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WO2014138795A1 - Détection d'agent pathogène infectieux et de maladie contagieuse - Google Patents

Détection d'agent pathogène infectieux et de maladie contagieuse Download PDF

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Publication number
WO2014138795A1
WO2014138795A1 PCT/AU2014/000254 AU2014000254W WO2014138795A1 WO 2014138795 A1 WO2014138795 A1 WO 2014138795A1 AU 2014000254 W AU2014000254 W AU 2014000254W WO 2014138795 A1 WO2014138795 A1 WO 2014138795A1
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Prior art keywords
individual
enzyme
pathogen
activity
disease
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English (en)
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Nicholas Jonathan Cole KING
Shane Ross THOMAS
Amanda Wing Shee YEUNG
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University of Sydney
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University of Sydney
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Priority claimed from AU2013900950A external-priority patent/AU2013900950A0/en
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Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the invention relates to the detection of infectious pathogen and contagious disease, especially sexually transmitted disease and related pathogens, and to devices and kits for enabling detection of same.
  • STD sexually transmitted diseases
  • Herpes simplex virus -1 HSV-1
  • Herpes simplex virus -2 HSV-2
  • virus particles may generally be detected in epithelia, and an infected individual may present with epithelial ulceration, headaches, malaise and muscular aches, in addition to localised itching, swollen lymph nodes, neuralgic pain and difficulty urinating.
  • HSV-2 infection serious complications of HSV-2 infection include meningitis and encephalitis, as well as the development of neonatal herpes in babies if the virus is transmitted to the infant during vaginal delivery. HSV-2 infection also increases the risk of HTV acquisition by 3- to 4-fold.
  • Most STDs, or the relevant symptoms, may be treated by various drugs. For example, genital herpes can be managed by acyclovir, valaciclovir and famciclovir, all of which suppress viral replication.
  • STDs that are not manifestly apparent in an individual until well after the relevant aetiological agent has had opportunity to establish widespread infection. It would be particularly useful to determine whether an individual is likely to develop symptoms of disease long before the symptoms become apparent, as this would allow early therapeutic intervention, potentially minimising the duration or extent of a symptomatic phase of the STD.
  • the invention seeks to minimise one or more of the aforementioned problems and/or to provide means and/or methods for addressing an aforementioned need and in one embodiment provides a method for determining whether an individual comprises an infectious pathogen including:
  • a method for determining whether an individual has an infectious pathogen in the form of infectious HSV-2 including: - providing a test sample of vaginal mucus in the form of a sample obtained from an individual in whom the presence of infectious HSV-2 is to be determined;
  • a method for determining whether an individual has contagious genital herpes including:
  • a method for preventing the appearance of symptoms of disease in an individual including the step of:
  • a device for use in detennining whether an individual contains a contagious disease or infectious pathogen including means for determining whether the individual contains an increased expression or activity of an enzyme of the individual that has substrate specificity for an amino acid.
  • a device including: - a solid phase
  • reagent attached to the solid phase for detecting the expression or activity ot an enzyme of an individual that has substrate specificity for an amino acid.
  • reagent attached to the solid phase for detecting the expression or activity of an enzyme of an individual that has substrate specificity for an amino acid
  • a device comprising means for use in executing a method according to any one of the above described embodiments.
  • infectious pathogen' generally refers to a disease-causing agent that exists in a form in an infected individual that enables the agent to establish disease in a non-infected individual (i.e. a recipient) when transmitted from an infected individual to the recipient, for example by skin, mucous membrane, or body fluid contact, for example, as may occur during intercourse.
  • Non-infectious pathogen' generally refers to an agent that exists in a form in an infected individual that does not enable the agent to be transmitted from infected individual to a recipient, or if transmitted, does not enable the agent to establish a viable infection or disease in the recipient.
  • a single type of disease-causing agent may exist as an infectious pathogen, or as a non- infectious pathogen in an infected individual.
  • a pathogen is generally an infectious pathogen when the infected individual has active disease. Where the individual has non-active disease, the pathogen is generally non-infectious.
  • HSV-2 Herpes simplex virus type 2
  • HSV-2 particles represent an infectious pathogen because they are transmissible as between infected individual and recipient and they are capable of establishing infection and one or more symptoms of disease in the recipient.
  • non-active disease i.e. remission
  • the HSV-2 particles represent a non-infectious pathogen because the particles are not transmitted between infected individual and non-infected individual, or otherwise the particles do not establish disease in the latter.
  • a disease-causing agent exists in the form of an infectious pathogen or non-infectious pathogen.
  • One factor may be tissue location.
  • some pathogens, such as HSV-2 are present in the epithelium during active disease only. In non-active disease, HSV-2 is found in neuronal bodies only. Location in the epithelium is relevant as to whether the pathogen is infectious because the epithelium is the primary point of contact between individuals when there is skin to skin or mucous membrane to membrane contact.
  • Another factor may be whether the disease-causing agent has expression of the necessary complement of genes for enabling the agent to exist in viable form extracellularly, and/or to infect the cells of a recipient tissue.
  • some disease-causing agents that are noninfectious pathogens may not contain an essential protein for infecting a target cell through a specific cell surface receptor, or might not otherwise contain the necessary proteins for preventing extracellular degradation.
  • the invention described herein is concerned with determining whether an individual contains an infectious pathogen.
  • the invention enables an infected individual to determine whether, at the time of practice of the invention, the individual has a disease-causing agent that will be transferred to, and establish infection in an individual should there be contact between the individual and the recipient shortly after practice of the invention.
  • the invention also enables an infected individual to determine whether contact with another individual (i.e. a recipient) prior to practice of the invention is likely to have transmitted a disease-causing agent, or established disease in the recipient.
  • the invention is distinguished from merely detecting whether an individual contains a pathogen, infectious or otherwise.
  • the invention is distinguished from detecting whether an individual contains a pathogen that at some later time could become an infectious pathogen. Consistent with the invention, generally the invention will find application among individuals who, prior to practice of the invention, are known to be infected with a disease-causing agent, and the objective among these individuals is to determine whether they are likely to transmit disease to another individual.
  • the inventors have found that enzymes that degrade amino acids are useful as a biomarker for the presence of infectious pathogens. While not wanting to be bound by hypothesis, the inventors have recognised that either or both of the expression and activity of these enzymes is induced in an infected individual when a disease-causing agent transitions from a non-infectious pathogen to an infectious pathogen. It is believed that the induction of these enzymes constitutes one element of the host response to activation of a pathogen. In one example the aforementioned transition requires protein synthesis and it is considered that by increasing the activity or expression of these enzymes, the host is able to deprive the pathogen of supply of essential amino acids for transition from a non-infectious to infectious state.
  • the above finding is particularly important where the individual is asymptomatic for active disease and yet contains an infectious pathogen, because it ostensibly establishes the level of expression or activity of the relevant enzyme as a biomarker of the infectious state of the disease-causing agent in the individual.
  • the invention it becomes possible to determine whether an individual - who may not present with any symptoms of the relevant disease - is nonetheless likely to transmit or otherwise establish disease in another. It is believed that with practice of the invention it will become possible to minimise the spread of transmissible disease and the underlying infectious agent, thereby decreasing the incidence of disease in a given population. In this context it is believed that the invention will be particularly advantageous for minimising the prevalence of STDs.
  • Another advantage from the practice of the invention is to minimise the symptoms and potentially serious complications of disease by facilitating earlier therapeutic intervention.
  • diseases there is a connexion between the disease state (in terms of development of symptoms) and infective potential of the disease-causing agent.
  • individuals who have infectious pathogens may have asymptomatic disease that, without therapeutic intervention, could develop into the full spectrum of symptoms of the disease.
  • the identification of an asymptomatic individual as having infectious pathogen also identifies the individual as a candidate for therapeutic intervention, to prevent the development of symptoms or complications of disease in the individual, long before these hallmarks of disease are manifested.
  • a method for determining whether an individual has an infectious pathogen, or whether an individual has contagious or transmissible disease is a disease that is capable of spreading from individual to individual by infection with a disease-causing agent.
  • an individual having contagious or transmissible disease is generally an individual who has an infectious pathogen.
  • Individuals who have non-infectious pathogen generally do not have contagious disease. These individuals may develop contagious disease should they develop infectious pathogen.
  • an individual in remission for genital herpes develops contagious, transmissible disease when the individual develops infectious pathogen in the form of HSV-2 particles that arc located in epithelial cells.
  • the method includes the following steps: providing an individual in whom the presence of infectious pathogen is to be determined; determining whether the individual contains an increased expression or activity of an enzyme of the individual that has substrate specificity for an amino acid; wherein the individual is determined to have an infectious pathogen where the individual is determined to contain an increased expression or activity of the enzyme.
  • an important advantage of the invention is that the methods described herein may be applied to an individual irrespective of whether the individual contains symptoms of the disease controlled by the relevant pathogen. Therefore, in certain embodiments, the individual is asymptomatic for the disease i.e. does not have a symptom that is normally associated with an individual that is infected with the pathogen. In other embodiments, the individual may have some or all of the symptoms or complications of the disease.
  • the method is applied for the purpose of determining whether the individual has contagious genital herpes, or for determining whether an individual has infectious HSV -2.
  • the individual may have none of, or some of, or all of the following symptoms: epithelial ulceration, headaches, fever, malaise and muscular aches, localised itching, swollen lymph nodes, neuralgic pain and difficulty urinating.
  • 'Direct detection ⁇ refers to detection of the pathogen itself.
  • Examples of direct detection include detection of the pathogen or component thereof by an agent that binds to one or more components of the pathogen, for example by an agent that is an antibody, polynucleotide or some other chemical that binds to the pathogen.
  • 'indirect detection' refers to the detection of a chemical species, generally a host factor, that does not form a component of the pathogen itself.
  • the pathogen is contained in the individual in an amount that is insufficient to enable direct detection of the pathogen in the individual.
  • the pathogen may be detected in tissue regions where the pathogen is generally considered to be non-infectious, but not directly detected in tissue regions where, if present, the pathogen would be considered to be infectious.
  • infectious pathogen is detected according to the method of the invention in an infected individual in circumstances where the pathogen cannot be directly detected in epithelium.
  • induction of the relevant amino acid degrading enzyme occurs prior to synthesis of infectious pathogen in the epithelial tissue.
  • the enzyme that is to be measured or detected according to the invention is one that has substrate specificity for an amino acid that is an essential amino acid.
  • An essential amino acid is one which the body does not produce or has limited capacity to produce.
  • These amino acids are provided in the body through diet. These enzymes are considered to be particularly important, because as discussed above, it is believed that these form part of the host response to re-activation of disease as a pathogen transitions from a non-infectious to an infectious phenotype. More particularly, it is believed that these enzymes deplete the host pool of otherwise available amino acid residues that are essential for formation of infectious pathogen. By depleting amino acids that are essential amino acids, the host response tends to minimise the number of infectious particles produced during disease reactivation. Examples of enzymes are those having substrate specificity for tryptophan and arginine.
  • the invention recognises that the connexion between expression and activity of the amino acid-metabolising enzymes and the formation of infectious pathogen means that the former can be used as a biomarker of the latter - i.e. the induction or increase in expression or activity of the enzyme is a marker of the formation or presence of infectious pathogen.
  • the invention recognises that the change in expression profile of these amino acid - scavenging enzymes, as disease reactivation occurs during transition from non-infectious to infectious phenotype, long precedes the development of symptoms that generally present in contagious disease such as an STD.
  • the enzyme has substrate specificity for tryptophan or for arginlne.
  • the enzyme is human indolcamine 2,3-dioxygenase (IDO).
  • IDO indolcamine 2,3-dioxygenase
  • the enzyme may be NOS2, otherwise known as inducible nitric oxide synthase.
  • the enzyme may have substrate specificity for other than an amino acid.
  • an enzyme within this embodiment is myeloperoxidase (MPO) and the relevant assay determines for whether the expression or activity of MPO is enhanced,
  • the method of the invention may involve measuring the expression or activity of the relevant enzyme, or both expression and activity may be measured.
  • the invention includes: determining whether the individual contains an increased amount of the enzyme; to determine whether the individual contains an increased expression of the enzyme.
  • Expression of the enzyme' generally refers to the amount of enzyme produced in the individual or sample. Generally this is a measure of the amount of enzyme in the individual or sample at the time of the application of the method, although in some embodiments, it may also be possible to alternatively, or additionally measure the rate of expression of the enzyme.
  • the amount of enzyme in the relevant sample can be determined by methods that directly detect the level of protein enzyme, for example by serology are preferred, although where there is an understood linkage as betvveen the amount of enzyme protein and amount of polynucleotide, for example RNA, it may be possible to additionally or alternatively determine the amount of RNA from which the enzyme is translated. Examples of these methods are further described below.
  • the invention includes the steps of: determining whether the individual contains an increased amount of a cataboHte of the enzyme; thereby determining whether the individual contains an increased activity of the enzyme.
  • a ⁇ cataboHte* is generally understood as being a molecular species arising from reaction of an enzyme with substrate.
  • the cataboHte may be an ⁇ immediate species' i.e. a species arising immediately from the reaction of enzyme with substrate.
  • the cataboHte may be another molecular species arising from metabolism or other modification of the immediate species.
  • the immediate species is N-formylkynurenine.
  • Other molecular species arising from metabolism of N-formylkynurenine that could be measured or detected are formic acid or kynurenine.
  • the cataboHte may be nitrite or related nitrite ion.
  • Nitrite-indicator polymer matrices for identifying nitrites in urine are well known in the art.
  • the method includes the following steps: determining whether the individual contains an increased amount of kynurenine; thereby determining whether the individual contains an increased activity of the enzyme.
  • the method includes the step of determining whether the individual contains an increased amount of a molecule, the expression of which is induced on exposure to a cataboHte of IDO,
  • a cataboHte of IDO One example is the aryl hydrocarbon receptor (AHR), which binds to kynurenine.
  • the method includes the follow steps: providing a sample of body fluid obtained from the individual and thereafter; determining whether the sample contains an increased expression or activity of an enzyme of the individual that has substrate specificity for an amino acid; wherein the individual is determined to contain an infectious pathogen where the sample is determined to contain an increased expression or activity of the enzyme.
  • the sample of body fluid is made available before commencement of the method of the invention.
  • the skilled worker is aware of many methods for obtaining a sample of body fluid.
  • One particularly preferred method particularly where the infectious pathogen or contagious disease of interest is HSV-2 and genital heipes respectively, is that which produces a sample of vaginal mucus. This method might include scraping or wiping inside the vagina to obtain a sample.
  • the method includes the step of providing a sample of body fluid.
  • body fluid Any body fluid that is expected to contain infectious pathogen and that is transmissible from one individual to another as a consequence of contact of skin or mucous membranes could be used in a method described herein. Examples include blood, plasma, serum, lymph, semen, saliva, sputum, sebum, uterine and vaginal secretions and related mucous and tear.
  • the body fluid is typically cell-free although in some circumstances it may contain residual cells or fragments thereof.
  • the sample contains vaginal mucus.
  • the methods described herein are applicable to the assessment of whether an individual, particularly an asymptomatic individual is at risk for spreading a sexually transmissible disease.
  • the individual may be aware of whether he/she has an infection with a relevant pathogen, but unaware, for example, prior to intercourse, as to whether he/she is likely to contain infectious pathogen capable of being transmitted during intercourse to a partner, thereby establishing disease in the latter.
  • diseases of interest include genital herpes, Chlamydia, Gonorrhoea, Syphilis and Candidiasis.
  • the STD is herpes.
  • infectious pathogen the subject of the methods described herein may be a virus, bacteria, fungus or other micro-organism. Particularly examples include HSV-1, HSV-2, HIV, HPV.. In a particularly preferred embodiment, the infectious pathogen is HSV-2.
  • the presence of a given protein, or level of expression of a given protein in a sample, such as an enzyme or protein or protein catabolite thereof can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry.
  • Immunoassays i.e. assays involving an element of the immune system are particularly preferred. These assays may generally be classified into one of:
  • purified antigen is used to detect an antibody in host serum.
  • purified antigen is bound to solid phase by adsorption or indirectly through another molecule and host serum or other body fluid is applied followed by another antibody for detecting presence or absence of host antibody;
  • ELISA, RIA or like assay involving the application of a liquid sample to an assay system.
  • the sample may or may not be processed prior to contact with an antibody.
  • an immunoassay format may not be appropriate.
  • a further assay format which does not require formation of an immune complex is one in which an assay output is the result of catalysis of a substrate and the output is observed for example by measuring a change in optical density.
  • antibodies that arc useful in the invention are those that bind selectively or specifically to IDO, NOS2 or to catabolites of these enzymes.
  • the catabolite is a small molecule, it may be conjugated to a hapten to increase molecular weight, thereby enabling an appropriate humoral response for antibody production.
  • the antiserum may be monoclonal or polyclonal.
  • capture agents include those which bind to a catabolite of IDO, NOS2.
  • the capture agent is a receptor.
  • the capture agent may be the AHR receptor.
  • the capture agent is an enzyme.
  • the capture agent may be kynurenine-3-hydroxylase.
  • assay formats may involve various types of chromatography. Examples include chromatographic methods that are not based on serology. In one example, the chromatographic method may employ a colour change to indicate the presence or absence of infectious agent or contagious disease. In another embodiment, the assay format may involve a particular chemical reaction, such as an Ehrlich reaction, and the assay detects for the presence or absence of the relevant reaction product.
  • chromatographic methods that are not based on serology.
  • the chromatographic method may employ a colour change to indicate the presence or absence of infectious agent or contagious disease.
  • the assay format may involve a particular chemical reaction, such as an Ehrlich reaction, and the assay detects for the presence or absence of the relevant reaction product.
  • a nucleic acid that encodes the enzyme, or that is complementary to a nucleic acid that encodes the enzyme is measured.
  • the nucleic acid may be one which can be used to determine the presence of a given protein, or level of expression of a given protein in a host as per a molecular genetic approach.
  • a polynucleotide that is complementary to a nucleic acid (DNA, UNA, cDNA) that encodes a target protein is hybridised to the nucleic acid and hybridisation is detected.
  • DNA, UNA, cDNA DNA, UNA, cDNA
  • Another example is quantitative PCR. Others include quantitative Northern and Southern blotting, and microarray.
  • hybridisation of nucleic acid molecules may be controlled by the type of buffer used for hybridisation and the temperature of the buffer, 'High stringency conditions' are conditions in which the buffer includes about 0.1 x SSC, 0.1% SDS and the temperature is about 60°C.
  • the enzyme or catabolite is directly detected. This is otherwise known, as a ' direct detection' of the enzyme or catabolite to measure the level of expression or activity of the enzyme. This may involve detection of the whole enzyme or fragment of it.
  • the level of expression of a molecule, the expression of which is modulated in accordance with the up-rcgulation of expression or activity of the enzyme is measured. This is otherwise known as an * indirect detection* of the enzyme to measure the level of expression or activity of the enzyme.
  • the method includes the step of: comparing the expression or activity of the enzyme of the individual with the expression or activity of the enzyme of an individual that does not contain the pathogen (the latter being a negative control), thereby determining whether the individual contains an increased expression or activity of the enzyme.
  • the level of expression or activity of the relevant enzyme in the individual is assessed by the steps of: measuring the level of expression or aotivity of the enzyme; and comparing the measured level with an uninfected control that describes the level of expression or activity of the enzyme as observed in the same tissue of a subject that has been determined as not having an infection with the relevant pathogen.
  • An 'uninfected control' may be derived by measuring the level of expression or activity of the enzyme in a tissue or fluid of a subject that, but for an absence of a relevant infeotion, is generally the same or very similar to the tissue or fluid of the individual selected for assessment for likelihood of infectious pathogen (the latter otherwise known as the 'test sample').
  • the uninfected control is derived by measuring the level of expression or activity of the enzyme in a cell line or an engineered tissue.
  • the method includes measuring the level of expression or activity of the enzyme in the uninfected control to compare the measured level in the test sample with the level in the uninfected control.
  • an internal standard is applied. This may be used to ensure that the method operates within accepted decision limit quality control criteria.
  • the assay then provides a value/number/result or other output for each test sample. That output is then deemed to represent infection or non-infection based on independent data derived from frequency distributions of results from an uninfected control.
  • This control may describe distributions of results obtained from more than one uninfected subject, for example, from an uninfected population which may be of the same specios, geographic origin, age, sex as the test sample.
  • a positive-negative cut- point for the method is determined from these distributions to provide defined levels of diagnostic sensitivity and specificity for the method.
  • the internal standard or reference may obviate the need to physically provide an uninfected control in the form of uninfected cells or fluid or otherwise to physically measure the level of the target in an uninfected control.
  • the level of expression in an uninfected control has been predetermined and may be provided for example in the form of written information that is supplied with a diagnostic kit.
  • the measurement of the level of expression or activity of the enzyme in the fluid or tissue of the subject for deriving the uninfected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.
  • the level of expression or activity of the enzyme in the individual is assessed by the steps of: measuring the level of expression or activity of the enzyme in a fluid or tissue of the individual; and comparing the measured level with an infected control that describes the level of expression or activity of the enzyme as observed in the same tissue of a subject that has been determined as having infection with the relevant pathogen.
  • An 'infected control' is generally derived by measuring the level of expression or activity of the enzyme in a fluid or tissue of a subject that is generally the same or very similar to the fluid or tissue of the test sample.
  • the measurement of the level of expression or aotivity of the enzyme in the fluid or tissue of the subject for deriving the infected control is generally done using the same assay format that is used for measurement of the target protein in the test sample.
  • the method includes measuring the level of expression or activity of the enzyme in the infected control to compare the measured level in the test sample with the level in the infected control.
  • an internal standard may be applied that obviates the need to provide an infected control or otherwise to measure the level of the target in an infected control.
  • the measurement of the level of expression or activity of the enzyme in the fluid or tissue of the subject for deriving the infected control is generally done using the same assay format as that that is used for measurement of the enzyme in the test sample. However, again it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.
  • the method includes: measuring the level of expression or activity of the enzyme in a fluid or tissue of the individual; and comparing the measured level with an uninfected control and an infected control.
  • the amount of enzyme may be variously described in terms of specific units of activity, weight ⁇ volume or weight of sample, or moles per volume, depending on the method used for measurement or detection of amount of enzyme.
  • the assay format further includes a means for detecting for the presence of a relevant pathogen.
  • the purpose of this means is to determine the reason underlying the increased expression or activity of the relevant enzyme. Accordingly, the assay format identifies whether there is a pathogen in an activated state, or a disease in contagious form and identifies the relevant pathogen.
  • the assay format includes means for detecting for the presence of HSV-2.
  • the assay format includes means for detecting the presence of more than one pathogen.
  • the assay may include single means for detecting more than one pathogen, or multiple means each for detecting a specific pathogen only.
  • IDO indoleamine 2,3-dioxygenase
  • Kyn in the mucus is a novel diagnostic marker of recrudescent HSV-2 infection in humans, since the expression of IDO after the first infection is even higher upon secondary exposure.
  • mice were anaesthesised via intraperitoneal injection of Avertin.
  • the vagina was washed three times with 50 ⁇ > sterile phosphate-buffered saline using a flame-polished bevelled-edge 200 ⁇ , plastic pipette tip to remove excess mucus.
  • HSV-2 (strain 183; 10 s plaque- forming units) was then introduced intravaginally using a sterile 200 ⁇ iL pipette tip in a final volume of 20 ⁇ ,.
  • Mock-infected mice were inoculated with 20 sterile phosphate-buffered saline (vehicle).
  • the mice were laid supine with their pelvis raised for at least 30 min before transfer into their respective cages.
  • mice On day 1, 3, 5, 7 and 10 post-infection, mice were euthanised and the vaginal lumen was washed 3 times with 50 ⁇ , phosphate-buffered saline to collect the mucus.
  • the mucus was stored at -80°C until analysis.
  • the vaginal wash samples were thawed and mixed with 20% (w/v) ice-cold trichloroacetic acid in a 3;1 ratio and centrifuged at 16100 x g for 15 min at 4°C.
  • the clarified supernatant was stored at -80°C until analysis by HPLC using a Hypcrsil 3 ⁇ ODS CI 8 column.
  • Samples were eluted with 100 mM chloroacetic acid and 9% acetonitrile (pH 2.4) at a rate of 0.5 mL/min.
  • L-Trp and Kyn were detected by UV absorbance at 280 and 364 nm, with retention times of -10-12 min and 6-7 min, respectively.
  • peak areas were compared to authentic L-Trp and Kyn standards of known concentrations that are included in each run.

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  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
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Abstract

L'invention concerne des méthodes et des dispositifs pour déterminer si un individu présente un agent pathogène infectieux ou une maladie contagieuse, notamment, mais de façon non limitée le VHS-2 et l'herpès génital.
PCT/AU2014/000254 2013-03-13 2014-03-13 Détection d'agent pathogène infectieux et de maladie contagieuse Ceased WO2014138795A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034014A1 (fr) * 1996-03-15 1997-09-18 Yale University Procedes et moyen de detection d'une synthase d'oxyde nitrique
US20060099628A1 (en) * 2004-11-10 2006-05-11 Wei-Mei Ching Diagnostic assay for rickettsia prowazekii disease by detection of responsive gene expression

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034014A1 (fr) * 1996-03-15 1997-09-18 Yale University Procedes et moyen de detection d'une synthase d'oxyde nitrique
US20060099628A1 (en) * 2004-11-10 2006-05-11 Wei-Mei Ching Diagnostic assay for rickettsia prowazekii disease by detection of responsive gene expression

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AZARDARYANY, M. K. ET AL.: "Modulation of immunity against gential Herpes simplex virus'.", CYTOKINE, vol. 63, September 2013 (2013-09-01), pages 245 *
BECERRA, A. ET AL.: "Increased activity of indoleamine 2,3-dioxygenase in serum from acutely infected dengue patients linked to gamma interferon antiviral function'.", JOURNAL OF GENERAL VIROLOGY, vol. 90, 2009, pages 810 - 817 *
RAPOPORT, M. I. ET AL.: "Studies of tryptophan metabolism in experimental animals and man during infectious illness'.", THE AMERICAN JOURNAL OF CLINICAL NUTRITION, vol. 24, no. 7, 1971, pages 807 - 814 *
REINHARD, J. F.: "Altered tryptophan metabolism in mice with herpes simplex virus encephalitis: increases in spinal cord quinolinic acid'.", NEUROCHEMICAL RESEARCH, vol. 23, no. 5, 1998, pages 661 - 665 *
SANNI, L. A. ET AL.: "Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria'.", AMERICAN JOURNAL OF PATHOLOGY, vol. 152, no. 2, 1998, pages 611 - 619 *

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