with corresponding peaks of signal intensity observed across the whole chromatogram. FIGURE 4 shows the calibration curves for ddhC acquired in water and SPE-treated serum. The residual plot and response plots for water are shown in panels A and B, respectively. The residual plot and response plots for SPE-treated serum are shown in panels C and D, respectively. The X-axes represent ddhC concentration in ng / mL, ranging from 0 to 2000 ng / mL in intervals of 200ng / mL. Regression analysis shows very high r and r2values from water (r = 0.998944; r2= 0.997888) and SPE-treated serum (r = 0.999361 ; r2= 0.998722). Additionally, the results show that the calibration curves acquired in water and in SPE-treated serum samples yielded almost identical slope and intercept values. Water had slope and intercept values of 0.024 and 0.005, respectively, and SPE-treated serum had slope and intercept values of 0.026 and 0.004, respectively. This indicates that the calibration is not significantly affected by the differences in matrices.

[0017] FIGURE 5 shows the results of a parallelism study of standard addition (black diamonds) and surrogate matrix (black circles) calibration curves. The X-axis represents ddhC concentration range from 0 to 65ng / mL. The Y-axis of the graph is the response value measured in Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) analysis as the ratio of the peak area of ddhC calibration solution and the peak area of the13Cs ddhC used as an internal standard added to each reference and study sample at a fixed concentration.

[0018] FIGURE 6 shows the chemical structure of a suitable ddhC labelled analogue to use as an internal standard when determining the quantity of ddhC present in a biological sample. The internal standard may have one or more atoms in the ribose ring (numbered 1’-5’ and marked with an asterisk) labelled with a heavy carbon isotope (i.e.,13Cs). Example 2 details the use of ddhC consisting of13Cs atoms in all asterisked positions in the ribose ring.

[0019] FIGURE 7 shows ddhC concentrations measured in influenza A and SARS-CoV-2 human challenge participants presented as aggregate data. Each graph displays average data for six participants - symptomatic (top right when viewed in landscape orientation), asymptomatic (bottom right when viewed in landscape orientation), and uninfected (left when viewed in landscape orientation). ddhC concentrations increased in the first few days of viral infection, and then decreased to baseline levels. This pattern was observed in symptomatic as well as asymptomatic / paucisymptomatic infection. In uninfected participants, no ddhC response was measured. Data are presented as mean +- SEM.

[0020] FIGURE 8 shows ddhC concentrations measured in serum or plasma samples from several different viral and bacterial infections (4 patients with influenza A, 2 patients with influenza B, 6 patients with adenovirus, 2 patients with dengue virus, 2 patients with HSV1 , 3 patients with measles, 4 patients with parainfluenza, 3 patients with respiratory syncytial virus, 9 patients with SARSC0V2, 16 patients with Escherichia coli, 5 patients with Enterococcus sp, 2 patients with Pseudomonas sp, 9 patients with Staphylococcus aureus, 7 patients with Streptococcus sp, and 14 uninfected patients). Samples were obtained from infection biobanks. Data are presented as mean +- SEM.

[0021] FIGURE 9 shows ddhC concentrations measured in cerebrospinal fluid samples obtained from a clinical diagnostic laboratory. N=26 had a viral pathogen detected, n=26 had no viral pathogen detected (of which n=13 had a bacterial pathogen detected). ddhC concentrations were measured. Viral samples had higher ddhC concentrations than non-viral samples. Data are presented as mean +- SEM.

[0022] FIGURE 10 shows ddhC concentrations which were measured in cerebrospinal fluid samples from participants with different pathogens. Lower ddhC levels were observed in cerebrospinal fluid samples with bacterial pathogens, or with no pathogen detected. Data are presented as mean +- SEM.

[0023] FIGURE 11 shows ddhC concentrations which were measured in urine samples. The urine samples were obtained from a clinical diagnostic laboratory (n=5 viral infections including influenza B, SARSCOV2 and respiratory syncytial virus; n=5 non-viral including Moraxella sp, Escherichia coli, Streptococcus sp, and uninfected). Urinary ddhC concentration was measured and normalized for urinary creatinine concentration and expressed as a urine ddhC : creatinine ratio (ng / pmol). Data are presented as mean +- SEM.

[0024] FIGURE 12 shows ddhC concentrations which were measured in saliva samples from n=6 viral infections (3 influenza A and 3 influenza B) and n=3 uninfected controls. Data are presented as mean +- SEM.

[0025] FIGURE 13 shows ddhC concentrations which were measured over time in n=11 pigs challenged with H1 N1 influenza A. ddhC concentrations were observed to rise initially and then fell to near baseline. Data are presented as mean +- SEM.

[0026] Detailed Description

[0027] As can be appreciated in the art, the various aspects and embodiments described below can be used in any combination without limitation. The mass spectrometry is typically liquid chromatography tandem mass spectrometry (LC-MS-MS). LC-MS / MS methods are highly desirable because LC-MS / MS methods provide both absolute structural specificity for the target biomarker and relative or absolute quantification of the target biomarker concentration when suitable heavy isotope internal standards are employed. In contrast to immunoassays, LC-MS / MS does not generally require the manufacturing of biologies.

[0028] The assay works with readily accessible body fluids, e.g. blood plasma, serum, urine or saliva. A desirable assay needs to have sufficient throughput and be sufficiently cost-effective to allow it to be used for routine diagnosis at a population level and to be used for clinical studies where multiple assays need to be carried out for individual patients over the course of treatment, which the current assay provides. The developed chromatographic method, based on hydrophilic interaction liquid chromatography (HILIC), efficiently retains and elutes the target molecule, avoiding interferences from other constituents of the sample, such as for instance phospholipids in blood plasma and serum. The methods of the invention are therefore particularly suitable for high-throughput screening of large numbers of samples. Time-consuming and expensive preparation of samples (e.g. a separate filtration step to remove lipids) is not required. Other methods known in the art, even if they do not employ a separate filtration step to remove lipids, are not suitable for use in high-throughput screening. For example, related methods known in the art (specialised methods for characterising the severity of a specific viral infection e.g. COVID- 19), require reversed-phase chromatography for separation of the panel of analytes including ddhC. Importantly, reversed-phase chromatography can be unsustainable for lipid- and proteinrich samples such as blood due to the (unpredictable) accumulation of lipids on the reversed- phase column unless additional steps for their removal are applied during sample preparation. Lipids can accumulate in reversed-phase columns, block them, and prevent them from functioning properly. Lipids are strongly retained under reversed-phase chromatography conditions and are highly ionizable in electrospray ionization (ESI). Consequently, complex and neutral lipid species can accumulate in a reversed-phase chromatography system during analyses of blood serum and plasma, eluting unpredictably from the column and exhibiting suppressive effects on the ionization of other metabolites. The hydrophilic interaction chromatography (HILIC) methods of the present invention solve this problem by retaining and eluting efficiently lipids containing in the samples. Proteins are removed by precipitation with organic solvent. This makes the methods of the present invention particularly suitable for high-throughput screening of large numbers of samples. Reversed-phase chromatography methods known in the art are also problematic because (polar) ddhC elutes early on, making it extremely difficult to separate from other polar analytes that are often found in biological samples, e.g. biological metabolites. The simultaneous elution of multiple compounds in a short retention time window in reversed-phase chromatographic methods known in the art can result in interference in their mass spectrometric detection such as MS / MS crosstalk - an undesirable phenomenon leading to the interferences in measurement of analytes when the ions from one multiple reaction monitoring (MRM) transition are not cleared completely from collision cell before next transition is monitored. Preferably, the sample may not be processed to remove lipids prior to assaying the sample for the presence of ddhC. The present invention efficiently retains and elutes ddhC while avoiding interferences from other abundant constituents of blood serum and plasma (such as phospholipids) and can be applied to the analysis of different types of biofluids (e.g., blood products, CSF, urine, saliva). The method uses mobile phases that can be easily prepared using common LC-MS grade reagents or the solvents already containing buffer and acid modifiers that can be purchased from several chemical suppliers. This targeted method is highly flexible regarding the samples to which it can be applied without modifying the method setup. This makes the method significantly more robust and easier to implement in a wide range of applications.

[0029] The sample preparation consists in addition of three parts of organic solvent (acetonitrile) for one part of sample leading to protein precipitation and their removal upon centrifugation. Since HILIC retains and elutes lipid species, their removal is not required for the analysis of any biological samples that may contain them. A sample according to the present invention may therefore be an unprocessed sample which is obtained from a subject and used in a method of the invention without any processing steps other than a dilution (e.g. with acetonitrile) as described above. A sample according to the present invention may be subjected to one or more processing steps prior to use in a method of the invention, but typically said one or more processing steps does not involve removal of lipids from the sample prior to use in said method. The ability to use samples without removing lipids makes the methods of the invention compatible with a broad range of different biological sample types. The sample is biofluid from a subject. The biofluid may be blood (e.g. blood serum or plasma), urine, saliva and cerebrospinal fluid. The sample is typically either blood serum or plasma. The method avoids interference from other constituents of blood and plasma such as phospholipids. The avoidance of interference from other constituents of the biological sample from the subject is particularly important to the sensitivity (and accuracy) of the method. For example, if ddhC were to elute in the same retention time window as other metabolites, e.g. cytidine, this would result in interference between the two metabolites in MS / MS quantification, thereby reducing the sensitivity (and accuracy) of the method, because ddhC and cytidine share the same daughter (target) m / z value. The methods of the invention advantageously use HILIC to avoid co-elution of ddhC with other metabolites such as cytidine, and this means that a wide range of biological samples are compatible. The use of HILIC enables the methods of the invention to diagnose a viral infection rapidly, such as via a high-throughput screening method, and using a wide range of different biological samples,

[0030] In all aspects of the invention, the biological sample from a subject can be selected from whole blood, plasma or serum, Cerebrospinal Fluid, Urine, Sweat, Saliva, bronchiolar lavage, pleural fluid, joint fluid and drain fluid. The biological sample can be selected from Whole Blood, Blood Plasma, Serum, Cerebrospinal Fluid, and Saliva. The biological sample can be selected from Whole Blood, Blood Plasma, Serum, Cerebrospinal Fluid, Saliva and Urine. The biological sample can be selected from Whole Blood, Blood Plasma, Serum, Cerebrospinal Fluid, Saliva, Urine, and plant extract. The biological sample can be selected from bronchiolar lavage, pleural fluid, joint fluid and drain fluid. The biological sample from a subject can be selected from blood plasma or serum, Cerebrospinal Fluid, Urine and Saliva. The biological sample can be serum. For single celled organisms, such as bacteria, the biological sample can be cell lysates and supernatants. For plants, the biological sample can be plant extract.

[0031] The subject can be mammal (such as a human or a pig). The subject can be a human. The subject can be a pig. The subject can be a bird. The subject can be selected from the group consisting of human, bird, pig, rabbit, cow, primate, non-human primate, and mammal. The subject can be a plant. The subject can be an annual plant. The subject can be a perennial plant. The subject can be a deciduous plant. The subject can be a coniferous (evergreen) plant. The subject can be a plant food crop. The subject can be cassava plant. The subject can be single celled organisms such as bacteria.

[0032] The method for determining the quantity of ddhC present in a biological sample from a subject can be used in a method for diagnosing a viral infection in a subject. A viral infection can be diagnosed if ddhC is greater than a particular threshold. A suitable threshold is from about 30ng / ml to 100 ng / ml, from 40 ng / ml to 80 ng / ml, from 45 ng / ml to 55 ng / ml. Typically a viral infection can be diagnosed if the amount of ddhC present in a sample greater than at least 30ng / ml, at least 40ng / ml, at least 50 ng / ml, at least 60 ng / ml, and least 70 ng / ml, at least 100 ng / ml. Preferably, a viral infection can be diagnosed if the amount of ddhC present in a sample greater than at least 50 ng / ml. More preferably, a viral infection can be diagnosed if the amount of ddhC present in a sample greater than at least 100 ng / ml. The concentration of ddhC may differ between sample types, and hence the diagnostic threshold may also vary depending on sample type.

[0033] As will be appreciated by a person of ordinary skill in the art, the particular threshold of ddhC for diagnosing a viral infection may require normalization relative to a metabolite. For example, when the biological sample is urine, the particular threshold of ddhC for diagnosing a viral infection may be normalized for urinary creatinine concentration and expressed as a urine ddhC : creatinine ratio. Dividing the ddhC concentration (ng / mL) by the creatinine concentration (mM) in the same urine sample affords a urine ddhC:Cr ratio (ng / pmol). Preferably, a viral infection may be diagnosed if the amount of ddhC present in a urine sample (expressed as a urine ddhC : creatinine ratio) is greater than at least 1000 ng / pmol. More preferably, a viral infection may be diagnosed if the amount of ddhC present in a urine sample (expressed as a urine ddhC : creatinine ratio) is greater than at least 2000 ng / pmol.

[0034] The biological sample may be blood plasma or serum, and the particular threshold of ddhC for diagnosing a viral infection may be at least 100 ng / mL. The biological sample may be cerebrospinal fluid, and the particular threshold of ddhC for diagnosing a viral infection may be at least 90 ng / mL, e.g. at least 100 ng / mL.

[0035] The biological sample may be urine, and the particular threshold of ddhC (expressed as a urine ddhC : creatinine ratio) for diagnosing a viral infection may be at least 1000 ng / pmol, e.g. at least 2000 ng / pmol.

[0036] The biological sample may be saliva, and the particular threshold of ddhC for diagnosing a viral infection may be at least 5 ng / mL, e.g. at least 10 ng / mL.

[0037] The biological sample may be a plant extract, and the particular threshold of ddhC for diagnosing a viral infection may be at least 50 ng / mL, e.g. at least 60 ng / mL.

[0038] As a person of ordinary skill in the art will appreciate, a viral infection may be diagnosed if ddhC is greater than a particular threshold in one or more biological samples (which may be different depending on the sample, as outlined above). Adding an internal standard can account for matrix effect in the absolute quantification of ddhC in biological samples.

[0039] As used herein, the term “diagnosing” may refer to a binary diagnosis (i.e. a “yes” / ”no” diagnosis) of a viral infection. The methods described herein for diagnosing a viral infection in a subject are particularly well suited to high-throughput screening of large numbers of samples without requiring time-consuming and expensive preparation (e.g. a separate filtration step to remove lipids) before being subjected to the method of the invention. Rapid diagnostic binary diagnostic tests are particularly important because it is desirable to obtain a binary diagnosis as soon as possible not only to begin treatment of the patient without delay, but also to reduce the risk of transmission of the viral infection to other subjects. Also described herein are methods to determine the severity of a viral infection, but these are different to methods described herein for diagnosing a viral infection in a subject. As one of ordinary skill in the art will appreciate, although methods for diagnosing a viral infection differ to methods for determining the severity of a viral infection, the various aspects and embodiments described herein can be used in any combination without limitation. As mentioned above, the invention detects ddhC as a pan-viral marker. Accordingly, a method for diagnosing a viral infection in a subject is typically a method for diagnosing a pan-viral infection in a subject. By this it is meant that the method can diagnose any viral infection in a subject, rather than being limited to the detection of one or more specific virus. Since ddhC is a non-specific biomarker of active viral infection, the method does not need to be adjusted to be able to detect new types or strains of viral infections - unlike existing techniques. This makes the method an invaluable tool in applications ranging from point of care diagnostics and pandemic preparedness to agricultural applications. ddhC is a marker of active viral infection. Thus, unlike conventional markers of viral infection, ddhC does not detect latent infection, nor will it return a “false positive” result from subjects who have cleared a previous viral infection, but in whom remnants from old infections may still be present. The methods of the invention may therefore be useful in the diagnosis and / or detection of active viral infection.

[0040] As described herein, ddhC is a pan-viral marker, rather than being specific to one or more individual virus. Accordingly, any active viral infection may be detected according to the present invention. Non-limiting examples of viral infections that may be detected according to the present invention include Adenovirus, Coronavirus OC43, Dengue, Ebola virus (EBV), Enterovirus, Metapneumovirus, particularly Human metapneumovirus (HMPV), HSV1 , Influenza A, Influenza B, Parainfluenza type 1 , Parainfluenza type 2, Parainfluenza type 3, Parainfluenza type 4, Parechovirus, Rhinovirus, Rotavirus, SARS-CoV-2, respiratory syncytial virus (RSV) and Varicella zoster virus (VZV), and any combination thereof. The viral infection may be selected from influenza A, influenza B, adenovirus, dengue, HSV1 , measles, parainfluenza, respiratory syncytial virus (RSV), SARS-CoV-2, and any combination thereof. The viral infection may be HIV. The viral infection may be HSV2. The viral infection may be Cassava brown streak virus (CBSV). In some embodiments, the viral infection is not SARS-CoV-2. Alternatively or in addition, in some embodiments, the viral infection is not RSV.

[0041] Preferably, the method is a method for diagnosing a viral infection in a subject comprising: a) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the method comprising:

[0042] (i) preparing the sample for mass spectrometry analysis;

[0043] (ii) adding internal standard comprising isotope labelled ddhC to the sample;

[0044] (iii) obtaining one or more reference samples; (iv) assaying the sample and the one or more reference samples for the presence of ddhC using liquid chromatography - tandem mass spectrometry (LC-MS-MS) and thereby determining the quantity of ddhC present in the biological sample; and b) diagnosing a viral infection if ddhC is greater than a particular threshold, wherein the liquid chromatography is hydrophilic interaction chromatography (HILIC). In some preferred embodiments, a zero matrix such as described herein may be used in said method. As described herein, ddhC is a pan-viral marker for active viral infection. Accordingly, said method may be useful as a pan-viral diagnostic method, and particularly for the detection of an active viral infection. Said method may provide a binary (yes / no) diagnosis. Such diagnostic methods may also be used as part of other methods as described herein, such as methods for treating viral infections and / or methods for assessing the efficacy of a treatment for a viral infection.

[0045] The invention also provides a method for determining by mass spectrometry the quantity of ddhC present in a biological sample from a subject comprising:

[0046] (i) preparing the sample for mass spectrometry analysis;

[0047] (ii) adding internal standard comprising isotope labelled ddhC to the sample;

[0048] (iii) obtaining one or more reference samples;

[0049] (iv) assaying the sample and the one or more reference samples for the presence of ddhC using liquid chromatography - tandem mass spectrometry (LC-MS-MS) and thereby determining the quantity of ddhC present in the biological sample; wherein the liquid chromatography is HILIC. In some preferred embodiments, a zero matrix such as described herein may be used in said method. Such methods may also be used as part of other methods as described herein, such as methods for treating viral infections and / or methods for assessing the efficacy of a treatment for a viral infection.

[0050] The internal standard comprises or consists of a stable isotope labelled ddhC (Figure 6). One or more atoms in ddhC may be heavy isotopes (heavy atom labels). The reference may comprise stable heavy isotopes selected from13C,15N,2H or18O. The carbon atoms can be labelled with carbon 13. The nitrogen atoms can be labelled with nitrogen 15. The hydrogen atoms can be labelled (e.g. using deuterium). The C, N and H or O labels can be used combination. The internal standard may comprise one or more heavy atom labels. The carbon atoms in the ribose ring can be labelled. A suitable labelled analogue of ddhC can contain13Cs atoms in the ribose ring (in Figure 6 atoms numbered 1’-5’ marked with *). As used herein the term matrix refers to the components of a sample other than the analyte of interest. The matrix can have a considerable effect on the quantitative measurement of an analyte. Such effects are called matrix effects. The isotope labelled ddhC can be used to account for matrix effect in the absolute quantification of ddhC in biological samples. A stable isotope labelled (SIL) internal standards (IS) (SIL-IS) can be used to correct for instrument variability, ionisation efficiency and / or matrix effect in a particular sample. Stable isotope labelled ddhC is a structural analogue of endogenous ddhC. It is distinguishable by mass and fragmentation transitions from ddhC in MS but otherwise has identical physicochemical properties. The internal standard can further comprise a solvent. The internal standard is typically prepared as a solution in the same solvent as the calibration solutions and spiked QC samples with varied and controlled concentration of an analyte. The concentration of the SIL-IS should be within the linear calibration range. The solution of SIL-IS is added to each reference and study sample at fixed concentration and fixed volume. A requirement for methods intended for use in clinical analysis is that calibration standards and quality control samples should be prepared in the same matrix as the samples to be analysed. In the case of endogenous analytes, it is often not possible to obtain analyte-free matrix, and accepted options include either using surrogate matrix, using matrix stripped of the analyte (zero matrix) or doing background subtraction.

[0051] The reference sample can be prepared using a substitute matrix such as a surrogate matrix. A surrogate matrix is a solution with a chemical composition and physicochemical properties that are identical or as close as possible to the analysed sample type. The reference sample can be a serum reference sample (e.g. National Phenome Centre’s long-term reference (LTR) serum) or a plasma reference sample (e.g. National Phenome Centre’s LTR plasma or NIST SRM 1950). The reference sample can be prepared using a zero matrix. The replacement matrix and / or the zero matrix is typically used to prepare calibration solutions which are used to calibrate the assay. The zero matrix can be prepared from reference study samples such as (i) a patient sample or (ii) pooled aliquots of the subjects’ samples (for a specific sample cohort) using SPE protocol described earlier. Reference samples may comprise a known concentration of ddhC. One or more reference samples may be used.

[0052] A zero matrix is a sample where the analyte of interest, such as ddhC, has been selectively removed. The zero matrix may be obtained by performing solid phase extraction. The solid phase can be Hydrophilic-Lipophilic Balanced (HLB) medium such as (OASIS HLB). The stationary phase can comprise N-vinylpyrrolidine and divinylbenzene. It is possible to selectively retain ddhC, yielding metabolic profiling of reference samples practically unchanged compared to the untreated samples after removal of ddhC. The method has been validated for use with a surrogate matrix by comparing the results obtained using a zero matrix to results obtained with possible surrogate matrices. The surrogate matrix can be prepared using common reagents, facilitating simple method transfer between laboratories. The preferred matrix would be surrogate matrix (Bovine Serum Albumen (BSA) in Phosphate Buffered Saline) as it is easy to prepare from common laboratory reagents. The method using such a surrogate matrix can be easily standardised and transferred between different laboratories. The parallelism study provided herein proves that surrogate matrix yields accurate and true measurement of ddhC as compared to the method of standard addition in authentic sample and zero matrix. A suitable matrix comprises bovine serum albumin (BSA) in phosphate buffered saline (PBS), (e.g. at about pH 7.4). The surrogate matrix can comprise 1 % to 3% BSA, 1.5 to 3% BSA, 1.5 to 2.5 %BSA, where the % is in w / v. BSA can be about 2% w / v. The zero matrix obtained by solid phase extraction is particularly advantageous because the use of this target-free matrix in the methods described herein, e.g. in the method of diagnosing a viral infection in a subject, means that the methods are validated to a clinical standard - this is in contrast to (significantly less useful) methods of e.g. analysing disease severity that are only validated to research standard.

[0053] A method for validation of a substitute matrix can comprise preparing a dilution series of one or more aliquots of sample using the substitute matrix and preparing a dilution series of one or more aliquots of sample with zero matrix (e.g. an SPE-treated matrix), and measuring the concentration of ddhC in the sample at each dilution point in the dilution series for each matrix. A suitable dilution series can be dilution two more, three or more, or four or more times by an appropriate dilution factor. It is within the routine practice of one of ordinary skill in the art to select a suitable dilution factor. Non-limiting examples of suitable dilution factors include 1.5, 2, 2.5, 5 or 10, etc. The substitute matrix can be a surrogate matrix as described herein. The sample can comprise ddhC. The sample can comprise 13C isotope-labelled ddhC. Validating a substitute matrix can comprise comparing the concentration of ddhC in the sample at one or more (such as 2, 3 or 4, or 4 or more) dilution points in the dilution series for the substitute matrix and the zero matrix. A substitute matrix can be used in preparation of a reference sample.

[0054] The choice of the LC column and solvent system was guided by several factors including retention of ddhC and its resolution from the other constituents of biological samples, linear range of targeted molecule detection, retention of major metabolite classes present in blood products, such as phospholipids, their chromatographic behaviour and the effect on chromatographic separation and LC system pressure.

[0055] The liquid chromatography may be hydrophilic interaction liquid chromatography (HILIC). The use of HILIC is associated with advantages as described herein, and may therefore be preferred. The chromatographic medium can comprise polyethoxysilane (such as BEH HILIC, e.g. BEH HILIC 1.7 urn, 100 x 2.1 mm column (Waters) 186003461). A suitable chromatographic medium is Ethylene Bridged Hybrid (BEH) silica particles (polyethoxysilane, BPEOS). Ethylene Bridged Hybrid (BEH) columns possess high chemical and mechanical stability achieved by modification of silica particles with tetraethoxysilane (TEOS) and bis(triethoxysilyl)ethane (BTEE, which incorporates the pre-formed ethylene bridge).

[0056] Sample preparation of blood product for HILIC-based methods consists in simple protein precipitation by organic solvents. Suitable organic solvents include methanol, ethanol, isopropanol, acetone and acetonitrile. The organic solvent can be acetonitrile. Sample preparation can comprise 1) addition of organic solvent to the sample, followed by 2) centrifugation to separate the soluble extract (solute) from the pellet (e.g. comprising precipitated protein and optionally other particulates).

[0057] As used herein, term “Absolute” as used in e.g. absolute quantity, absolute concentration, or absolute amount, refers to measuring very precisely the amount of the metabolite ddhC by comparison with a pre-determined amount of a corresponding heavy reference metabolite that is spiked into the sample and that is measured simultaneously with the unlabelled metabolite. A key advantage of the present invention is the ability to determine absolute quantities of the metabolite in a range of biological samples.

[0058] The stable isotope labelled ddhC (SIL-IS) can be added in a fixed amount to each study and reference sample. To generate a linear calibration curve, the ratio of ddhC peak area (for a selected quantifier transition) to its corresponding labelled ddhC IS peak area can be plotted against the nominal concentration of each calibration solution. The ddhC concentrations in each study sample and QC sample can be back-calculated from constructed calibration curves using the peak area ratio of ddhC to labelled ddhC IS.

[0059] The targeted method for quantification of ddhC is validated according to established FDA and EMA guidelines for bioanalytical method validation. The main characteristics of the method assessed and essential for the acceptability of its performance and the reliability of the results are: Method linearity; Lower limit of quantitation (LLOQ); Method accuracy and precision; Carryover; Selectivity; Matrix effect; Stability. The method has a coefficient of variation >.95. Preferably the method has a coefficient of variation >0.99. %CV (coefficient of variation) is defined as (standard deviation / mean * 100%).

[0060] The method for determining the quantity of ddhC present in a biological sample can be used in a method of diagnosis where i) the quantity of ddhC in a sample from the subject is determined, ii) the quantity of ddhC is used to determine whether the patient has a viral infection. The method for determining the quantity of ddhC present in a biological sample can be use in a method of treatment where i) the quantity of ddhC in a sample from the subject is determined, ii) the quantity of ddhC is used to determine whether the patient has a viral infection, iii) if the patient is determined to have a viral infection the patient is treated with a therapeutic agent for treatment of a viral infection. The treatment can be an antiviral agent such as Nirmatrelvir plus ritonavir, molnupiravir and remdesivir and / or an antibody based treatment. If the patient is determined not to have a viral infection the patient may be treated with an antibacterial agent.

[0061] The method for determining the quantity of ddhC present in a biological sample from a subject can be used in a method of managing the subject. Managing the subject can include withholding certain treatments from the subject, such as not treating the subject with antibacterial agents such as antibiotics. Managing the subject can include isolating the subject.

[0062] The method for determining the quantity of ddhC present in a biological sample can be used in a method for assessing the efficacy of a treatment for a viral infection in a subject previously treated for a viral infection wherein i) the quantity of ddhC in the sample from the subject previously treated for a viral infection is determined, ii) the quantity of ddhC is used to assess the efficacy of the treatment for a viral infection. The viral infection may be HIV. The subject may have HIV (i.e. HIV positive). The subject may have HIV and be an HIV elite controller.

[0063] The method for determining the quantity of ddhC present in a biological sample can be use in a method of determining the severity of a viral infection wherein i) the quantity of ddhC in a sample from the subject is determined, ii) the quantity of ddhC is used to determine the severity of a viral infection. Typically if ddhc > 400ng / mL, (e.g. if ddhc >300ng / mL) then the viral infection is severe. If 400ng / mL > ddhC > 50ng / mL, the viral infection is not severe. In some embodiments, if ddhc < 50ng / mL the patient does not have a viral infection. In some embodiments, if ddhc < 30ng / mL the patient does not have a viral infection. As discussed above, the threshold levels of ddhC for diagnosing a viral infection may vary depending on sample type. The invention detects ddhC as a pan-viral marker, so the method for determining the severity of a viral infection in a subject may be a method for determining the severity of a pan-viral infection in a subject. By this it is meant that the method can determine the severity of any (active) viral infection in a subject. Since ddhC is a non-specific biomarker of active viral infection, the method does not need to be adjusted to be able to determine the severity of new types or strains of viral infections - unlike existing techniques.

[0064] The viral infection may be an active viral infection; and / or the method may be a pan-viral diagnostic method. As a person of ordinary skill in the art will appreciate, an active viral infection is one in which the virus is replicating within the subject. In this context the term “active viral infection” means a viral infection where the virus is actively replicating. An “active viral infection” is also not the same as transient viral carriage or viral remnants from old infections. For example, as described herein, ddhC concentrations in samples taken from subjects were found to increase in the days following viral infection and subsequently falling back to baseline as infection is overcome. An “active viral infection” could therefore be defined as a viral infection in which ddhC concentrations are elevated above baseline concentration.

[0065] As disclosed herein in the following Examples, ddhC acts as a virally-induced acute phase reactant, rising in the days following viral infection and subsequently falling back to baseline as infection is overcome. This is a particularly important finding because it allows for the use ddhC as an active viral infection biomarker in a diagnostic clinical setting. This utility is provided based on understanding the temporal changes in ddhC concentration in biological samples during a viral infection as described and exemplified herein.

[0066] The viral infection may be diagnosed between about 1 and about 15 days, between about 1 and about 14 days, between about 1 and about 13 days, between about 1 and about 12 days, between about 1 and about 11 days, between about 1 and about 10 days, between about 1 and about 9 days, between about 1 and about 8 days, between about 1 and about 7 days, between about 1 and about 6 days, between about 1 and about 5 days, between about 1 and about 4 days, between about 1 and about 3 days, and / or between about 1 and about 2 days, post viral infection. The viral infection may be diagnosed between about 2 and about 10 days, between about 2 and about 9 days, between about 2 and about 8 days, between about 2 and about 7 days, optionally between about 3 and about 7 days post viral infection. The viral infection may be SARS-CoV-2 or influenza A. As one of ordinary skill in the art will appreciate, the time periods described above may be equally applicable to other aspects and embodiments of the present invention, including but not limited to methods for determining the severity of a viral infection. Clearly, a host-derived viral biomarker (ddhC) that peaks in the first few days of infection and subsequently returns to baseline is of great use in acute healthcare settings as an indicator of an active acute viral infection. Currently, in the absence of widespread rapid diagnostic capability, many patients presenting with non-specific symptoms of infection (e.g., fever, cough) are given antibiotics, even when they have a viral infection, driving antibiotic resistance. Therefore, methods of the present invention may be used to decrease the unnecessary administration of antibiotics, and hence to reduce antibiotic resistance. Secondly, during the early stages of a novel viral pandemic, before pathogen-specific diagnostics can be made available, a (binary) pan-viral method for diagnosing a viral infection would be invaluable. This could also help clinicians to isolate patients with viral infections, thereby reducing further transmission. Thirdly, the diagnostic methods of the invention would also provide a useful ‘rule-out’ diagnostic that could exclude active viral infection. This would be particularly helpful because highly sensitive molecular tests known in the art can detect the presence of viral RNA or DNA and potentially mislead clinicians into assuming that a patient’s illness is related to an active viral infection.

[0067] Advantageously, the methods of the invention may be applied to biological samples from symptomatic, asymptomatic, and paucisymptomatic subjects. Advantageously, the subject may be asymptomatic or paucisymptomatic. One of ordinary skill in the art will readily appreciate that e.g. methods for diagnosing a viral infection may be performed on infected or healthy (i.e. not infected, or those who did not develop infection post-challenge) subjects - these subjects may also be symptomatic, asymptomatic, or paucisymptomatic subjects).

[0068] Kits are provided that are suitable for carrying out the method for determination of the concentration of ddhC and the other methods described herein that include the method for determination of the concentration of ddhC. The kit can comprise one or more stably isotopically labelled internal standard (SIL-IS) as described herein. The kit can comprise instructions for use. The kit for treatment of a viral infection, and / or the kit for assessment of the efficacy of an antiviral agent can comprise an antiviral agent. Preferably the kits also contain machine readable media containing mass spectrometric transitions or target mass spectra for ddhC (e.g. as found in Table 1) and the stably isotopically labelled standards thereof. A kit may contain one or more or all of: reference samples, or instructions protocol for preparing biological samples and reference samples before analysis, instructions for preparation of surrogate and zero matrix as described herein from lab reagents, labelled ddhC, analysis order and analytical plate layout, parameters for the assessment of data quality. The reference samples can be present (such as calibrators and QC samples) in the types and number required for quantification of ddhC in a cohort of biological samples.

[0069] The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination. EXAMPLES to determine most reliable LC column ddhC was detected in the global metabolic profiles of human serum samples acquired using hydrophilic interaction liquid chromatography (HILIC) assay in positive ionisation mode. The molecule can be detected using a reversed-phase chromatographic (RPC) assay, eluting earlier than in HILIC assay. We tested an RPC method using both C8 and C18 columns. However, in the analysis of long-term reference samples of urine and plasma, it was observed that ddhC was co-eluting with some major constituents of biofluids, such as acetylcarnitine and uric acid, which could potentially compromise the measurements by suppressing ionisation of ddhC. In addition, the preparation of blood product samples for a sustained long-term large-scale RPC analysis requires removal of phospholipids, increasing the complexity and duration of the analysis. The advantages of using HILIC stationary phase include ddhC retention time, the absence of coeluting metabolites, high ionisation in selected LC conditions, and also the easiness of sample preparation based only on protein precipitation. Phospholipids are easily eluted from a HILIC column thus there is no need for their removal.

[0070] Ethylene Bridged Hybrid (BEH) HILIC (Waters) column proved to be the optimal choice providing acceptable retention of ddhC and its separation from the other metabolites. Phospholipids can be easily eluted from HILIC columns without accumulating on a column and increasing system backpressure. Sample preparation of blood product for HILIC-based methods consists of simple protein precipitation by organic solvents. The systematic LC assessment using BEH HILIC 1.7 urn, 100 x 2.1 mm column (Waters) yielded the optimal set of LC conditions (flow rate, column temperature) and gradient that provided symmetrical and narrow peak of ddhC well resolved from other metabolites in tested serum, plasma, and urine samples. Aiming to shorten the method, a shorter BEH HILIC column 1.7 urn, 50 x2.1 mm was assessed yielding the following observations:

[0071] 1. Calibration curves on a Time-of-Flight (TOF) mass spectrometer using 100 mm and 50 mm columns were almost identical.

[0072] 2. The retention time (RT) of ddhC on the 50 mm column was ca. 0.6-0.7 min and on the 100 mm column it was of 1 .28 min. The peak shape was narrower using 100 mm column.

[0073] 3. The analysis of the reference samples on both columns yielded different results, with the values being lower using 50 mm column. Ion suppression by co-eluting metabolites present in biofluids was suspected.

[0074] The 100 mm column was therefore used for further method development, validation and ddhC detection and quantification in biological samples. The labelled analogue of ddhC containing13Cs atoms in the ribose ring (Atoms with a star in Figure 6) was synthesized to account for matrix effect in the absolute quantification of ddhC in biological samples.

[0075] Raw UHPLC-ESI-MS / MS spectral data were processed using the TargetLynx application package within MassLynx (v4.1) software (Waters Corporation). Microsoft Excel was used for the validation data assessment.

[0076] Example 2 - Determining final LC-MS / MS method parameters

[0077] The LC instrument setup consisted of a Waters Acquity LIHPLC solvent management system and a Waters 2777C external autosampler (Waters, Wilmslow, U.K.). For the final LC method using a BEH HILIC 1.7 urn, 100 x 2.1 mm column (Waters), mobile phase A consisted of 20 mM ammonium formate with 0.1% formic acid in water, and mobile phase B was 0.1 % formic acid in acetonitrile. Both mobile phases can be easily prepared using common LC-MS grade reagents or the solvents already containing buffer and acid modifiers can be purchased from several chemical suppliers.

[0078] The gradient was shortened to allow efficient wash and equilibration of the chromatographic column after ddhC elution, resulting in a total length for the method of 7.5 minutes. The weak and the strong washes were 1 :3 water / acetonitrile (v / v) and 100% isopropanol, respectively. During method development, a carryover was observed, so an extensive needle wash cycle in autosampler was employed.

[0079] MS detection was performed with a Waters Xevo TQ-S tandem quadrupole instrument (Waters, Wilmslow, U.K.) using electrospray ionization (ESI) in positive ion mode. Multiple reaction monitoring (MRM) was used for the quantification of ddhC; the specific metabolite and labelled standard transitions are presented in Table 1. Nitrogen was used as the desolvation gas, and argon was used as the collision gas. Figure 1 shows extracted ion chromatograms of the quantifier transitions of ddhC and 13C5-ddhC. Table 1. MS / MS transitions used for ddhC and its labelled 13C5-ddhC standard.

[0080] Example 3 - Generating a zero matrix for ddhC

[0081] We found that ddhC is present in low concentration in serum, plasma, and urine samples from healthy controls. Therefore, to account for this we developed a solid phase extraction (SPE) approach to generate ddhC-free (zero) sample matrix. Performing assessment of different solid phase extraction (SPE) cartridges, we observed that HLB Oasis cartridges (Waters) were able to selectively retain ddhC contained in reference serum and plasma samples (the National Phenome Centre’s long term reference (LTR) serum and LTR plasma) during the loading step of the sample onto the cartridge (Figure 2). The collection of the loading solution provided a serum / plasma sample depleted from ddhC but with preserved otherwise general metabolic profile (Figure 3). Calibration curves acquired in water and in SPE-treated serum samples yielded almost identical slope and intercept values (Figure 4)

[0082] This approach of creating zero matrix has not been reported for this metabolite previously and it facilitates generation of a zero matrix for each specific sample set. The method enabled us to have a matrix as close as possible to the native matrix, which we then validated our surrogate matrix against - see Examples 4 and 5 below.

[0083] Example 4 - Surrogate matrix for method validation

[0084] We validated the method using a surrogate matrix generated from easily available reagents to allow standardisation of the method’s use and transfer across different analytical and clinical labs.

[0085] The surrogate matrix proposed for serum and plasma was bovine serum albumin (BSA) dissolved in PBS at pH 7.4. Method validation in water was additionally assessed for its application as a matrix for the analysis of other biofluids (e.g. urine, saliva, Cerebrospinal fluid (CSF). A stable isotope-labelled ddhC analogue was used in all applications to account for any matrix effects, such as ion suppression / enhancement and / or differences in recovery between the native and surrogate matrices.

[0086] Example 5 - Parallelism study

[0087] As part of the Method validation for biofluid samples we include evidence that the surrogate matrix is suitable for ddhC absolute quantification when compared to native matrix, (a parallelism study).

[0088] Parallelism study design

[0089] A parallelism study was conducted to assess the ability of the surrogate matrix to perform as the native matrix, to show that 2% BSA in PBS can be used for the full method validation and all future applications for the analysis of ddhC in human serum / plasma.

[0090] Surrogate matrix 2% BSA in PBS, LTR-plasma (untreated), and SPE-treated LTR-plasma were prepared in a volume needed for the preparation of replicates of calibration curves and QC samples to assess accuracy and precision. To create the LTR-plasma, 10 L of bulk plasma were purchased from Seralab (Product Code: PLH-123-V Batch Number: XX6072313), homogenised, centrifuged and aliquoted for long term storage. Calibration curves were then prepared in triplicate in each of these three matrices. The calibration curves included zero point which was the corresponding matrix spiked with the labelled ddhC only.

[0091] Dilution series of the three aliquots of untreated LTR plasma were prepared using surrogate matrix and an additional three aliquots were diluted with SPE-treated matrix. Triplicate dilution series and calibration curves were analysed in LTR-plasma along with triplicate calibration curves in surrogate matrix and SPE-treated LTR-plasma.

[0092] Three methods (spike-recovery, dilution linearity and standard addition) were used to prove the suitability of surrogate matrix and SPE-treated plasma for ddhC measurements in plasma samples.

[0093] Linearity was assessed along with the method’s accuracy and precision in three separate days. The linear fit with 1 / X2weighting was applied to the response calculated as a ratio of ddhC peak area to the13Cs-ddhC SIL-IS peak area for each calibration concentration level. LLOQ was accepted from the tested linear range based on the 20% deviation of the back-calculated standard concentration with the relative standard deviation of the response value across all replicates of calibration curves not exceeding 20%. Intra-day method accuracy and precision were assessed using three series of QC samples prepared at five concentration levels (QC1-QC5 described above). For acceptance, the QC were required to be within 15% (20% for QC1 = LLOQ QC) of their nominal concentration and 15% (20% for QC1) of %CV between the analysed replicates.

[0094] Standard addition method performed in untreated LTR-plasma provided an extrapolated concentration of LTR-plasma that was used to calculate concentrations of dilution series plasma samples (diluted four-times using the factor of 1 .5) as well as concentration of each addition of calibrant to the LTR plasma (spiked concentration).

[0095] Surrogate and zero matrix calibration curves were used to measure concentrations at each dilution and addition point of the standard addition calibration curves providing “interpolated” concentrations. Interpolated concentrations were compared to the concentrations of the standard addition calibration curves calculated by adding extrapolated concentration of the LTR-plasma samples obtained to the concentration of the spiked in standard. Deviation of each interpolated concentration value from the “extrapolated” and “spiked” concentrations were calculated for both surrogate and zero matrix measurements along with the precision presented as %CV (coefficient of variation = (standard deviation / mean * 100%). The applicability of the method for serum samples was demonstrated by the analysis of paired serum / plasma samples.

[0096] Results

[0097] Standard addition calibration curves were generated in untreated plasma LTR samples and an extrapolated concentration of ddhC was calculated (Table 2). The concentrations of diluted LTR plasma samples were obtained by applying dilution factor of 1.5 used to prepare dilution series from LTR plasma (Table 3).

[0098] Table 2. Parameters of three intra-day and one inter-day standard addition calibration curves and extrapolated concentration of LTR plasma which was used as matrix for standard addition. %CV = coefficient of variation.

[0099] Table 3. Calculated concentrations of dilution series prepared from the LTR plasma and serially diluted four times using a dilution factor of 1.5. LTR plasma concentration was an average obtained from the four replicates of the standard addition curves.

[0100] Calibration curves were generated in surrogate matrix -2% BSA in PBS (Table 4). Table 5 shows associated concentrations, including the assessment of the precision and closeness of each interpolated value to the extrapolated value, measured using the standard addition methods above. Figure 5 shows the results of a parallelism study of standard addition (black diamonds) and surrogate matrix (black circles) calibration curves.

[0101] Table 4. Parameters of three intra-day and one inter-day standard addition calibration curves and extrapolated concentration of LTR plasma which was used as matrix for standard addition. %CV = coefficient of variation.

[0102] Table 5. Comparison of the concentration values calculated from the standard addition curve for the dilution series and spiked standard concentration to the concentration values measured for each point of the standard addition calibration curve and dilution series using the surrogate matrix method - interpolated concentrations.

[0103] Extrapolated concentration of ddhC (LTR-P(from std add) + spiked in

[0104] Similar comparisons were performed for the calibration curves obtained in zero matrix (SPE- treated LTR plasma sample) and standard addition calibration curves. The interpolated concentration values were compared to the concentration values calculated from the standard addition curve (Table 6). These values were generated using the methods described in Example 5.

[0105] Table 6. Comparison of the concentration values calculated from the standard addition curve for the dilution series and spiked standard concentration to the concentration values measured for each point of the standard addition calibration curve and dilution series using the zero matrix method (using SPE-treated plasma matrix)- interpolated concentrations.

[0106] Extrapolated concentration of ddhC (LTR-P(from std add) + spiked in)

[0107] The results obtained from the interpolation (zero matrix method) of each concentration and dilution point obtained for LTR plasma matrix to the values calculated using the standard addition method prove parallel behaviour of two types of calibration curves and the accuracy of the measurements obtained using the former method, as well as absence of matrix effect in the measurement of ddhC using a native matrix.

[0108] Given the parallel behaviour of the surrogate matrix and its general accessibility and availability, the surrogate matrix was chosen for further method validation and analysis of biological samples.

[0109] Example 6 - Further validation of the surrogate matrix

[0110] The assessment of the method’s accuracy and precision using water as a matrix for calibration curves and QC samples was carried out to enable analyses of various biofluid sample types such as urine, saliva and cerebrospinal fluid.

[0111] Briefly, the calibration curves and QC samples were prepared in water and analysed on three separate days. First day analysis was done using five replicate QC samples sets prepared at six concentration levels from the linear range of the ddhC calibration curve. The lowest QC level was equal to LLOQ and the highest was prepared at 80% of IILOQ (upper limit of quantitation). On the second and third days, the analysis was done for freshly prepared calibration curves and one set of QC samples prepared freshly on each day. On all three days, replicate samples of LTR plasma and serum were analysed along with the spiked QC samples. Accuracy and precision were assessed for each QC sample intra- and inter-day and for the LTR plasma and serum samples precision only as shown in Table 7 and Table 8. Table 7. Intraday precision and accuracy assessment of QC samples and calibration curves prepared in water and analysed on the first day of validation study.

[0112] Table 8. Interday precision and accuracy assessment of QC samples and calibration curves prepared in water and analysed in three consecutive days.

[0113] Inter-day accuracy and precision were assessed in separate days with the total number of analysed QC samples (across day 1 and all additional days). The same acceptance criteria were used for the inter-day accuracy and precision assessment (Table 9). Table 9. Intra- (n=3) and Inter-day (n=13) % accuracy and precision as (%CV) for five QC concentrations and precision for LTR-P and LTR-S.

[0114] Example 7 - Additional validation of the assay

[0115] Carryover

[0116] Carryover was assessed by injecting DB sample after IILOQ and QC5. Carryover acceptance criteria required that the MRM transitions of ddhC in the DB sample were less than 20% of LLOQ, and the MRM transitions of SIL-IS were less than 5% of the SIL-IS response detected in SB solution.

[0117] Selectivity

[0118] Selectivity was assessed using six individual sources of analyte-free matrix. These were generated using the described earlier SPE approach to remove endogenous ddhC from six healthy control plasma samples. The samples were prepared for selectivity analysis without spiking SIL-IS in. Absence of interfering components was accepted when the response was less than 20% of LLOQ for ddhC and less than 5% for the SIL-IS.

[0119] Matrix effect

[0120] Was assessed using six individual sources of analyte-free (zero) matrix prepared as described in Selectivity assessment using SPE to remove ddhC from six healthy control plasma samples. Each source of matrix was spiked with analyte and SIL-IS at concentration levels of 3.9 ng / mL (LLOQ QC) and 1200 ng / mL (high QC). The same concentrations were spiked into surrogate matrix (2% BSA in PBS). Matrix effect (ME) was calculated for each concentration as ME% = peak area zero matrix / peak area BSA. ME normalised by IS was calculated for each matrix source and each concentration as MEIS%=ME%(analyte) / ME%(IS), CV of MEIS% should be less than 15%.

[0121] Matrix Effect (ME %) was measured using six individual ddhC-free (zero) plasma samples, obtained from healthy controls, that were treated with SPE to remove endogenous ddhC and spiked with known concentration of ddhC corresponding to LLOQ QC (3.9 ng / mL) and high QC (1200 ng / mL) and SIL-IS13Cs-ddhC. Each spiked QC sample was measured in triplicate. Each peak area of ddhC measured in the zero matrix were referenced to the same concentrations of ddhC standard spiked in surrogate matrix (2% BSA in PBS). The results of ME assessment are shown in Table 10.

[0122] Table 10. Matrix effect assessment results.

[0123]

[0124] Stability

[0125] Stability was assessed using LLOQ QC and high QC prepared in three replicates for both concentration levels for each test. Quantification of the replicates of the QC samples and subsequent stability assessment was made following sample storage at 4°C (in the autosampler) for 24 and 72 hours, at -80°C for 24 hours, 72 hours, and 14 days, and at -20°C for 14 days. The acceptance criteria required accuracy and precision of the determined concentrations of LLOQ QC and high QC after each storage condition to be within 20% and 15%, respectively (Table 11). Table 11. Stability assessment results. Accuracy and precision of the ddhC quantification were assessed at two different concentration levels after storing the prepared solutions under different temperature conditions for different time periods.

[0126] Dilution linearity

[0127] Dilution linearity was assessed by preparing a calibration solution at concentration of 2xllLOQ (4000 ng / mL) and diluted 4 and 16 times using the diluent matching the composition of the single blank sample (SB). Each dilution was prepared in five replicates which were quantified using ddhC calibration curve. The acceptance criteria required accuracy and precision of the determined concentrations of diluted 2xllLOQ to be within 15% (Table 12).

[0128] Table 12. Dilution integrity assessment results.

[0129] Example 8 - Same timepoint comparison of serum and plasm samples in water and SPE- treated LTR plasma.

[0130] Serum and plasma samples taken at the same time were compared (Table 13).

[0131] Table 13. ddhC quantification in paired serum and plasma samples obtained at the same time point from the same patients and replicate analysis of LTR plasma and serum samples against calibration curves generated in two different matrices - water and SPE-treated LTR plasma (zero matrix)

[0132] Example 9 - ddhC quantification in serum, plasma, urine, and saliva samples

[0133] Additionally, the method was tested for the analysis of four different biofluid samples collected at various timepoints from one individual, showing method applicability for different sample types (Table 14). Serum and plasma samples can be directly measured from the calibration curve since they are prepared in the same way (using the same dilution factor and the same proportion of organic solvent to aqueous part of the sample) as calibration and QC solutions. Urine samples contain higher levels of ddhC and had to be diluted 20 times prior to the preparation for the analysis. Saliva samples were initially treated with acetonitrile in the 1 :3 sample: acetonitrile proportion. The supernatant was collected after protein precipitation and dried under nitrogen stream before reconstituting in water and preparation for the analysis in the same way as it is done for blood product samples.

[0134] Table 14. ddhC quantification in urine, saliva, plasma and serum samples collected at the same time points on different days from one individual. Calibration curves and QC samples were prepared using surrogate matrix - 2% BSA in PBS. All QC samples passed QC criteria established in published guidelines.

[0135] Example 10 - Extrapolation of original ddhC-discovery samples

[0136] The method has been used to analyse a subset of samples that underwent initial untargeted metabolomic profiling leading to the discovery of ddhC (Mehta et al., Med, 2022 Mar 11 ;3(3):204- 215), showing an excellent correlation with an r2value of 0.9881.

[0137] Example 11 - ddhC kinetics

[0138] Using the new method, serial measurements over time of individuals infected with viral infections have shown that ddhC is an acute phase reactant, peaking at approximately day 3-5 post infection, after which its levels fall back to baseline. This underlies its reliability as a marker of acute viral infection - if levels did not fall back to baseline after infection, its use in identifying patients with active, as opposed to recovered, viral infection would be limited.

[0139] Specifically, healthy human participants were inoculated with SARS-CoV-2, or influenza A virus (H3N2). The new method was used to quantify ddhC concentrations in serial plasma samples collected pre- and post-inoculation.

[0140] The LC-MS / MS assay method

[0141] Chemicals and materials

[0142] LC-MS grade acetonitrile with formic acid (0.1 %, v / v), LC-MS grade water with formic acid (0.1%, v / v), and LC-MS grade water were purchased from Fisher Scientific (Loughborough, U.K.), LC- MS grade acetonitrile was purchased from VWR International Ltd (Leicestershire, UK). Ammonium formate, CHROMASOLV LC-MS Ultra was obtained from SLS Scientific Laboratory Supplies Limited (Fairham, UK). Oasis® HLB Vac Cartridge (30 mg, 1cc) for zero matrix generation were from Waters (Wilmslow, UK). For generating the surrogate matrix, bovine serum albumin (BSA) and phosphate buffered saline (PBS, pH 7.4) were obtained from Sigma-Aldrich (Gillingham, UK).

[0143] LC-MS / MS setup

[0144] Serum and plasma samples for method validation

[0145] For the assay validation various serum and plasma samples were used. Pooled long-term reference (LTR) human serum and plasma samples, LTR-S and LTR-P respectively, maintained by the National Phenome Centre (NPC) and used as independent sample references (Lewis, M., et al. An open platform for large scale LC-MS-based metabolomics. preprint ChemRxiv (2022)) were purchased from Seralab (BiolVT) as bulk human serum and plasma (10L of each that were homogenised, centrifuged and aliquoted for long-term storage).

[0146] Surrogate and zero matrix

[0147] Surrogate matrix was prepared by weighing BSA and dissolving it in PBS (pH 7.4) to prepare a solution of 2% [w / v]. Surrogate matrix was used in all analyses for the preparation of calibration and quality control (QC) solutions.

[0148] Zero matrix - a sample matrix lacking the analyte of interest or analyte-free matrix - was used to assess matrix effect and selectivity. It was prepared by solid phase extraction (SPE) using an Oasis® HLB Vac Cartridge (30 mg, 1cc) conditioned with LC-MS grade acetonitrile (1 mL) and then equilibrated with LC-MS water (1 mL). After the conditioning and equilibration, an aliquot of 300 pL of plasma samples (LTR-P and healthy control plasma) diluted 1 :1 with LC-MS grade water was loaded onto the cartridge. The loading solution was collected in an Eppendorf tube. The cartridge was washed twice with LC-MS grade water (300 pL) collecting the wash solution in a separate Eppendorf tube. Following this step, the elution of the retained material was done sequentially using 1 :1 acetonitrile:water twice (300 pL) and then acetonitrile twice (300 pL). The loading solution lacked ddhC but retained the global plasma / serum metabolic profile as the untreated sample when analysed by hydrophilic interaction liquid chromatography (HILIC) assay used at the NPC as shown in (Figure 3). The SPE process was repeated using the same cartridge by passing through the collected loading solution. The new loading solution (after the second SPE) was collected into a new Eppendorf tube and was used in the further validation analyses as a zero matrix (with removed endogenous ddhC).

[0149] Preparation of the calibration and quality control working solutions

[0150] Stock solution of ddhC of 1 mg / mL was prepared in LC-MS grade water and stored at -80°C in 200 pL aliquots. SIL-IS13Cs-ddhC stock was prepared at 1 mg / mL in LC-MS grade water, then it was subaliquoted at a concentration of 600 ng / mL. The multiple aliquots of 600 ng / mLwere stored at -80°C, and the needed number of aliquots were thawed prior to each analysis.

[0151] A set of ten calibration working solutions (WS) of ddhC in the defined validated linear range (3.9- 2000 ng / mL) was prepared from the stock solution of ddhC by serial dilution with LC-MS grade water using a dilution factor of two. WS of the quality control (QC) samples were prepared separately, diluting the ddhC stock solution with LC-MS grade water to five concentration levels of ddhC, that were within the linear range but different from any calibration WS concentration levels, except for the lower limit of quantification (LLOQ): 3.9 ng / mL (LLOQ QC), 50 ng / mL, 300 ng / mL, 625 ng / mL, and 1200 ng / mL (high QC).

[0152] Calibration solutions and QC samples preparation for LC-MS / MS analysis

[0153] Preparation of the calibration solutions and QC samples for the LC-MS / MS analysis was done using the same procedure as for the preparation of serum and plasma samples starting with aliquots of 40 pL of each WS in LC-MS water, which were diluted 1 : 1 with surrogate matrix of 2% BSA in PBS. This was followed by the addition of 16 pL of 600 ng / mL aqueous solution of13Cs- ddhC SIL-IS. Three volumes of ice-cold acetonitrile (288 pL) were added to 96 pL of calibration WS or QC solution mixed with the surrogate matrix and SIL-IS for protein precipitation. After the mixing at 1400rpm for 2h at 4°C and centrifugation for 10 minutes at 3486xg and 4°C, the supernatant was collected for the targeted LC-MS / MS analysis.

[0154] Double blank (DB) solution was prepared as method diluent containing 1 :3 LC-MS grade wateracetonitrile. Single blank (SB) was prepared as described above for calibration solutions but using water instead of the calibration WS diluting 1 : 1 with the surrogate matrix and adding the same amount of SIL-IS and ice-cold acetonitrile. SB sample was mixed and centrifuged as described for the calibration and QC solutions. It was analysed as “zero point” calibration solution before the LLOQ solution but not included in the calibration linear fit regression.

[0155] Samples formatting and run order

[0156] Clinical samples were analysed as randomised blocks of samples collected from each patient in different days of infection. Additionally, the established run order randomized the samples from different days of infection within those sample blocks to avoid any confounding effects of clinical variables to the analytical run order.

[0157] The samples were extracted as described above and aliquoted onto two analytical plates. The first analytical plate comprised 40 study samples and the second 33 study sample. Each plate included a set of ten calibration solutions with SB solution preceding it, and two full set of QC samples. LTR-P samples were added to the run to monitor the precision of ddhC quantification in a repeat analysis of a biological sample. DB solutions were aliquoted in the column 12 of the analytical plate.

[0158] The analytical was initiated by the triple injection of the DB solution to equilibrate the column followed by a SB injection, calibration set and one full QC series. The study samples from two analytical plates were analysed continuously in subsets of eight samples. LTR-P sample was injected before each subset to monitor the precision of ddhC quantification across the full run. The calibration and analytical QC set were repeated at the end of the analysis of all study samples. DB was injected in duplicate after each IILOQ solution and high QC to avoid any carryover in the subsequent samples.

[0159] For method validation, performed on different days, each analytical plate contained the required number of calibration and QC sets according to the requirements for each validation parameter described in the next section. ddhC measurement

[0160] The ddhC analytical standard and13Cs-ddhC stable isotope labelled internal standard (SIL-IS) was prepared. The ddhC analytical standard was also purchased from Berry & Associates (cat. no. PY 7790). Detailed information regarding other chemicals and materials, method validation, clinical samples used in validation, the surrogate and zero (analyte-free) matrix used for calibration and quality control, and data processing is described above.

[0161] The LC instrument setup consisted of a Waters Acquity LIPLC solvent management system and a Waters 2777C external autosampler (Waters, Wilmslow, U.K.). Chromatographic separation was performed on a Waters BEH HILIC 2.1 x 100 mm, 1.7 urn column (Waters, Wilmslow, U.K.). Mobile phase A consisted of 20 mM ammonium formate with 0.1 % formic acid in water (v / v), and mobile phase B was 0.1% formic acid in acetonitrile (v / v). The weak and the strong washes were 1 :3 water / acetonitrile (v / v) and 100% isopropanol, respectively. To avoid carryover observed during the method development, the needle wash cycle in the autosampler method was extended to three washes prior and after the injection.

[0162] The chromatographic column was maintained at 55°C during the run, and the LC gradient was performed at 0.8 mL / min starting at 3% A for 0.1 min followed by an increase to 20% A at 1.5 minutes, which was maintained for the next 0.25 minutes. This was followed by an increase to 50% A at 2 minutes and maintained until 3.5 minutes to elute all sample material from the column. At 3.6 minutes, it was returned to the initial LC conditions of 3% A for re-equilibration, ending at 7.5 minutes.

[0163] MS detection was performed with a Waters Xevo TQ-S tandem quadrupole instrument (Waters, Wilmslow, U.K.) using electrospray ionization (ESI) in positive ion mode. Multiple reaction monitoring (MRM) was used for the quantification of ddhC; the specific metabolite and labelled standard MRM transitions are presented in Table 1 . The cone voltage for all transitions was of 4 V. Nitrogen was used as desolvation gas, and argon was used as collision gas. The following source conditions were used for the run: capillary voltage of 2.5 kV; source offset of 30 V; desolvation temperature of 600°C; source temperature of 150°C, desolvation gas flow of 1200 L / h; cone gas flow 250 L / h; nebulizer gas of 7.0 bar; collision gas of 0.18 mL / min.

[0164] For ddhC quantification, study plasma samples stored at -80°C were thawed overnight at 4 °C and then vortex-mixed. Aliquots of 40 pL of each sample were added to 96-deep-well polypropylene plates (2 mL, Eppendorf). Subsequently, samples were diluted 1 :1 with LC-MS grade water, and 16 pL of 600 ng / mL aqueous solution of13Cs-ddhC SIL-IS were spiked into each sample. Three parts of ice-cold acetonitrile (288 pL) were then added to one part (96 pL) of diluted sample for protein precipitation. Each plate was sealed prior to mixing at 1400 rpm for two hours at 4 °C (MixMate, Eppendorf). The plates were then centrifuged for ten minutes at 3486xg and 4 °C, and the supernatants (125 pL) were transferred into 96-well polypropylene plates (Eppendorf), which were heat sealed and centrifuged for five minutes at 3486xg and 4 °C prior to the LC- MS / MS analysis. Preparation of the calibration solutions and QC samples followed the same protocol, starting with aliquots of 40 pL of each concentration level working solution in LC-MS water and were diluted 1 :1 with surrogate matrix of 2% BSA in PBS.

[0165] Study design

[0166] Samples were sourced from two independent viral human challenge studies conducted in the United Kingdom.

[0167] Healthy volunteers were screened and recruited for intranasal challenge with the respective virus, followed by quarantine in a clinical research facility. Daily recording of symptoms, measurement of nasal viral loads (via lavage for influenza A virus, flocked swabs for SARS-CoV-2), and collection of plasma samples were undertaken.

[0168] Statistics

[0169] Analysis was done in Excel v16.9, GraphPad Prism and R (R Core Team. R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria., 2023)). Mean ddhC concentrations and their standard errors were plotted using GraphPad Prism. An unpaired two-tailed t-test was used to compare peak ddhC levels between symptomatic and asymptomatic / paucisymptomatic groups and between viral challenge and typhoid challenge groups. For RNA sequencing data analysis, Pearson correlation coefficients and p-values were calculated using the cor and cor.test packages in R with default parameters.

[0170] Results ddhC as a viral acute phase reactant

[0171] Sequential plasma samples from eighteen participants challenged with SARS-CoV-2 or H3N2 influenza A virus underwent ddhC measurement using the targeted LC-MS / MS method described above.

[0172] A clear ddhC response was detected in human challenge participants who developed PCR- confirmed infection following challenge with SARS-CoV-2 and influenza A virus. ddhC concentrations remained under 20 ng / mL in all participants that did not develop infection post challenge. The ddhC response in all patients infected with SARS-CoV-2 and influenza A virus followed an acute phase reactant pattern, rising to a maximum between day 3 and 7 post viral inoculation, and falling to baseline between day 10 and 14. In SARS-CoV-2 and influenza A virus infections, ddhC was elevated in both symptomatic, and paucisymptomatic or asymptomatic participants. These results are shown in Figure 7.

[0173] These data demonstrate that in infection following SARS-CoV-2 and H3N2 influenza A viral challenge, ddhC concentration follows an acute phase pattern over time, rising in the first few days of infection and falling to baseline between day 10 and 14 post inoculation. Thus, using human challenge infection models, it has been shown for the first time that ddhC, a pan-viral biomarker, acts as an acute phase reactant in SARS-CoV-2 and H3N2 influenza A virus infection. This pattern was seen in both symptomatic and asymptomatic / paucisymptomatic infected individuals. The data supports to the use of ddhC as a biomarker for viral infection e.g. acute viral infection, and serves as a salutary reminder that sample timing is critical when researching infection biomarkers.

[0174] Example 12 - ddhC levels are increased in a wide range of different viral infections, but not bacterial infections

[0175] The method described in Example 11 was evaluated in patients that had been infected with a range of infections selected from: influenza A, influenza B, adenovirus, dengue virus, HSV1 , measles, parainfluenza, respiratory syncytial virus, SARSC0V2, Escherichia coli, Enterococcus sp, Pseudomonas sp, Staphylococcus aureus, and Streptococcus sp. ddhC concentration was also measured in serum or plasma of uninfected patients. The results are shown in Figure 8.

[0176] As shown by the data in Figure 8, ddhC is present at elevated levels in the serum and plasma of patients with a broad range of different viral infections. The concentrations of ddhC in serum and plasma of patients with viral infections were higher than those patients with bacterial infections, and those patients that were uninfected. The methods described herein are therefore able to detect (and quantify) ddhC as a pan-viral biomarker of viral infection. As one of ordinary skill in the art will appreciate, the methods described herein are particularly useful in rapidly diagnosing active viral infection in patients suffering from new viral infections e.g. as may be the case in an epidemic or pandemic. The methods described herein are highly sensitive and are capable of detecting very low ddhC concentrations. The methods can quantify ddhC concentrations in biological samples from patients with a broad range of different viral infections, in addition to patients with bacterial infection, and uninfected patients. This allows for patient classification on the basis of viral / bacterial infection or uninfected.

[0177] Example 13 - ddhC can be detected in a range of bodily fluids

[0178] The method of the invention is capable of performing rapid, high-throughput diagnosis of active viral infection based on samples of a range of bodily fluids. This makes the method particularly well suited to applications in e.g. the clinic or even the home. In this Example, ddhC concentrations were measured in CSF, urine, and saliva from patients with various infections. Urine data was obtained using the method described in Example 11. CSF and saliva data was obtained using the method described in Example 11 with a different pre-treatment process as described below:

[0179] Pre-treatment: a. 3 parts of iced cold ACN were added to 1 part of sample. b. The mixture was mixed for 2 minutes at 1400rpm at 4 °C c. The mixture was incubated for 2 hours at 4 °C d. Samples were centrifuged for 10 minutes at 4500 rpm at 4 °C e. Supernatant was collected f. All supernatants were dried under nitrogen. g. The dried extracts were reconstituted with LC-MS grade water. h. The remainder of the pre-treatment was performed as per the ‘direct preparation’ protocol described in Example 11. The results are shown in Figures 9 to 12. Specifically, Figures 9 and 10 show ddhC concentrations measured in cerebrospinal fluid samples for viral and non-viral participants, and for different pathogens, respectively. Figure 11 shows ddhC concentrations measured in urine samples from viral and non-viral participants. N.B. urinary ddhC concentrations were measured, normalized for urinary creatinine concentration, and expressed as a urine ddhC : creatinine ratio (ng / pmol). Figure 12 shows ddhC concentrations measured in saliva samples from viral and non-viral participants. These results show that ddhC can be detected in a range of biological samples, including serum / plasma, CSF and urine. Increased ddhC levels were observed in a range of viral infections in both plasma / serum (Figure 8) and CSF (Figure 10). Further, ddhC levels were distinguishable between viral / non-viral participants in saliva and urine, indicating the flexibility of ddhC as a pan-viral marker across different sample types.

[0180] Example 14 - ddhC as a pan-viral marker in pigs

[0181] Rather than being limited to use in humans, the methods of the invention are capable of performing rapid, high-throughput diagnosis of active viral infection based on samples taken from mammals such as pigs. This makes the method particularly well suited to applications in animal husbandry and agriculture. In this Example, ddhC concentrations were measured in serum from pigs with influenza A - H1 N1 , using the method with the different pre-treatment process used to obtain the CSF and saliva data which is described in Example 13. The pigs were infected with the virus at day 0. The results are shown in Figure 13.

[0182] Example 15 - ddhC as a pan-viral marker in plants

[0183] The methods of the invention are applicable even beyond mammalian use, and can perform rapid, high-throughput diagnosis of active viral infection based on samples taken from plants. This makes the method particularly well suited to applications in horticulture and agriculture. In this Example, ddhC concentrations were measured in plants with and without CBSV (cassava leaf banana virus). The following procedure was followed: a. 500 mg of leaves were weighed b. Extracted with grinding (Solvent systems for extraction were MeOH / FW 4:1 and ACN / MeOH / H2O 2:2:1) - 5ml total c. Sonication d. Centrifugation e. The extracts were filtered through syringe filter 0.45 urn followed by 0.22 urn f. Dried in Speedvac or under nitrogen g. Dried extracts were stored at -80 °C h. The dried extracts were reconstituted with LC-MS grade water. i. Then ddhC concentrations were measured using the method described in Example 11 . The results are shown in Table 15.

[0184] Table 15. Plant extract ddhC concentration (ng / mL) measured in plants with CBSV.

[0185] All documents referred to in this application are hereby incorporated by reference in their entirety.

Claims

Claims1) A method for determining by mass spectrometry the quantity of ddhC present in a biological sample from a subject comprising:(i) preparing the sample for mass spectrometry analysis;(ii) adding internal standard comprising isotope labelled ddhC to the sample;(iii) obtaining one or more reference samples;(iv) assaying the sample and the one or more reference samples for the presence of ddhC using mass spectrometry and thereby determining the quantity of ddhC present in the biological sample.

2. The method of claim 1 wherein the mass spectrometry is liquid chromatography - tandem mass spectrometry (LC-MS-MS).

3. A method for diagnosing a viral infection in a subject comprising: i) determining by mass spectrometry the absolute quantity and / or concentration of ddhC present in a biological sample from a subject according to the method of claim 1 or claim 2, ii) diagnosing a viral infection if ddhC is greater than a particular threshold.

4. The method according to claim 3, wherein: a) (i) the biological sample is blood plasma or serum, and the particular threshold of ddhC for diagnosing a viral infection is at least 100 ng / mL; and / or(ii) the biological sample is cerebrospinal fluid, and the particular threshold of ddhC for diagnosing a viral infection is at least 90 ng / mL, e.g. at least 100 ng / mL; and / or(iii) the biological sample is urine, and the particular threshold of ddhC (expressed as a urine ddhC : creatinine ratio) for diagnosing a viral infection is at least 1000 ng / pmol, e.g. at least 2000 ng / pmol; and / or(iv) the biological sample is saliva, and the particular threshold of ddhC for diagnosing a viral infection is at least 5 ng / mL, e.g. at least 10 ng / mL; or b) the biological sample is plant extract, and the particular threshold of ddhC for diagnosing a viral infection is at least 50 ng / mL, e.g. at least 60 ng / mL.

5. The method according to any preceding claim, wherein the step of preparing a biological sample from the subject comprises:(i) mixing the sample with organic solvent, such as acetonitrile.(ii) centrifuging the sample to separate a solute from a solid phase;(iii) retaining the solute for mass spec analysis.

6. The method according to any preceding claim, wherein: a) the reference sample is prepared by performing solid phase extraction on(i) an aliquot of the subject sample; or(ii) a sample of reference serum (such as the National Phenome Centre’s long term reference (LTR) serum and LTR plasma); thereby extracting ddhC, optionally when the chromatographic medium is (OASIS HLB), or comprises N-vinylpyrrolidine and divinylbenzene or b) a surrogate reference sample comprising BSA and phosphate buffered saline (PBS).

7. The method for use according to any preceding claim, wherein the ddhC is 13C isotopelabelled ddhC, wherein one or more carbon atom in ddhC is a 13C atom, optionally wherein every carbon atom in ddhc is a 13C atom, preferably wherein every ribose carbon is 13C.

8. The method for use according to any preceding claim wherein assaying the sample for the presence of ddhC using mass spectrometry comprises performing LC-MS-MS wherein the liquid chromatography is hydrophilic interaction chromatography, preferably wherein the chromatographic media comprises polyethoxysilane.

9. The method for use according to any preceding claim wherein the absolute quantity of ddhC present is determined.

10. The method for use according to any one of claims 3 to 9 wherein:(i) the viral infection is an active viral infection; and / or(ii) the method is a pan-viral diagnostic method.

11. A method for validating a substitute matrix for use in the method of any preceding claim comprising:(i) preparing a dilution series of one or more aliquots of sample using the substitute matrix;(ii) preparing a dilution series of one or more aliquots of sample with zero matrix (e.g. an SPE-treated matrix);(iiii) measuring the concentration of ddhC in the sample at each dilution point in the dilution series for the substitute matrix and the zero matrix, optionally wherein the reference sample comprises substitute matrix and ddhC.

12. The method according any of the preceding claims wherein the biological sample from the subject is Whole Blood, Blood Plasma, Serum, Cerebrospinal Fluid, Saliva, Urine, or plant extract.

13. The method according any of the preceding claims wherein, wherein the sample is not processed to remove lipids prior to assaying the sample for the presence of ddhC.

14. A kit for diagnosing a viral infection comprising one or more stably isotopically labelled standards, optionally wherein the kit comprises one or more of: i) protocol for preparing biological samples and reference samples before analysis, ii) instruction for preparation of surrogate and zero matrix, iii) analysis order and analytical plate layout; iv) parameters for the assessment of data quality.

15. The kit according to claim 14 wherein the kit contains machine-readable media containing mass spectrometric transitions or target mass spectra for ddhC and the SIL-IS, optionally wherein the mass spectrometric transitions are selected from Table 1.

16. A method for treating a viral infection in a subject, comprising i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the method of any one of claim 1 to claim 13, ii) diagnosing a viral infection if absolute quantity of ddhC is greater than a particular threshold; (iii) administering an antiviral agent to the subject.

17. A method for determining the severity of a viral infection, comprising i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the method of any one of claim 1 to claim 13, wherein i) the quantity of ddhC in a sample from the subject is determined, ii) the severity of the viral infection is determined using the quantity of ddhC.

18. A method for assessing the efficacy of a treatment for a viral infection in a subject previously treated for a viral infection comprising: i) determining by mass spectrometry the absolute quantity of ddhC present in a biological sample from a subject according to the method of any one of claim 1 to claim 13, ii) the quantity of ddhC present in the sample from the subject is used to assess the efficacy of the treatment for a viral infection.

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