WO2009094212A1 - Essai de diagnostic pour la détection d'une tuberculose active - Google Patents

Essai de diagnostic pour la détection d'une tuberculose active Download PDF

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WO2009094212A1
WO2009094212A1 PCT/US2009/000478 US2009000478W WO2009094212A1 WO 2009094212 A1 WO2009094212 A1 WO 2009094212A1 US 2009000478 W US2009000478 W US 2009000478W WO 2009094212 A1 WO2009094212 A1 WO 2009094212A1
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tuberculosis
glutamine synthetase
sample
serum
bacterial
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PCT/US2009/000478
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Marcus A. Horwitz
Guenter Harth
Debabrata Nag
Chittaranjan Maity
Dipak Kumar Chattopadhyay
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The Regents Of The University Of California
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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
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • 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
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Tuberculosis continues to ravage civilization with an estimated 8.8 million new cases and 1.6 million deaths per year. The incidence is increasing and is estimated to reach 10 million cases by 2015.
  • the TB pandemic is exacerbated by the HIV pandemic, with HIV victims being two orders of magnitude more sensitive to TB than the general population, and by the emergence of multi-drug resistant TB.
  • About 2 billion people are believed to be latently infected with M. tuberculosis, the primary causative agent of TB, and at risk of developing disease at a later point in life. Further, most cases of TB occur in economically poor regions of the world where resources for diagnosing and treating TB are limited.
  • the tuberculin skin test identifies persons or animals previously or currently infected with Mycobacterium tuberculosis, but does not provide information as to the current activity of tuberculosis, i.e. whether it is currently active or inactive. The disease is inactive in the great majority of persons who have a positive tuberculin skin test.
  • Another type of test used to assess tuberculosis in humans and animals is based on the detection of interferon gamma in blood samples in vitro after lymphocytes in the sample are incubated with Mycobacterium tuberculosis (or Mycobacterium bovis) antigens such as PPD (purified protein derivative) or various proteins.
  • PPD purified protein derivative
  • An example is the Quantiferon Gold assay.
  • Tuberculosis is one of the most wide spread diseases of humans on earth.
  • An assay that allows the diagnosis of active tuberculosis would be a boon to the control of tuberculosis by allowing rapid, inexpensive identification of active cases so that treatment and other control measures can be instituted rapidly and effectively.
  • An assay that allowed the monitoring of tuberculosis would also be a boon to the treatment and control of tuberculosis by allowing rapid, inexpensive identification of potentially drug-resistant cases so that treatment with alternative antibiotics could be rapidly instituted and control measures to prevent the spread of drug-resistant tuberculosis could be rapidly instituted.
  • Tuberculosis is also a problem in domesticated animals including cattle and sheep. Current policy involves culling animals with a positive skin or Quantiferon test; however, many of these animals do not have active disease. Use of the techniques set forth herein would allow the culling of only animals with active disease as well as allow use of BCG vaccination in domesticated animals, further reducing the incidence of active disease.
  • BRIEF DESCRIPTION [0010] Described herein is a method for rapidly diagnosing active tuberculosis, including both pulmonary and extra-pulmonary tuberculosis, in humans and animals using a blood or serum sample.
  • M. tuberculosis glutamine synthetase (GS; GInAI) is an abundant and omnipresent biomarker in the serum of guinea pigs and humans with TB. Further, applicants have found that an early, highly sensitive and specific diagnosis of TB can be made by the detection of M. tuberculosis glutamine synthetase in the selected body fluids of infected individuals.
  • GS is one of the abundantly released proteins of M. tuberculosis as well as other pathogenic mycobacteria. Its presence in high amounts in the culture medium of pathogenic mycobacteria has been correlated with the presence of a poly L-glutamate/glutamine component in the cell wall of these pathogenic organisms. Nonpathogenic mycobacteria lack this component (Harth G, Clemens DL, Horwitz MA "Glutamine Synthetase of Mycobacterium Tuberculosis: Extracellular Release and Characterization of its Enzymatic Activity", Proc Natl Acad Sci USA.
  • Glutamine synthetase is a dodecamer of identical 53 kDa subunits that has a central role in every cell's nitrogen metabolism; in M. tuberculosis, GInAI (product of the g/nA1 gene, Rv2220 (Cole ST et al.
  • the GS inhibitor L-methionine-S,f?-sulfoximine (MSO) selectively inhibits enzymes of bacterial origin, including M.
  • tuberculosis GS Harmonic tuberculosis GS
  • Harth G, Horwitz MA "An Inhibitor of Exported Mycobacterium Tuberculosis Glutamine Synthetase Selectively Blocks the Growth of Pathogenic Mycobacteria in Axenic Culture and in Human Monocytes: Extracellular Proteins as Potential Novel Drug Targets", J Exp Med. 189. pp1425-1435 (1999)
  • MSO tuberculosis
  • M. tuberculosis GS which is a large multimeric molecule, is highly stable in the culture medium of M. tuberculosis (Tullius M, Harth G, Horwitz MA "The High Extracellular Levels of Mycobacterium Tuberculosis Glutamine Synthetase and Superoxide Dismutase are Primarily Due to High Expression and Extracellular Stability Rather Than to a Protein Specific Export Mechanism", Infect Immun. 69, pp 6348-6363 (2001 )). It appears that its high stability largely accounts for its abundance in the extracellular medium of broth cultures. This molecule also appears to be stable in the infected host and persist in host tissues and fluids.
  • M. tuberculosis GS is present in the serum of infected guinea pigs and have now demonstrated that humans with both pulmonary and extrapulmonary TB, but not control patients, have readily detectable GS activity in their sera. Moreover, with antibiotic treatment, the levels of GS fall in both humans and animals. Based thereon applicants have determined, and confirmed by laboratory analysis that GS in serum is a biomarker of active TB and its level in serum can be used as a means of monitoring the success of treatment. The abundance and enzymatic activity of GS also allows for the development of a simple inexpensive assay for diagnosing TB.
  • the new test described herein in its various embodiments is positive only when tuberculosis is active.
  • the tuberculin skin test requires 2 days from the time tuberculin is injected into the skin until the time the test is read, requiring two patient visits.
  • the new test described herein requires only a sample of the patient's serum and a single patient visit. Further, the assay only requires a few minutes to perform and therefore the results can be available while the patient is at the medical facility which is particularly advantageous in developing nations where access to medical care is limited and the patient does not have the ability or capability of making multiple visits to the medical facility.
  • the tests and procedures described herein can also be used as an assay to monitor the progress and success of the treatment of tuberculosis.
  • the assay can determine if treatment is successful or not and indicate whether a change in antibiotic therapy is indicated.
  • the test Since the great majority of tuberculosis cases occur in developing countries, it is important that the test be inexpensive.
  • there is no simple rapid diagnostic test for active tuberculosis no licensed serological test for tuberculosis and no simple serologic test for monitoring the course of tuberculosis.
  • Set forth herein is a simple, rapid, inexpensive serologic diagnostic test for active tuberculosis.
  • the assay can be used to monitor the course of tuberculosis. With treatment, the level of the enzyme measured by the test falls. Thus, a drop in the test level of the enzyme measured after antibiotic treatment can confirm that antibiotic treatment is successful. A failure to observe a drop in enzyme level would indicate that antibiotic treatment is not working. The test may thus serve as an early indication of multi-drug resistant tuberculosis (MDRTB).
  • MDRTB multi-drug resistant tuberculosis
  • a small amount of patient serum is obtained and then assayed for Glutamine Synthetase (GS) Activity or for the presence of M. tuberculosis GS.
  • the assay for GS activity may include a selective inhibitor of bacterial GS, e.g. D.L-Methionine-S ⁇ -Sulfoximine.
  • Applicants have previously shown that M. tuberculosis GS is 100 times more sensitive to this inhibitor than mammalian GS.
  • the assay for M. tuberculosis GS can also be made specific for M. tuberculosis GS by using monoclonal or polyclonal antibodies specific to M. tuberculosis GS, which differs significantly from mammalian GS.
  • GS catalyzes the production of glutamine from glutamate and ammonia via the following reaction:
  • GS activity is typically assayed by the so-called "Transfer Reaction” to avoid possible contaminating phosphatase activity.
  • the transfer reaction catalyzed by GS is as follows:
  • the GS activity can be measured in the presence of a selective inhibitor of M. tuberculosis GS such as L- Methionine-S,R-Sulfoximine (only the L-S isomer is an active inhibitor) or alpha- ethyl-D,L-Methionine-S,R-Sulfoximine (only the L-S isomer is an active inhibitor).
  • concentration is selected so that it is sufficient to inhibit bacterial GS activity but not mammalian GS activity. If the sera shows GS activity in the absence of the inhibitor but not in its presence, this indicates that the GS was M. tuberculosis rather than mammalian in origin.
  • tuberculosis whether pulmonary or extrapulmonary, in humans or animals can be performed.
  • old infection with tuberculosis can be distinguished from current active disease.
  • the procedures and tests set forth herein can distinguish previous exposure to BCG vaccination from active tuberculosis.
  • the test also has application in detecting TB in domesticated animals. Because the test can distinguish between previous exposure to tuberculosis resulting in inactive disease and active disease, animals with active disease can then be separated from those not having active TB.
  • BCG vaccination interferes with the tuberculin skin test, yielding false positives.
  • the cost of culling all domesticated animals with a positive skin test or positive Quantiferon test, which does not distinguish active from inactive disease, is tremendous and burdensome in developing nations. Because the test would identify only animals with active tuberculosis, there is a huge economic benefit. Previous BCG vaccination would not interfere with a test for GS or GS activity as set forth herein.
  • BCG vaccination in domesticated animals precludes the use of the current tuberculin skin test or Quantiferon test (which measures blood cell interferon response to tuberculin or other M. tuberculosis antigens rather than a skin test response to tuberculin) because it often results in a false positive test.
  • the procedures set forth herein allow vaccination of domesticated animals with BCG or recombinant BCG vaccines because vaccination will not interfere with an assay for serum GS activity. By allowing vaccination of herds, the incidence of tuberculosis in the herds would be minimized thus reducing the likelihood of disease spread from infected animals.
  • the invention By allowing a reduction in the incidence of tuberculosis via vaccination with BCG or recombinant BCG and the identification of only animals with active disease, the invention would allow the culling of fewer animals from herds. Use of the procedures set forth herein would have a major beneficial economic impact and, at the same time, augment the food supply as a result of a reduction in the destruction of food animals based on these false positive readings. Further, there currently is no blood test for monitoring the course of tuberculosis. BRIEF DESCRIPTION OF DRAWINGS
  • Figure 1 shows the protein profile following each major step in the purification of Glutamine Synthetase from infected guinea pig sera.
  • Figure 2 illustrates the reactivity of Glutamine Synthetase from M. tuberculosis infected guinea pig sera.
  • M. tuberculosis is unusual among bacterial species in that it secretes or otherwise releases a large number of proteins in considerable quantities into its extracellular milieu. Such extracellular proteins are released both by organisms growing in broth medium and by organisms growing intraphagosomally in human mononuclear phagocytes, the bacteria's primary host cells. Approximately 100 proteins are released into broth medium by growing M. tuberculosis bacteria, eleven of which are released in great abundance and comprise about 90% of the total protein released (Horwitz, M. A., B. W.
  • Glutamine synthetase is a dodecamer of identical 53 kDa subunits that has a central role in nitrogen metabolism, catalyzing the synthesis of L- glutamine from L-glutamate, ammonia, and ATP.
  • pathogenic mycobacteria such as M. tuberculosis and M. bovis release large amounts of glutamine synthetase extracellularly, while nonpathogenic mycobacteria such as M. smegmatis and M. phlei and non mycobacterial microorganisms such as Legionella pneumophila and E. coli do not.
  • GS in culture filtrates correlates with the pathogenicity of mycobacteria.
  • Applicants also subsequently found that the release of glutamine synthetase by pathogenic mycobacteria can be correlated with the presence of a poly L-glutamate/glutamine component in the cell wall of these organisms; nonpathogenic mycobacteria lack this component. Accordingly, it appears that extracellular glutamine synthetase is involved in the synthesis of poly L- glutamate/glutamine and that the enzyme's extracellular presence is significant to virulence.
  • GS is an essential enzyme for M. tuberculosis as well as being essential for the survival and growth of M. tuberculosis (Tullius MV, Harth G, Horwitz MA "Glutamine Synthetase GInAI is Essential for Growth of Mycobacterium Tuberculosis in Human THP- 1 Macrophages and Guinea Pigs" Infect Immun. 71.. pp3927-3936 (2003)).
  • GS is an essential enzyme for M. tuberculosis as well as being essential for the survival and growth of M. tuberculosis (Tullius MV, Harth G, Horwitz MA "Glutamine Synthetase GInAI is Essential for Growth of Mycobacterium Tuberculosis in Human THP- 1 Macrophages and Guinea Pigs" Infect Immun. 71.. pp3927-3936 (2003)).
  • GS is an essential enzyme for M. tuberculosis as well as being essential for the survival and growth of M. tuberculo
  • the mutant was completely avirulent in vivo.
  • the glnA genes (glnA2-4) other than glnA 1 are glutamine synthetase homologs and are either not expressed or expressed at such low levels that they are undetectable in both the cell extract and the culture filtrate.
  • MSO L-methionine-S,R-sulfoximine
  • antisense oligodeoxyribonucleotides specific to M. tuberculosis GS mRNA inhibits formation of the poly-L-glutamate/glutamine cell wall structure
  • MSO selectively blocks the growth of pathogenic mycobacteria in broth culture, including M. tuberculosis, M. bovis, and M. avium, but has no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms. The inhibitor also blocks the growth of M.
  • the inhibitor MSO is an effective antibiotic in vivo as demonstrated in studies utilizing the demanding guinea pig model of pulmonary tuberculosis, where it reduces the burden of tuberculosis in the lung and spleen of guinea pigs challenged by aerosol with virulent M.
  • tuberculosis (Harth G, Horwitz MA, Inhibition of MycobacteriumTtuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy in vivo" Infect Immun. 71., pp 456-464 (2003)).
  • M. tuberculosis GS is high expressed and has high stability in in vitro culture filtrates. Additionally, it was found that M.
  • tuberculosis GS is also highly expressed and highly stable in vivo and therefore measurable in patient sera. Based on the finding of GS activity in the sera of animals and humans with active tuberculosis but not in the sera of control animals and humans, and the demonstration that the GS activity in the sera of M. tuberculosis - infected animals is derived from M. tuberculosis GS rather than mammalian GS, applicants have now shown that the measurement of M. tuberculosis GS can be used as the basis for a diagnostic assay for active tuberculosis in humans and animals and as an assay for monitoring the success of treatment.
  • the rapid serologic test described herein can also be used to detect reactivation of tuberculosis in mammals with latent tuberculosis comprising by periodically obtaining a sample of patient's or subject's serum, assaying that sample for bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase and comparing the levels thereof with prior obtained samples from the same subject.
  • guinea pigs (age -13 months, weight ⁇ 1 ,500 g, male outbred Hartley strain animals from Charles River Laboratories) were infected with an aerosolized dose of M. tuberculosis Erdman strain, resulting in approximately 75 primary lung lesions. Challenged animals were housed in stainless steel single cage racks within a laminar flow enclosure for five weeks and weighed weekly.
  • the transfer reaction was used to detect and monitor glutamine synthetase activity in the various serum samples.
  • the transfer reaction is highly specific for glutamine synthetases and produces ⁇ -glutamylhydroxamate which, when acidified, forms a hexadentate complex with Fe 3+ in a color reaction that can be spectrophotometrically read at 540 nm.
  • reaction mixes containing one ml_ of reaction buffer and from about 100 to 125 ⁇ l_ of serum were incubated for various times up to 1 h at 37 0 C, and then read at 540 nm against the standard curve established with a pure sample of ⁇ -glutamylhydroxamate.
  • a stock of normal guinea pig serum served as a control.
  • the lower limit of glutamine synthetase activity detection is ⁇ 0.02 OD 540 nm mUnits equal to the synthesis of - 0.03 ⁇ moles or ⁇ 150 ng of ⁇ -glutamylhydroxamate.
  • Transfer reaction was used to detect and monitor glutamine synthetase activity in the serum samples. All reactions were standardized to conform to the definition of one enzyme unit as the amount of glutamine synthetase that produces one ⁇ mole of ⁇ -glutamylhydroxamate per min at 37 0 C (Woolfolk CA, Shapiro B, Stadtman ER, "Regulation of Glutamine Synthetase. I. Purification and Properties of Glutamine Synthetase from Escherichia CoIi", Arch Biochem Biophvs. 116, pp 177-192(1966)). The results are shown in Table 1C.
  • Table 1C GS activity in sera of infected guinea pigs
  • Table 1D GS activity in sera of uninfected control guinea pigs
  • Table 1E GS Activity in Sera of BCG-immunized Guinea Pigs 10 weeks after Immunization
  • GS Activity in Serum of Guinea Pigs Infected with M. tuberculosis is Abolished by Concentrations of the GS Inhibitor D, L-Methionine-S.R-Sulfoximine (MSO) that Inhibit Bacterial but not Mammalian GS -
  • MSO L-Methionine-S.R-Sulfoximine
  • the GS activity could represent a non-specific response to M. tuberculosis infection. If bacterial in origin, the GS activity would be derived from M. tuberculosis.
  • the first study evaluated the differential effect of MSO on bacterial vs. mammalian GS.
  • MSO is a well-characterized GS inhibitor with much greater specificity for bacteria, including M. tuberculosis GS, than for mammalian GS (K, for sheep brain GS ⁇ 110 ⁇ M; K 1 for M. tuberculosis GS ⁇ 1 ⁇ M).
  • Table 2A shows the selectivity of MSO for M. tuberculosis GS vs. Human GS at two concentrations. The data clearly show that M. tuberculosis GS is much more sensitive to MSO than eukaryotic GS, in this case human GS.
  • Table 3A Precipitation of GS activity in sera of M. tuberculosis infected guinea pigs with either anti-M. tuberculosis GS or anti-human GS antibodies
  • tuberculosis-] nfected guinea pigs [0071] The pooled sera from the 9 guinea pigs infected with M. tuberculosis was filtered through membranes with 10 and 30 kDa exclusion limits to eliminate many of the small molecules and break down products of larger proteins, which are normally present in sera. Additional protein degradation was minimized by the addition of a protease inhibitor cocktail that inhibited all major types of proteases. The entire volume was then dialyzed against imidazole-manganese chloride, chromatographed on an AfTi gel blue column in the same buffer, and eluted with increasing concentration of ADP.
  • Active fractions were pooled and immediately chromatographed on hydroxyapatite or Q-Sepharose in imidazole- manganese chloride buffer, eluted with increasing imidazole and salt concentrations and directly filtered again either through size exclusion membranes or a Sepharose CL-6B column. After these steps, the purified GS was ⁇ 95% homogeneous. It was concentrated in an Amicon Diaflo unit to a small volume, assayed for its specific activity and analyzed for its N-terminal amino acid sequence.
  • the mean weights of the guinea pigs are shown in Table 4A.
  • the weight pattern was typical for guinea pigs challenged with M. tuberculosis. After M. tuberculosis challenge, the guinea pigs gained weight for two weeks after challenge and then lost weight at week 3, which coincides in time with the dissemination of the infection from the primary site of infection in the lung to other organs. The animals then recovered and gained weight until week 8 when they again lost weight, presumably as a result of the high burden of M. tuberculosis at that time.
  • Table 4A Mean weight of guinea pigs after challenge
  • Table 4B CFU in lungs and spleen and mean number of liver lesions in guinea pigs 10 weeks after challenge
  • Rabbit polyclonal anti-M tuberculosis GS (Horwitz lab stock) or rabbit polyclonal anti-human GS antibodies (obtained from Owen Griffith, Dept. of Biochemistry, Medical College of Wisconsin, Milwaukee, Wl) were used as antibodies.
  • both antibodies failed to detect a eukaryotic type GS in the GS preparation from infected guinea pig sera, providing evidence that the potential contribution of such an enzyme to the total measurable GS activity is at most very minimal.
  • Vl The Sequence of Serum GS in M. tuberculosis-Infected Guinea Pigs Matches M. tuberculosis GS but not Mammalian GS - To confirm that the GS in the sera of M. tuberculosis- ' mfected guinea pigs was derived from M. tuberculosis rather than from the guinea pig, partial amino acid sequences of the isolated protein were determined. [0085] A. Method
  • the first analysis showed a mixed sequence for the GS sample, as the protein suffered some break down in the process of amino acid analysis, generating secondary N-terminal amino acids which served as starting points for the sequencing procedure. Sequences were obtained for three fragments of the protein. All three sequences matched the sequence of the M. tuberculosis GS sequence and not any mammalian GS sequence, confirming that the protein was derived from M. tuberculosis GS. A comparison with other sequences (see sequence alignments below) confirmed that none of the eukaryotic GS molecules showed any significant sequence homology to the sequence derived from the sera of guinea pigs infected with M. tuberculosis. [0090] 2. Sample #2
  • a homology search of the human GS amino acid sequence yielded 35 eukaryotic species whose GS is ⁇ 90% similar to the human GS. Including the 50 most homologous species in the search retrieved enzymes that are still up to 88% similar to the human enzyme (Fasta and Clustal W programs at the European Bioinformatics Institute at www.ebi.ac.uk). Most of these molecules are ⁇ 373 amino acids in length, much smaller than their prokaryotic counterparts which average ⁇ 470 amino acids (the M. tuberculosis mature enzyme has 477 amino acids).
  • the Chinese hamster GS is among the twelve most homologous GS molecules and shows an 89.7% identity and 96.9% similarity to the human GS sequence. The sequences determined for Samples 1 and 2 are listed below.
  • the glycine residues in sequence #3 of the first sample appear in parentheses because they were only tentatively assigned to the two positions as shown due to the high glycine background on the membrane.
  • the Arg-Thr residues of the human and Chinese hamster homologs of sequence #3 are followed by a gap of 14 amino acid residues; the last, carboxy-terminal cluster of 15 residues in eukaryotic GS molecules containing three residues similar to the M. tuberculosis GS were then found.
  • the first amino acid of the M. tuberculosis GS coding region is valine, encoded by a gtg codon. This codon, in addition to atg, is recognized in M. tuberculosis as a start codon.
  • VII. The level of GS in the Sera of Guinea Pigs Infected with M. tuberculosis is Orders of Magnitude Higher than the Level of GS in Normal Human Serum
  • the levels of GS in M Assuming the levels of GS in M.
  • tuberculosis- ' mfected human sera is comparable to the level in the guinea pig, the levels of GS protein are orders of magnitude higher than the level in normal human serum (50 ⁇ 19) (Tumani, H., G. Q. Shen, J. B. Peter, and W. Bruck, "Glutamine Synthetase in Cerebrospinal Fluid, Serum and Brain", Arch. Neurol.. 56. pp 1241-1246 ( 1999.)).
  • the elevation in GS level in humans with Alzheimer's disease 61 ⁇ 32) (Takahashi, M. E. Stanton, J.I. Moreno, and G.
  • GS was measured by the method of Woolfolk et al. (Woolfolk, C.A., B. Shapiro, and E. R. Stadtman, "Regulation of Glutamine Gynthetase. I. Purification and Properties of Glutamine Synthetase from Escherichia CoIi", Arch. Biochem Biophvs., 116, pp 177-192 (1966.)).
  • the assay system was contained in a total volume of 2 ml. A volume of 0.2 ml of patient serum was added to 1.8 ml of a solution containing:
  • Group 3 Normal Control Subjects
  • Group 4 Disease Control Subjects
  • Applicants describe herein a rapid serologic test for detecting or monitoring in mammals the presence or absence of active tuberculosis, both pulmonary and extra-pulmonary.
  • the rapid serological test provides to the clinician a broad range of capabilities regarding the identification in humans and animals of active tuberculosis, the treatment of the disease, the control of subjects exposed to the disease and the follow up of patients exposed and/or treated for the disease.
  • the test can also be used as an aid in monitoring the effectiveness of treatment and in adjusting treatment dosages as the disease progresses or is being eliminated as a result of treatment as a result of obtaining serum samples during as well as after treatment.
  • the rapid serological test can also be used following other tuberculosis testing procedures to distinguish over false positives as a result of the presence of inactive disease or a the incidence of prior treated disease.
  • the rapid serological test can also be provided as part of a kit containing some or all of the components required to test for active tuberculosis and deliver a treatment modality.
  • the kit would contain the necessary devices for obtaining a sample of blood from the mammal and separating a sample of serum therefrom and the means for assaying the serum sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.
  • the kit would also include one or more doses of antibiotic suitable for treating active tuberculosis.
  • the kit could also include additional blood collection devices and assaying devices to track the efficacy of the treatment.

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Abstract

L'invention porte sur un test sérologique rapide pour la détection ou la surveillance chez les mammifères de la présence ou de l'absence d'une tuberculose active, à la fois pulmonaire et extrapulmonaire, d'une tuberculose de réactivation chez des mammifères infectés de façon latente, la distinction d'une tuberculose active par rapport à une tuberculose inactive chez les mammifères et la détection de la présence d'une tuberculose sensible aux médicaments ou de l'émergence d'une tuberculose résistante aux médicaments chez des mammifères, consistant à doser la présence ou le taux d'activité de glutamine synthétase bactérienne ou de glutamine synthétase de M. tuberculosis chez ce mammifère.
PCT/US2009/000478 2008-01-23 2009-01-22 Essai de diagnostic pour la détection d'une tuberculose active WO2009094212A1 (fr)

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CN105548545A (zh) * 2016-02-17 2016-05-04 遵义医学院附属医院 一组活动性结核病诊断标志物及其用途

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013112103A1 (fr) * 2012-01-27 2013-08-01 Peas Institut Ab Procédé de détection de la tuberculose
CN105548545A (zh) * 2016-02-17 2016-05-04 遵义医学院附属医院 一组活动性结核病诊断标志物及其用途
CN105548545B (zh) * 2016-02-17 2017-07-28 遵义医学院附属医院 一组活动性结核病诊断标志物及其用途

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