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WO2004001070A1 - Marqueurs de substitution permettant de determiner l'etat de maladie d'une personne infectee par mycobacterium tuberculosis - Google Patents

Marqueurs de substitution permettant de determiner l'etat de maladie d'une personne infectee par mycobacterium tuberculosis Download PDF

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Publication number
WO2004001070A1
WO2004001070A1 PCT/GB2003/002630 GB0302630W WO2004001070A1 WO 2004001070 A1 WO2004001070 A1 WO 2004001070A1 GB 0302630 W GB0302630 W GB 0302630W WO 2004001070 A1 WO2004001070 A1 WO 2004001070A1
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Prior art keywords
seq
individual
tuberculosis
infected
disease status
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PCT/GB2003/002630
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English (en)
Inventor
Kenneth Duncan
Pauline Teresa Lukey
Rohitbhai Mistry
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Glaxo Group Limited
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Publication date
Priority claimed from GB0214251A external-priority patent/GB0214251D0/en
Priority claimed from GB0300928A external-priority patent/GB0300928D0/en
Application filed by Glaxo Group Limited filed Critical Glaxo Group Limited
Priority to AU2003241055A priority Critical patent/AU2003241055A1/en
Publication of WO2004001070A1 publication Critical patent/WO2004001070A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to surrogate markers, and in particular to methods for determining the disease status of an individual that has been or is infected with Mycobacterium tuberculosis.
  • the present invention further relates to diagnostic kits and to methods of treatment and clinical trial design utilizing the methods described herein.
  • Tuberculosis is a highly contagious airborne infectious disease caused by the bacterial pathogen M. tuberculosis. Worldwide about 3 million people die of tuberculosis every year, the biggest death toll from a single pathogen, and there are estimated to be about 8 million new cases each year. In 1993, the World Health Organization (WHO) described tuberculosis as a global health emergency.
  • WHO World Health Organization
  • Tuberculosis attacks the respiratory system and is easily spread through coughing and sneezing.
  • the clinical disease generally manifests as a pulmonary infection with progressive lung granuloma formation, liquefaction and cavitation. This is accompanied by weight loss, fever and a productive infectious cough containing acid fast bacilli.
  • the causative bacterium, M. tuberculosis is a facultative intracellular pathogen which can survive and replicate within the host macrophage.
  • latent infection latent TB
  • latent TB The infected individual remains at risk of developing active disease by activation of the latent infection, generally under conditions of immunosuppression, with a lifetime risk of 10%.
  • co-infection with HIV increases the risk of activation of latent tuberculosis to clinical disease, to 10% per annum.
  • a proof of concept study for a new therapy for tuberculosis requires administration of the novel therapy in combination with the existing therapy for six months, followed by a two- year follow-up period to detect relapse patients.
  • the length of study required is a serious hindrance to the development of new therapies for tuberculosis. It is estimated that between 5 and 20% of patients treated for tuberculosis will relapse with active disease within two years of completion of therapy. At present it is not possible to predict who these patients will be.
  • the only potential marker which has been used clinically is sputum conversion at two months.
  • the number of patients who are sputum negative (no bacilli detectable microscopically) after two months of therapy appears to correlate with the clinical outcome (cure vs recurrent).
  • the evidence is weak and due to poor predictability it has not been used in clinical trials aimed at testing drug efficacy.
  • the present invention provides one or more genetic markers or biomarkers which can be used (singly or in panels) to predict the disease status of an individual that has been or is infected with M. tuberculosis.
  • the present invention provides a method of determining the disease status of an individual that has been or is infected with M. tuberculosis comprising:
  • a preferred method comprises analysing the expression of:
  • each of the polynucleotide sequences comprising SEQ ID No:1 to SEQ ID No:8, or (ii) any other wild type polynucleotide sequence encoding the same protein or peptide as that encoded by the polynucleotide sequences defined in (i).
  • a particularly preferred method comprises analysing the expression of:
  • each of the polynucleotide sequences comprising SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:7, SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11 , or (ii) any other wild type polynucleotide sequence encoding the same protein or peptide as that encoded by the polynucleotide sequences defined in (i).
  • test expression pattern is compared with the expression pattern of an individual that has been or is infected with M. tuberculosis and having a known disease status.
  • the individual is classified into one of the following disease statuses: recurrent tuberculosis; latent tuberculosis; active tuberculosis and cured of tuberculosis to enable the disease status of the unknown individual to be determined.
  • the methods of the invention can be performed on any suitable body sample such as a body fluid sample.
  • a body fluid sample such as a body fluid sample.
  • the sample is a blood and/or urine sample.
  • the invention provides a method of performing a clinical trial for a new therapy for the treatment of an individual that has been or is infected with M. tuberculosis comprising:
  • the invention provides a method of treating an individual infected with M. tuberculosis comprising:
  • the invention provides a method of monitoring the effectiveness of an anti-mycobacterial therapy for the treatment of individuals infected with M. tuberculosis comprising:
  • step (b) after a prescribed period of treatment, determining the disease status of said individual in accordance with the methods described herein; and (c) continuing with, modifying or ceasing the therapy based on the disease status determined in step (b).
  • a further aspect of the invention is a diagnostic kit for determining the disease status of an individual that has been or is infected with M. tuberculosis comprising means for analysing the expression of one or more polynucleotide sequences selected from the group consisting of: (i) a polynucleotide sequence comprising any of SEQ ID No:1 to SEQ ID No: 11 ; and (ii) any other wild type polynucleotide sequence encoding the same protein or peptide as that encoded by the polynucleotide sequences defined in (i),
  • said means capable of generating an expression pattern.
  • said means comprises one or more nucleotide probes capable of specifically hybridizing to each of the polynucleotide sequences the expression of which it is desired to analyse.
  • said means comprises paired primers for each of the polynucleotide sequences the expression of which is to be analysed.
  • the present invention is based, in part, on the discovery that certain polynucleotide and/or gene sequences define expression patterns which associate with the disease status of individuals that have been or are infected with M. tuberculosis. Most significantly, by comparing a test individual's expression pattern of one or more of these polynucleotide sequences with a reference pattern which can be from uninfected individuals or preferably individuals that have been or are infected with M. tuberculosis and have a known disease status, one is able to predict the likely disease status of the test individual.
  • an “expression pattern” as used herein denotes the result of analysing or measuring (quantitatively or qualitatively) the expression of one or more of the polynucleotides identified in the present invention as biomarkers or surrogate markers.
  • the invention thus provides a powerful predictive tool for clinical testing and treatment of tuberculosis.
  • the present invention permits smaller, more efficient clinical trials by identifying individuals who require treatment or a particular treatment regimen and thereby reducing the length of the clinical trial and the amount of un- interpretable data.
  • a physician can prescribe a prophylactic or therapeutic treatment regimen customised to that individual's disease status.
  • a “disease status” as used herein refers to the clinical outcome of M. tuberculosis infection and/or therapeutic treatment for infection. Disease status includes without limitation one or more of the following clinical outcomes: Active tuberculosis evidence of the clinical disease of tuberculosis,
  • Re-current tuberculosis at least two episodes of clinical disease.
  • polynucleotide sequence denotes a contiguous nucleic acid sequence corresponding to a sequence present in the human genome or a part of a sequence present in the human genome (including any polymorphic and/or splice variants).
  • the polynucleotide sequences identified in the present invention may represent genes (or a fragment thereof) for particular proteins or peptides i.e. nucleotide sequences which comprise:
  • a coding (or transcribed) region which comprises exons, introns, sequences at the junction between exons and introns, and 5 and 3 un-translated regions;
  • polynucleotide sequences identified in the present invention as biomarkers or surrogates are described in further detail in Table 1.
  • a nucleotide "probe” refers to a nucleic acid e.g. a cDNA, gene, gene fragment, polynucleotide or preferably an oligonucleotide that forms a hybrid structure with a sequence in a sample or target polynucleotide due to complementarity of at least one sequence in the probe with a sequence in the target/sample polynucleotide sequence.
  • the probe may be labelled so it can be detected after hybridization.
  • a nucleotide "primer” refers to an oligonucleotide that is complementary to a sequence in a sample or target polynucleotide and functions as the starting point for nucleic acid synthesis.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule such as cDNA, genomic DNA or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T m of 55°C, can be used e.g.
  • 5x SSC 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS.
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g. 40% formamide, with 5x or 6x SSC.
  • High stringency hybridization conditions correspond to the highest T m , e.g. 50% formamide, 5x or 6x SSC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art.
  • RNA:RNA, DNA:RNA, DNA:DNA The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • T m The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • oligonucleotides For hybrids of greater than 100 nucleotides in length equations for calculating T m have been derived (see Sambrook et al., supra, 9.50 - 9.51).
  • the length of the oligonucleotide determines its specificity (see Sambrook et al., supra 11.7 - 11.8).
  • a minimum length for a hybridizable nucleic acid is at least 10 nucleotides: preferably at least about 15 nucleotides.
  • Oligonucleotide probes used in the diagnostic kits of the present invention should be at least 10 nucleotides, preferably at least 15 nucleotides and more, preferably 20 to 40 nucleotides in length.
  • the present invention provides surrogate markers for assessing the disease status of an individual that has been or is infected with M. tuberculosis.
  • the methods are carried out by comparing the "test expression pattern" obtained from analysing the expression of one or more of the polynucleotide sequences identified in the present invention with the expression pattern of an uninfected individual i.e. an individual that had never been infected with M. tuberculosis or with the expression pattern of an individual that has been or is so infected and exhibits a known disease status, (hereinafter "a reference expression pattern").
  • the polynucleotide sequence or combination of polynucleotide sequences to be analysed will depend on the disease status or statuses into which it is desired to classify an unknown patients and the required degree of accuracy of the prediction.
  • an individual's expression pattern of one or more of the polynucleotide sequences comprising SEQ ID No:1 to SEQ ID No:11 can be established at the RNA or protein level e.g. by obtaining the corresponding mRNA or protein from the individual and analysing the expression pattern.
  • RNA or protein can be obtained from any cell source.
  • ceil sources available in clinical practice include without limitation blood cells, brochoalveolar lavage, biopsy material, histological specimens containing cells involved in the immune response to TB (macrophages, lymphocytes, giant cells, pneumocytes, pericytes, epithelial cells) cells from lung tissue, epithelial cells from urine, or any cells present in tissue obtained by biopsy of other infected tissues (e.g. lymph nodes).
  • Cells from extrapulmonary TB e.g. pleural effusions, pericardial effusions or ascites
  • extrapulmonary TB e.g. pleural effusions, pericardial effusions or ascites
  • Cells may also be obtained from body fluids, including without limitation blood, saliva, sweat, urine and sputum.
  • blood is used as a source of RNA or protein.
  • RNA or protein is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art (protein purification applications, a practical approach, edited by E L V HARRIS and S ANGAL, Oxford University Press, 1999; Protein protocols, edited by J. M. Walker, Humana Press 1998).
  • Polynucleotide expression can be analysed using RNA (or cDNA prepared from the RNA) using known methods including without limitation Northern blots, RNA ase protection assays, differential display, SAGE and quantitative nucleic acid amplification such as PCR e.g. Taqman or SyBr Green PCR.
  • Quantitative nucleic acid amplication such as PCR is particularly suitable for analysising the transcription of single polynucleotide sequences or small panels of polynucleotide sequences under various conditions.
  • PCR is the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA using heat-stable DNA polymerase.
  • the polymerase chain reaction employs two primers, each complementary to opposite strands of the region of DNA, which have been denatured by heating.
  • the primers are arranged so that each primer extension reaction directs the synthesis of DNA towards the other. This results in the de novo synthesis of the region of DNA flanked by the two primers.
  • the requirements of the reaction include : deoxynucleotides to provide both the energy and nucleosides for the synthesis of DNA, DNA polymerase, primer, template, and buffer containing magnesium.
  • the deoxynucleotides and primers generally are present in large excess, so the synthesis step can be repeated by heating the newly synthesized DNA to separate the strands and cooling to allow the primers to anneal to their complementary sequences. After several cycles the predominant reaction product will be that piece of DNA which is flanked by the primers, and will include the primers themselves.
  • Labelled nucleotide probes or dyes such as a fluorogenic probe used in conjunction with PCR enables the measurement of an accumulating PCR product in real time. This allows rapid generation of quantitative data showing changes in transcript numbers in tissue samples.
  • Protein expression can be analysed using known methods including without limitation array based proteomics, radioimmuno assays, competitive binding assays, Western Blot analysis, ELISA assays, ELISpot assays, FACS analysis and bead-based multiplexing assays.
  • the expression pattern of one or more of the polynucleotide sequences identified herein is determined in a number of individuals exhibiting a particular disease status or that are uninfected, and compared with the expression pattern in patients that preferably have been matched for age, sex, and/or any other statistically or medically relevant parameters, who exhibit quantitatively or qualitatively different disease status. Associations are achieved using known statistical methods in the art, including linear discriminant analysis such as stepwise linear discriminant analysis, principal component analysis and cluster analysis. In this manner, it is possible to establish statistically significant associations between particular expression patterns and particular disease statuses. It is further possible to establish statistically significant associations between particular expression patterns and changes in disease status such as, could result, e.g. from a particular treatment regimen or as a consequence of immunodepression, for example, following HIV infection (cured TB to active TB).
  • a statistically significant association preferably has a "p" value of less than or equal to 0.05. Any standard statistical method can be used to calculate these values such as the normal student's T test, or Fischer's Exact Test.
  • the reference expression pattern may comprise the expression profile of any one or more and in any combination of the polynucleotide sequences defined hereinbefore.
  • the number of polynucleotide sequences to be analysed and included within a reference expression pattern depends on the predictive value of each polynucleotide sequence for any given disease status and its requirement for accuracy of prediction. The greater the predictive value of a particular polynucleotide sequence for a given disease status the lower the need for inclusion of more than one polynucleotide sequence in the reference expression pattern.
  • the number of polynucleotide sequences included in the reference expression pattern may be reduced accordingly.
  • test expression pattern from an individual can be compared to a reference expression pattern established for a predetermined disease status or an uninfected individual.
  • Identity between the test expression pattern and the reference expression pattern means that the tested individual has a probability of having the same disease status as that represented by the reference pattern. As discussed above, this probability depends on the statistical significance of its association or correlation with a disease status. Statistical methods and algorithms known in the art such as those described above can be used.
  • stepwise discriminant analysis is used to predict the probable disease status of an individual with an unknown disease status, based on that individual's expression pattern of one or more of the polynucleotides identified in the present invention.
  • Stepwise linear discriminant analysis is a technique that will build a linear discriminant model by selecting a subset of polynucleotide sequences that best discriminate between different disease statuses.
  • the linear discriminant model can discriminate between one or more of the following clinical outcomes: active TB; cured of TB; latent TB; and re-current TB.
  • the linear discriminant model will discriminate between the clinical outcomes re-current TB and cured of TB, and in a particularly preferred embodiment the linear discriminant model will discriminate between all four clinical outcomes.
  • the expression pattern of all 8 polynucleotide sequences comprising SEQ ID No:1 to SEQ ID No:8, or preferably all 9 polynucleotide sequences comprising SEQ ID No:2 to SEQ ID No:5 and SEQ ID No:7 to SEQ ID No:11 are used to discriminate between the four clinical outcome/disease statuses described hereinbefore.
  • test expression pattern may comprise the expression profile of any one or more and in any combination of the polynucleotide sequences defined hereinbefore.
  • the number of polynucleotide sequences to be analysed and included within a test expression pattern depends on the disease status or statuses into which it is desired to classify unknown patients, the predictive value of each polynucleotide sequence for any given disease status and its requirement for accuracy of prediction. The greater the predictive value of a particular polynucleotide sequence for a given disease status the lower the need for inclusion of more than one polynucleotide sequence in the test expression pattern. Also, if a lesser degree of predictability is required then the number of polynucleotide sequences included in the reference expression pattern may be reduced accordingly.
  • kits for determining the disease status of an individual comprise means for analysing the expression of one or more polynucleotide sequences comprising any of SEQ ID No:1 to SEQ ID No:11 and may optionally include data or an algorithm for comparing expression patterns.
  • the means for analysing expression may comprise suitable nucleic acid based reagents and, preferably suitable buffers and/or hybridization reagents where appropriate.
  • the kits may further comprise correlating means to enable comparison of the test expression pattern with a reference expression pattern and thereby to determine the particular disease status of an individual.
  • Such correlating means may comprise data or an algorithm which enables the relative expression levels of each polynucleotide to be analysed and outputs the likely disease status.
  • the diagnostic kit is array based and includes the following components:
  • Nucleotide probes nucleotide probes of known sequence capable of specifically hybridizing to one or more of the polynucleotide sequences defined hereinabove and which may be labelled are provided immobilsed onto a solid support such as a glass slide or bead to form a microarray;
  • Hybridization reagents the kit may also contain other suitably packed reagents and materials for the particular hybridization protocol including, if applicable, suitable standards.
  • the diagnostic kit is quantitative nucleic acid amplification based and may comprise:
  • primers for each polynucleotide sequence defined hereinabove the expression of which is to be analysed.
  • primers are oligonucleotides 15-40 nucleotides in length, more typically 20-30 nucleotides in length and may be labelled; and optionally (ii) a nucleotide probe: which can be pre-labelled with any suitable label which includes without limitation fluorescent, chemiluminescent, radioactive or dye labels. Fluorescent labels are preferred.
  • the probe can be unlabelled and the reagents necessary for labelling included in the kit in separate containers.
  • the diagnostic kits of the invention may further comprise one or more of the following:
  • a fluorescent dye such as SyBr Green 1 ;
  • correlating means to enable comparison of the test expression pattern with a reference expression pattern and thereby to determine the particular disease status of an individual.
  • the correlation means may include an algorithm or data which calculates the relative expression levels of each polynucleotide sequence analysed and outputs the likely disease status.
  • the present invention further provides methods of treating individuals infected with M. tuberculosis and methods of monitoring the effectiveness of an anti-mycobacterial therapy.
  • the methods comprise first determining the disease status of an individual that has been or is infected with M. tuberculosis in accordance with the diagnostic methods described herein.
  • the individual may then be treated as appropriate for that individual's disease status. For example, if it is determined that the individual is suffering from an active TB infection then a suitable anti-mycobacterial therapy can be prescribed for that individual. Similarly, if it is determined that an individual is susceptible to relapse following completion of a normal treatment regimen then a prophylactic treatment regimen can be prescribed or the existing therapy continued with for an extended period.
  • Suitable anti-mycobacterial therapies include, without limitation the antibiotics Isoniazed, Rifampicin, Ethambutol and Pyrazinamide.
  • Isoniazid and Rifampicin and optionally Ethambutol and Pyrazinamide are co-administered by which is meant the individual is prescribed both Rifampicin and Isoniazid optionally with Ethambutol and/or Pyrazinamide.
  • Second line drugs that may also form part of an anti-mycobacterial treatment regimen include, without limitation, Ethionamide and Streptomycin.
  • Suitable anti-mycobacterial therapies include without limitation the following drugs which may be used alone or more preferably in combination with another known anti- mycobacterial agent such as those described above: Rifapentine; Rifabutin; fluoroquinolones such as Moxifloxacin, Gatifloxacin and Ofloxacin; oxazolidinones such as Linezolid; and nitroimidazopyrans such as PA - 824 (ChironCorp).
  • Recurrent TB have had at least two episodes of pulmonary TB, but were healthy at time of sample collection
  • Cured of TB One episode of TB which was clinically cured at least 24 months prior to sample collection (adequate immune response).
  • M Tuberculin skin test (Mantoux) positive: have been infected with M. tuberculosis, but not had clinical TB (M. tuberculosis resistant/protective immunity).
  • A Active TB: Individuals with newly diagnosed active pulmonary TB. All individuals underwent a standard evaluation for the presence of tuberculosis including chest radiography, tuberculin skin test (Mantoux) and testing for the presence of M. tuberculosis in the sputum and confirmed to be zero negative for HIV.
  • Tuberculin skin test Mantoux
  • RNA stabilisation solution (Roche Molecular Biochemicals, East Wales, UK) and stored at - 70°C until required.
  • cDNA fragments were generated from bacterial clones by performing PCR across the cloning site of the library vector using T7 and Sp6 primers. A proportion (2%) of the PCR reactions were analysed by agarose gel electrophoresis.
  • Membranes were exposed to phosphor screens for 7d and the resulting hybridisation signals captured with a phospho-imager (Storm 860, Moldecular Dynamics, Buckinghamshire, UK) and quantified using a ImageQuant software (Molecular Dynamics, Buckinghamshire, UK).
  • ANOVA Analysis of variance
  • Stepwise linear discriminant analysis was used to identify polynucleotides that can individually or in groups discriminate between one or more of the four disease statuses of interest.
  • the columns represent the corresponding coefficients for the constant and adjusted log (base 10) intensity for the 8 polynucleotides sequences listed in the first column. These equations are used to both reveal the differences amongst the four disease statuses and also to predict to which disease status a given individual of unknown disease status might belong based on that individual's expression pattern for those eight polynucleotide sequences.
  • the probability of membership for each disease status for each individual can be determined using the functions in Table 2 and individuals are then classified into the disease status with the highest probability of membership.
  • Figure 1 shows the expression levels of the two polynucleotides SEQ ID No:1 and SEQ ID No:2 that can preferably be used to discriminate between different disease statuses.
  • 9 polynucleotide sequences (Group Y) were identified the expression profile of which could together perfectly discriminate between all four disease statuses, i.e. 100% of all patients were correctly classified.
  • the coefficients of the discriminant function are shown in Table 3.
  • the columns represent the corresponding coefficients for the constant and adjusted log (base 10) intensity for the 9 polynucleotides sequences listed in the first column. These equations are used to both reveal the differences amongst the four disease statuses and also to predict to which disease status a given individual of unknown disease status might belong based on that individual's expression pattern for those nine polynucleotide sequences.
  • the probability of membership for each disease status for each individual can be determined using the functions in Table 3 and individuals are then classified into the disease status with the highest probability of membership.
  • Quantitative PCR was used to measure the mRNA expression levels for nine genes (group Y:SEQ ID No: 2, 3, 4, 5, 7, 8, 9, 10 and 11) and three housekeeping genes ( ⁇ - actin, GAPDH and cyclophilin) in duplicate in the same RNA samples as used for the DNA array experiments.
  • mRNA was reverse transcribed using oligo-(dT) 12 . 18 (Life Technologies) and Superscript II reverse transcriptase (Life Technologies) according to the manufacturers protocol.
  • the cDNA produced was diluted so that 5 ⁇ l contained the equivalent of 1 ng of poly (A)+ RNA.
  • a master mix was utilized which included 900 nM each of the forward and reverse primers and 1 x SYBR Green PCR master mix (Applied Biosystems, Warrington, UK).
  • the total volume per reaction was 25 ⁇ l with 20 ⁇ l of the master mix and 5 ⁇ l of the cDNA.
  • the PCR reaction was carried out on an ABI7700 Sequence Detector (Applied Biosystems) using the PCR parameters: 95°C for 10 min, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Amplification was followed by melting curve analysis using the program run for one cycle at 95°C with 15 second hold, 60°C with 20 second hold, and a 20 minute ramp time ending at 95°C with 15second hold at the step acquisition mode.
  • the level of mRNA-derived cDNA in each sample was calculated from the fluorescent signal using duplicate human genomic DNA calibration standards included in each run.

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Abstract

La présente invention concerne une technique de détermination de l'état de maladie d'une personne qui a été infectée par M. tuberculosis. Cette technique consiste: (a) à obtenir un prélèvement d'une personne qui a été ou qui est infectée par M. tuberculosis, (b) à analyser l'expression d'au moins une séquence polynucléotidique sélectionnée dans le groupe constitué de: (i) une séquence polynucléotidique comprenant n'importe quelle séquence allant de SEQ ID No:1 à SEQ ID No:11 et, (ii) n'importe quelle séquence polynucléotidique de type sauvage codante pour la même protéine ou le même peptide que celle ou celui qui est codé(e) par les séquences polynucléotidiques définies en (i) de façon à générer une structure d'expression test, et (c) à comparer cette structure d'expression test avec une structure d'expression d'une personne non infectée ou avec une structure d'expression d'une personne qui a été ou qui est infectée par M. tuberculosis et dont l'état de maladie est connu, et à classer l'état de maladie de cette personne à partir de cette comparaison.
PCT/GB2003/002630 2002-06-20 2003-06-18 Marqueurs de substitution permettant de determiner l'etat de maladie d'une personne infectee par mycobacterium tuberculosis WO2004001070A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110129817A1 (en) * 2009-11-30 2011-06-02 Baylor Research Institute Blood transcriptional signature of active versus latent mycobacterium tuberculosis infection
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EP2836608A4 (fr) * 2012-04-13 2016-02-24 Somalogic Inc Biomarqueurs de la tuberculose et leurs utilisations
CN111650287A (zh) * 2020-04-16 2020-09-11 广东省结核病控制中心 用于检测活动性肺结核的粪便中小肽及其检测系统

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JP2011526152A (ja) * 2008-06-25 2011-10-06 ベイラー リサーチ インスティテュート マイコバクテリウム・ツベルクローシス感染症の血液転写サイン
EP2300823A4 (fr) * 2008-06-25 2012-03-14 Baylor Res Inst Signature transcriptionnelle du sang lors d'une infection par le mycobacterium tuberculosis
CN102844444A (zh) * 2009-11-30 2012-12-26 贝勒研究院 活性与潜伏性结核分枝杆菌感染的血液转录签名
JP2013511981A (ja) * 2009-11-30 2013-04-11 ベイラー リサーチ インスティテュート 活動性結核菌感染を潜在性結核菌感染と対比させる血中転写サイン
EP2519652A4 (fr) * 2009-11-30 2013-05-01 Baylor Res Inst Signature transcriptionnelle sanguine d'une infection active ou latente par mycobacterium tuberculosis
US20110129817A1 (en) * 2009-11-30 2011-06-02 Baylor Research Institute Blood transcriptional signature of active versus latent mycobacterium tuberculosis infection
AU2010325179B2 (en) * 2009-11-30 2015-03-12 Baylor Research Institute Blood transcriptional signature of active versus latent Mycobacterium tuberculosis infection
EP2825671A4 (fr) * 2012-03-13 2015-08-26 Baylor Res Inst Détection précoce d'une réponse au traitement de la tuberculose
EP2836608A4 (fr) * 2012-04-13 2016-02-24 Somalogic Inc Biomarqueurs de la tuberculose et leurs utilisations
US10408847B2 (en) 2012-04-13 2019-09-10 Somalogic, Inc. Tuberculosis biomarkers and uses thereof
WO2013190321A1 (fr) * 2012-06-22 2013-12-27 Nottingham Trent University Biomarqueurs permettant de déterminer l'état d'infection par m. tuberculosis
CN111650287A (zh) * 2020-04-16 2020-09-11 广东省结核病控制中心 用于检测活动性肺结核的粪便中小肽及其检测系统
CN111650287B (zh) * 2020-04-16 2022-10-25 广东省结核病控制中心 用于检测活动性肺结核的粪便中小肽及其检测系统

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