WO2009094212A1

WO2009094212A1 - Diagnostic assay for detection of active tuberculosis

Info

Publication number: WO2009094212A1
Application number: PCT/US2009/000478
Authority: WO
Inventors: Marcus A. Horwitz; Guenter Harth; Debabrata Nag; Chittaranjan Maity; Dipak Kumar Chattopadhyay
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2008-01-23
Filing date: 2009-01-22
Publication date: 2009-07-30
Anticipated expiration: 2010-07-23

Abstract

A rapid serologic test for detecting or monitoring in mammals the presence or absence of active tuberculosis, both pulmonary and extra-pulmonary, reactivation tuberculosis in latently infected mammals, distinguishing active from inactive tuberculosis in mammals and detecting the presence of drug-sensitive or the emergence of drug-resistant tuberculosis in mammals comprising assaying the presence or level of bacterial Glutamine Synthetase activity or of M. tuberculosis Glutamine Synthetase in that mammal.

Description

DIAGNOSTIC ASSAY FOR DETECTION OF ACTIVE TUBERCULOSIS

[0001] This application claims benefit of US Provisional Application 61/023,017 filed January 23, 2008. The invention set forth herein was made with Government support under Grant No. AI031338 and AI055352 awarded by the National Institutes of Health (NIH). The federal government may have certain rights in the invention. BACKGROUND

[0002] Tuberculosis (TB) continues to ravage humanity 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.

[0003] In order to limit the spread of disease by person-to-person transmission it is important to diagnose its presence in individuals and institute effective therapy as early in its course as possible because patients treated with effective antibiotics rapidly become non-contagious. Currently, however, only a minority of new TB cases are diagnosed. As a result, contagious cases are missed or diagnosed late, allowing further spread of the disease. A highly infectious person can spread the disease to 10-15 others in a year. Furthermore, in the absence of accurate diagnosis, precious resources in resource poor areas of the world are wasted on misdiagnosed cases; for every person with TB about 10 people have similar symptoms but from a different cause. Hence, one of the highest priorities in TB research is a simple, inexpensive, user-friendly, rapid, equipment-free, highly sensitive and specific test for active TB that can be used at the point of contact with the patient. It is estimated that such a test could save 400,000 lives per year.

[0004] Currently, the diagnosis of TB depends primarily upon identifying the organism in a sputum smear under the microscope. This test is only 40-60% sensitive under field conditions and requires a trained and motivated technician, a microscope, and repeat patient visits to provide serial sputum samples in settings where even one visit may entail economic or logistical hardship for the patient. The detection rate for smear microscopy is even lower (0-20%) in HIV- coinfected patients, children, and persons with extrapulmonary TB (~15% of cases). To detect M. tuberculosis in a sputum smear, the number of bacilli must be very large (>5,000/mL of sputum) (Shingadia, D. and Novelli, V., "Diagnosis and Treatment of Tuberculosis in Children", Lancet Infectious Diseases, 3, pp 624-32 (2003)). Hence, this test detects only advanced disease, typically long after the patient has spread the disease to others, especially close contacts. [0005] An alternative to smear microscopy is culturing M. tuberculosis from patient samples. This is a much more sensitive (80%) and specific (-98%) test than smear microscopy, requiring -10-100 bacilli/mL of sputum, and is the current gold standard for TB diagnosis. However, the culturing procedure requires specialized equipment, trained personnel, and a dependable supply of electricity and water. Hence culture facilities are typically lacking in the resource- limited regions pf the world where the burden of TB is highest. Moreover, growing a culture requires 2-6 weeks, which can further delay diagnosis and treatment.

[0006] Other diagnostic tests for active TB, such as those involving nucleic acid amplification are rapid, fairly sensitive (95% in smear-positive 60-70% in smear- negative culture-positive TB), and highly specific, but are expensive and highly sophisticated. Such tests are therefore essentially limited to developed countries where the burden of TB is very low. A mycobacteriophage-based assay is more suitable to resource-poor regions of the world, but requires technical expertise, and it has low sensitivity in smear-negative TB. [0007] In addition to the diagnostic tests discussed above, the tuberculin skin test is used to assess tuberculosis in humans and animals. 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. An example is the Quantiferon Gold assay. Like the tuberculin skin test, this assay does not distinguish between active and inactive infection. [0008] 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. A test based on this invention potentially would be used worldwide to test millions of people annually. [0009] 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.

[0011] Applicants have found that 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.

[0012] 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. 91 , pp9342-9346 (1994); Harth G, Horwitz MA "Expression and Efficient Export of Enzymatically Active Mycobacterium Tuberculosis Glutamine Synthetase in Mycobacterium Smegmatis and Evidence that the Information for Export is Contained Within the Protein". J Biol Chem. 272. pp 22728-22735(1997)). 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. "Deciphering the Biology of Mycobacterium Tuberculosis from the Complete Genome Sequence", Nature. 393. pp 537-544 (1998)) is an essential enzyme as glnA1 mutants are not viable in vitro or in vivo, and the other glnA'\ homologs in M. tuberculosis (glnA2-4) cannot replace the functional loss of gr/nAI (Harth G, Maslesa-Galic S, Tullius MV₁ Horwitz MA "All Four Mycobacterium Tuberculosis glnA Genes Encode Glutamine Synthetase Activities But Only GInAI Is Abundantly Expressed And Essential For Bacterial Homeostasis", MoI Microbiol. 58, pp1157-1172 (2005); 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)). The GS inhibitor L-methionine-S,f?-sulfoximine (MSO) selectively inhibits enzymes of bacterial origin, including M. 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)), but has little effect on mammalian glutamine synthetases. Treatment of guinea pigs infected with M. tuberculosis using MSO or its analogs reduces the mycobacterial burden in their lung and spleen.

[0013] Applicants previously discovered, unexpectedly, that 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. In fact, applicants discovered that 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.

[0014] In contrast to the tuberculin skin test, the new test described herein in its various embodiments is positive only when tuberculosis is active. In addition, 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. Currently, there is no assay available for monitoring the course of tuberculosis. [0015] Since the great majority of tuberculosis cases occur in developing nations, it is important that the test be inexpensive. Currently, 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.

[0016] In addition to utility as a diagnostic test for 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). [0017] In accordance with embodiments incorporating features of the invention set forth herein, a small amount of patient serum is obtained and then assayed for Glutamine Synthetase (GS) Activity or for the presence of M. tuberculosis GS. To insure that only M. tuberculosis GS is assayed, 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.

[0018] GS catalyzes the production of glutamine from glutamate and ammonia via the following reaction:

Glutamate + ATP + NH₃ → Glutamine + ADP + Pi

For convenience 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:

Glutamine + hydroxylamine — > gamma-glutamylhydroxamate

[0019]Gamma-glutamylhydroxamate is then assayed by its reaction with FeCb at acidic pH to form gamma-glutamylhydroxamate-Fe³⁺ complex (orange-brown in color), the absorbance of which can be measured at 540nm. [0020] It is contemplated that card assay techniques, which requiring only that a drop of serum from a patient be placed on the substrate for GS, can be utilized. A change in color would reveal a positive test. Alternatively, an ELISA or latex agglutination test can be developed to assay the level of GS. [0021] In a first embodiment of the invention serum is obtained from a patient and assayed, e.g. using a card assay impregnated with substrate such that it yields a color change if GS is present. At the same time 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). The 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.

[0022] Some of the advantages over existing practices is that a rapid assay of tuberculosis, whether pulmonary or extrapulmonary, in humans or animals can be performed. In contrast to the tuberculin skin test for tuberculosis, old infection with tuberculosis can be distinguished from current active disease. Further, in contrast to the tuberculin skin test, the procedures and tests set forth herein can distinguish previous exposure to BCG vaccination from active tuberculosis. [0023] 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. Further, 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.

[0024]The use of 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. In contrast, 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. 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

[0025] Figure 1 shows the protein profile following each major step in the purification of Glutamine Synthetase from infected guinea pig sera. [0026] Figure 2 illustrates the reactivity of Glutamine Synthetase from M. tuberculosis infected guinea pig sera. DETAILED DESCRIPTION

[0027] Set forth herein is a test for active tuberculosis based on measuring the amount of glutamine synthetase (GS) or GS activity in serum. M. tuberculosis, along with other pathogenic mycobacteria, 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. Lee, B. J. Dillon, and G. Harth, Protective immunity Against Tuberculosis Induced by Vaccination with Major Extracellular Proteins of Mycobacterium Tuberculosis", Proc. Natl. Acad. Sci. U.S.A.. 92, pp 1530-1534 (1995)). [0028] Applicants previous discovered and identified the enzyme GS as one of the abundantly released proteins of M. tuberculosis. This was a surprising finding since this enzyme is typically located in the bacterial cytoplasm and lacks a signal sequence for secretion. GS is released both by organisms growing in broth medium and by organisms growing intraphagosomally in human mononuclear phagocytes. 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. [0029] In subsequent studies, applicants also found that only 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. Hence, the presence of GS in culture filtrates correlates with the pathogenicity of mycobacteria. [0030] 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.

[0031]As reported by applicants, 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)). To further study the role of GS in pathogenicity, applicants constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity, and it was auxotrophic for L- glutamine both in broth culture and in human macrophages. Most importantly, the mutant was completely avirulent in vivo. In M. tuberculosis, 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.

[0032] Moreover, applicants' previous studies showed that treatment of M. tuberculosis with the GS inhibitor L-methionine-S,R-sulfoximine (MSO) or with antisense oligodeoxyribonucleotides specific to M. tuberculosis GS mRNA inhibits formation of the poly-L-glutamate/glutamine cell wall structure These agents also inhibit bacterial growth, indicating that the enzyme plays an important role in bacterial homeostasis (Harth, G., P. C. Zamecnik, J-Y. Tang, D. Tabatadze, and M. A. Horwitz, "Treatment of Mycobacterium Tuberculosis with Antisense Oligonucleotides to Glutamine Synthetase mRNA Inhibits Glutamine Synthetase Activity, Formation of the Poly-L-glutamine/glutamate Cell Wall Structure, and Bacterial Replication". Proc. Natl. Acad. Sci. USA. 97: pp 418-423. (2000)). 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. tuberculosis and M. avium within human mononuclear phagocytes, the primary host cells of these pathogens, and at concentrations that are completely nontoxic to the mammalian cells, likely reflecting the 100-fold greater sensitivity to MSO of bacterial GS than mammalian GS. Additionally, 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)).

[0033] Importantly, a key reason why glutamine synthetase is one of the most abundant proteins found in actively growing cultures of M. tuberculosis is that the enzyme is not only highly expressed but remarkably stable in culture medium (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 )). 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. Alternatively, 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.

[0034] I. Serum GS Activity Correlates with M. tuberculosis Infection in

Guinea Pigs - To determine if GS activity is present in the sera of guinea pigs infected with M. tuberculosis, guinea pigs were infected with M. tuberculosis by aerosol and their sera assayed five weeks later for GS activity.

[00351 Example 1

[0036] A. Procedures

[0037] 1. Infection of Guinea pigs with M. tuberculosis

[0038] Five 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.

One animal died two weeks after infection. The surviving animals were euthanized five weeks after infection and the burden of M. tuberculosis in the animals' lungs and spleens assayed by plating dilutions of organ homogenates on agar medium and enumerating CFU after a two week incubation period. At necropsy, in addition to assessing gross pathology, the lesions visible on the animals' liver lobes were counted. Control animals consisted of five uninfected guinea pigs and five guinea pigs immunized with BCG 10 weeks before assessment of GS activity.

[0039] 2. Serum collection and preparation of sera for GS assay

[0040] Five weeks after infection, blood was collected from each of the four surviving guinea pigs. The animals were anaesthetized with 50 mg/kg ketamine and 7.5 mg/kg xylazine, administered intraperitoneal^, and blood was obtained by heart puncture (cardiocentesis) with a 23-gauge needle. All blood samples were collected in sterile glass tubes and left undisturbed for 24 h at 4⁰C. Then the clear sera were collected and filtered through 0.45 μm and 0.22 μm membranes. For each guinea pig, approximately 1.5 ml_ of clear serum was stored at 4⁰C for protein analyses and assay for glutamine synthetase activity. A 100 μl_ aliquot was removed from each sample and plated on 7H11 (Middlebrook) agar media to ascertain whether the sera were free of M. tuberculosis.

[0041] 3. Glutamine synthetase assay

[0042] As the guinea pig sera are crude protein mixtures, the standard catalytic glutamine synthetase assay of glutamine synthesis could not be used. Instead, 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 γ-glutamylhydroxamate which, when acidified, forms a hexadentate complex with Fe³⁺ in a color reaction that can be spectrophotometrically read at 540 nm. The reaction mixes containing one ml_ of reaction buffer and from about 100 to 125 μl_ of serum were incubated for various times up to 1 h at 37⁰C, and then read at 540 nm against the standard curve established with a pure sample of γ-glutamylhydroxamate. For assay of sera from infected guinea pigs, BCG-immunized guinea pigs, and uninfected control guinea pigs, and for assay of commercial guinea pig serum, 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 μmoles or ~ 150 ng of γ-glutamylhydroxamate. [0043] B. Results

[0044] 1. Guinea pig infection with M. tuberculosis

[0045] All five guinea pigs lost weight beginning one week after challenge with M. tuberculosis; one animal died two weeks after challenge (Table 1A). At necropsy, five weeks after challenge, all surviving guinea pigs had evidence of infection with M. tuberculosis. All had large numbers of lung, spleen, and liver lesions. CFU of M. tuberculosis in the lung and spleen and the number of liver lesions are shown in Table 1B. Table 1A: Experiment 1: Guinea pig weight

Guinea Weight [g] pig Week and initial 1 2 3 4 5 weight [g]

#1 (1791) 1396 1382 1360 1332 1330

#2(1591) 1205 1163 1199 1171 1175

#3(1606) 1200 1192 1164 1137 1128

#4(1652) 1237 1224 1194 1164 1160

#5(1558) 1179 1085

Table 1B: Experiment 1: CFU in Lung and Spleen and Number of Liver Lesions at necropsy

[0046] 2. GS activity in guinea pig serum

[0047] 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 μmole of γ-glutamylhydroxamate per min at 37⁰C (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

[0048] In contrast, sera obtained from five control animals had no detectable GS activity (Table 1 D). The sera of the uninfected guinea pigs were cultured for CFU of M. tuberculosis and none had detectable CFU.

Table 1D: GS activity in sera of uninfected control guinea pigs

[0049] As an additional control, the sera of 5 guinea pigs was assayed 10 weeks after they were immunized with BCG Tice (1000 CFU). None of the five animals had detectable GS activity (Table 1 E). The sera of the BCG-immunized guinea pigs were cultured for CFU of M. tuberculosis and none had detectable CFU.

Table 1E: GS Activity in Sera of BCG-immunized Guinea Pigs 10 weeks after Immunization

[0050] Additionally, a sample of commercially obtained (Calbiochem) normal guinea pig serum was assayed and was found to have no detectable GS activity. [0051] II. 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 - In light of the above study showing that the sera of guinea pigs infected with M. tuberculosis had GS activity the question as to whether the GS was mammalian or bacterial in origin was explored. If mammalian in origin, 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. To differentiate between mammalian and M. tuberculosis GS in guinea pig sera, the following additional studies were conducted. The first study evaluated the differential effect of MSO on bacterial vs. mammalian GS.

[0052] 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 μM; K₁ for M. tuberculosis GS ~1 μM). 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 2A: Specificity of the inhibitor MSO

[0053] Given the greater sensitivity of M. tuberculosis GS to MSO than mammalian (human) GS, it was concluded that if incubation of the sera from M. tuberculosis-infected guinea pigs resulted in only a slight decrease in detectable GS activity, the GS activity was most likely due to the presence of a eukaryotic GS; in contrast, a large decrease in activity would indicate that the activity measured in the sera was most likely prokaryotic in origin, i.e. derived from the M. tuberculosis enzyme. To determine the sensitivity of the serum GS activity to MSO, the GS activity in the sera of M. tuberculosis-infected guinea pigs was assayed in the presence and absence of high or low concentrations of MSO. [0054] Example 2 [0055] A. Method

[0056] The sera from the infected guinea pigs (same as described in Example 1 , Tables 1A-C) were initially added to a modified transfer reaction mixture lacking the substrate L-glutamine but containing various concentrations of MSO (which is very soluble in water or reaction buffer), as indicated below, and preincubated for 15 min at 37⁰C. Each reaction mixture was then individually transferred to a standard transfer reaction mixture and further incubated for 60 min at 37⁰C, and processed for the detection of γ-glutamylhydroxamate as described above. [0057] B. Results

[0058] The results (Table 2B) showed that the GS activity in the sera of M. tuberculosis -infected guinea pigs is sensitive to low concentrations of MSO. At 2 μM, a concentration that has little effect on mammalian GS, the GS activity in sera from M. tuberculosis-\nfected guinea pigs was reduced by > 75% in each of the guinea pigs i.e. to < 25% of the initial level of GS activity. This indicated that the GS was of bacterial origin rather than mammalian origin. Table 2B: Inhibition of GS activity in sera of M. tuberculosis-Mected guinea pigs with MSO

Inhibitor & GS activity at week 5 in four infected guinea pigs (gp) Concentration [% of Initial GS Activity] gp#1 gp#2 gp#3 gp#4

None 100 100 100 100

MSO at 0.1 μM 95 97 92 90

MSO at 0.2 μM 75 83 85 70

MSO at 2 μM 18 25 16 19

MSO at 20 μM 12 9 8 13

MSO at 200 μM 6 5 5 7

[0059] III GS Activity in Serum of Guinea Pigs Infected with M. tuberculosis is Preferentially Precipitated by Antibody to M. tuberculosis GS vs. Antibody to Mammalian GS consistent with the majority of the GS activity in the sera deriving from M. tuberculosis GS - In a second assay aimed at differentiating mammalian and M. tuberculosis GS in the sera of M. tuberculosis-^'mfected guinea pigs anti- M. tuberculosis GS or anti-human GS antibodies were used to immuno- precipitate the detectable GS activity in the sera. Using Western blotting it was first determined that, at 1 :1 ,000 dilutions, both antibodies strongly recognized their cognate antigens, but also cross-reacted with the non-specific enzyme target, although at a much lower signal intensity (10 - 20% of the specific signal intensity; anti-M. tuberculosis GS antibodies showed less cross-reactivity than the anti-human GS antibodies). Based on the results of the enzyme activity assays, an estimate of the amount of GS per ml_ of serum was made. Each of the two antibody preparations were separately added to the serum samples at a 10-fold excess to quantitatively immunoprecipitate the GS molecules. The GS activity in the immunoprecipitate and in the filtrate could then be assayed. If both antibodies quantitatively precipitated all the GS activity it could be concluded that there would be no enzyme activity in the treated serum. On the other hand, if both antibodies were highly specific for their cognate protein, each GS enzyme could be selectively assayed and their proportional contribution to the total detectable GS activity in the serum of infected guinea pigs could be determined. [0060] Example 3 [0061] A. Methods

[0062] The sera from the infected guinea pigs were incubated with each antibody preparation overnight at 4O⁰C and centrifuged to pellet the immunocomplexes. The supernates were transferred to a standard transfer reaction mixture, incubated for 60 min at 37⁰C₁ and processed for the detection of γ-glutamyl-hydroxamate as described above. The pelleted immunocomplexes were treated with 4 M MgCI₂ (Stoeckel, K., Gagnon, C, Guroff, G., and Thoenen, H., "Purification of Nerve Growth Factor Antibodies by Affinity Chromatography", J. Neurochem. 26, pp 1207-1211 (1976)) to separate the GS molecules from the antibodies and fractionated through a Sephadex CL-6B column to yield intact GS molecules. The GS fraction was extensively dialyzed against 10 mM imidazole pH 7, 2 mM MnCb, and assayed in the transfer reaction as described above. [0063] B. Results

[0064] Based on the results listed in Table 3A, it was concluded that the GS activity in serum from M. tuberculosis-infected guinea pigs is derived from M. tuberculosis GS. Anti-M tuberculosis antibody immunoprecipitated > 85% of the GS activity in the sera whereas anti-mammalian GS precipitated <15% of the GS activity in the sera.

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

Specific antibody % Free / % Precipitated GS activity in guinea pig (gp) sera gp#1 gp#2 gp#3 gp#4

None 100 / 0 100 / 0 100 / 0 100 / 0 anti-M tuberculosis GS 6 / 92 7 / 90 5 / 88 8 / 85 anti-human GS 85 / 7 83 / 10 90 / 3 77 / 15 [0065] IV. Purification of GS from the Serum of M. tuberculosis-] nfected Guinea Pigs - To definitively determine the identification of the source of the GS in guinea pig sera, the enzyme from the pooled sera of nine M. tuberculosis- infected guinea pigs was isolated and purified and its amino acid sequence was determined. Because it is present in small quantities when compared with the major serum proteins, such as albumin, several different procedures were evaluated to isolate the protein. One of the procedures is set forth below. [0066] Example 4 [0067] A. Methods [0068] 1. Infection of guinea pigs

[0069] Nine guinea pigs (age ~5 months, weight -600 g, male outbred Hartley strain from Charles River Laboratories) were infected with a dose of aerosolized M. tuberculosis Erdman strain. This resulted in approximately 75 primary lung lesions. Ten weeks later the animals were euthanized, bled via cardiac puncture, and necropsied to determine the M. tuberculosis burden in the animals' lungs and spleens. A total of 170 ml_ of serum was obtained from the nine guinea pigs. [0070] 2. Purification of GS from sera of M. 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.

[0072] B. Results

[0073] 1. Guinea pig infection

[0074] 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.

[0075] At 10 weeks after challenge, the guinea pigs had a high burden of M. tuberculosis in the lung and spleen and numerous lesions in these organs as well as in the liver (Table 4B).

Table 4A: Mean weight of guinea pigs after challenge

Mean Weight [g] at week:

0 1 2 3 4 5 6 7 8 9 10

637 660 747 727 736 767 787 812 823 818 810

Table 4B: CFU in lungs and spleen and mean number of liver lesions in guinea pigs 10 weeks after challenge

[0076] 2. Purification of serum GS activity

[0077] In several small scale tests, it was ascertained that the Affi gel blue chromatography is a very critical step in the procedure and that care must be taken to adjust the size of the bed volume such that it is highly effective in removing the large quantities of serum albumin and other proteins in serum. The chromatography on Q-Sepharose or hydroxyapatite eliminated a large number of proteins that do not bind tightly to the multitude of charges present on these resins, and the final sizing steps eliminated all small molecules and single subunits that had accumulated during the purification process. Fractions were then cut very narrowly, as the intact GS molecules are so much larger than all the remaining proteins in the preparations. The presence of protease inhibitors and a fast purification schedule did not completely prevent breakdown of GS molecules. The procedure yielded 125 μg of GS from 170 ml_ of guinea pig sera. The protein profile following each major step in the purification is depicted in Figure 1.

[0078] V. Immunoreactivity of Purified Serum GS - As a first step towards characterizing the GS activity of the GS isolated from the sera of guinea pigs infected with M. tuberculosis, we determined the protein's reactivity with anti-M. tuberculosis GS and anti-mammalian GS. [0079] A. Method

[0080] 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. GS isolated from the sera of guinea pigs infected with M. tuberculosis and a sample of human GS was subjected to SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and each set was separately probed using either an\\-M. tuberculosis GS or anti-human GS antibodies. All blots were developed with horse radish peroxidase substrates (Super Signal chemiluminescent substrates, Pierce) and digitized. [0081] B. Results [0082] A quantitative evaluation of the results of the immunoblots (Figure 2) showed that both antibody preparations used at the same dilution reacted with both GS proteins loaded at the same amounts. However, in the case of both M. tuberculosis GS and human GS, the reaction with the cognate antigen was much stronger than with the non-specific GS protein. The anti-/W. tuberculosis GS antibodies were more specific (< 10% cross-reactivity based on signal intensities) than the anti-human GS antibodies (~ 20% cross-reactivity based on signal intensity).

[0083] Importantly, 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.

[0084] 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

[0086] Two samples of the purified enzyme were subjected to denaturing polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and the stained membrane strips submitted were forwarded to the HHMI Keck Foundation Biotechnology Resource Laboratory at Yale University for amino acid sequence analysis. [0087] B. Results: [0088] 1. Sample #1

[0089] 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

[0091] For the second analysis a larger amount of purified GS was used and the N-terminal eight amino acids of the intact GS molecule were determined. As the genome of the guinea pig (Cavia porcellus) is not yet fully sequenced and annotated, the amino acid sequence of the Chinese hamster GS, a close relative, was used for comparative sequence alignments. Again, the sequence was an exact match for the M. tuberculosis GS and had no similarity to any eukaryotic GS.

[0092] 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.

Sample 1

Sequence #1

M. tuberculosis GS residues 248-253: Ile-lle-Lys-Asn-Thr-Ala Human GS homolog 224-229: Ile-Leu-His-Arg-Val-Cys

Chinese hamster homolog 224-229: Ile-Leu-His-Arg-Val-Cys

Sequence #2

M. tuberculosis GS residues 263-267: Met-Pro-Lys-Pro-Leu Human GS homolog 239-243: Asp-Pro-Lys-Pro-lle

Chinese hamster homolog 239-243: Asp-Ser-Lys-Pro-lle Sequence #3

M. tuberculosis GS residues 385-392: Met-Ala-(Gly)-Leu-Asp-(Gly)-lle

Human GS homolog 357-358: Arg-Thr

Chinese hamster homolog 357-358: Arg-Thr

Sample 2

Sequence #1

M. tuberculosis GS residues 2-9: Thr-Glu-Lys-Thr-Pro-Asp-Asp-Val Human GS homolog 9-16: Asn-Lys-Gly-lle-Lys-Gln-Val-Tyr

Chinese hamster homolog 9-16: Asn-Lys-Gly-lle-Lys-Gln-Met-Tyr

[0093] 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. [0094] 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

[0095] A. Quantitation of GS in the sera of guinea pigs infected with M. tuberculosis

[0096] Densitometry analysis of the purified protein band derived from the sera of guinea pigs infected with M. tuberculosis, demonstrated by sequence analysis to be M. tuberculosis GS, extrapolated to 170 mL of guinea pig serum, indicated that 125 μg of purified GS was obtained from 170 mL of serum, or 0.735 μg of purified GS/mL of serum. The yield was estimated to be 43%, based on the total GS activity in the 170 mL of guinea pig serum (5258 mUnits) and the total GS activity recovered after purification (2270 mUnits) (2270/5258 x 100 = 43%), suggesting that the serum contained ~1.7 μg of GS/mL of serum (100%/43% x 0.735 μg GS/mL of serum = 1.7 μg of GS/mL of serum). [0097] 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. Jackowski, "Immunoassay for Glutamine Synthetase in Serum: Development, Reference Values, and Preliminary Study in Dementias", Clin. Chem.. 48, pp 375-378 (2002)) is trivial by comparison. Hence disease states that raise GS levels in humans are unlikely to interfere with the diagnosis of tuberculosis using an assay for GS protein or activity. [0098] VIII. Serum from Patients with Tuberculosis but Not from Control Persons Has GS Activity - To determine if serum from patients with active tuberculosis has measurable GS activity, we assayed GS in patient serum using the Transfer Assay. GS levels in serum of patients newly diagnosed and treated for tuberculosis (0-15 days) and of the same patients after an additional 30 days of treatment were studied. Patients with both pulmonary TB as well as patients with extra-pulmonary tuberculosis were studied. Persons without tuberculosis who were either not sick or had other diseases were used as controls. [0099] A. Methods

[0100] Assay of GS (Transfer Assay) in serum

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:

0.03 M L-glutamine

0.02 M sodium arsenate (Na₂AsO₄) 0.003 M manganese chloride

0.06M hydroxylamine hydrochloride [neutralized with sodium hydroxide

(NaOH)]

4 X iO^-4 M adenosine diphosphate (ADP)

0.015 M Tris hydrochloric acid (HCI), pH 7.0

The solution was incubated at 37 ⁰C for 15 minutes and the reaction was terminated by the addition of 0.5 ml_ of a mixed reagent containing equal volumes of 24% trichloroacetic acid, 6(N)HCI and 10% ferric chloride in 0.02 (N) HCI. The mixture was centhfuged to remove the denatured protein and the intensity of the color of the supernate was measured at an OD of 540nm. A standard curve was established with a pure sample of gamma- glutamylhydroxamate to determine the amount of serum GS activity in ml⁾. One enzyme unit was defined as the amount of glutamine synthetase that produces one μmole of gamma-glutamylhydroxamate per min at 37⁰C. [0101] 2. Patients

[0102] All subjects were evaluated at the Out-Patient Department or the Isolation Ward of the B. S. Medical College & Hospital, Bankura, India. Four groups of patients were studied: 1) patients with pulmonary tuberculosis (n = 10 subjects, ages 17-45, 8 males and 2 females, of which 6 were sputum smear-positive and 2 sputum smear-negative, and 2 not assessed); 2) patients with extrapulmonay tuberculosis (n = 10 subjects, ages 18-40, 8 males and two females); 3) normal control subjects who were relatives of the patients with TB but who had no clinical signs or symptoms of tuberculosis, negative chest x-rays for tuberculosis, and sputum that was negative for tuberculosis (n = 14 subjects, age 20-56 years old); and 4) disease control subjects who were patients with respiratory tract infection or bronchogenic carcinoma, and with no evidence of tuberculosis (n = 12 subjects, age 15-40 years old).

[0103] Approximately 2.5mL of blood was obtained from each subject by venipuncture; the blood was allowed to clot and the serum was separated and stored at 2-4⁰C until assayed, usually on the same day the serum was obtained. [0104] B. Results [0105] Table 7A below shows the results from 10 patients with pulmonary tuberculosis, 10 patients with extrapulmonary tuberculosis, 14 normal control subjects and 12 disease control subjects. All of the patients with active tuberculosis, whether pulmonary or extra-pulmonary, had GS activity in their sera, whereas none of the normal control patients or patients with diseases other than tuberculosis had detectable GS in their sera. In all patients with tuberculosis, both pulmonary and extra-pulmonary, GS activity declined with antibiotic treatment, as reflected by the lower values of GS activity in a second sample of patient serum obtained after 30 days of additional antibiotic treatment compared with the level in the initial sample of patient serum obtained within the first 15 days of diagnosis and antibiotic treatment.

Table 7A. GS activity in sera of patients and controls

Group 1 : Pulmonary Tuberculosis

ND, Not determined Group 2: Extra-pulmonary Tuberculosis

ND, Not determined

Group 3: Normal Control Subjects

Group 4: Disease Control Subjects

[0106] 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. However, the invention set forth herein is not so limited. 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. This includes, but is not limited to testing for reactivation of tuberculosis in latently infected mammals, distinguishing active from inactive tuberculosis in mammals and detecting the presence of drug-sensitive or the emergence of drug-resistant tuberculosis in mammals by assaying for the presence or level of bacterial glutamine synthetase activity or of M. tuberculosis Glutamine Synthetase in that mammal. 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.

[0107] 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. In one embodiment, 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. In a second embodiment 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.

[0108] In light of the teachings herein, one skilled in the art will recognize other suitable applications of the rapid serological test set forth herein.

Claims

We claim:

1. A rapid serologic test for active tuberculosis in mammals or a rapid serological test to monitor the efficacy of treatment for active tuberculosis in mammals comprising: obtaining a sample of patient serum assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.

2. The rapid serologic test of claim 1 wherein the site of tuberculosis is pulmonary.

3. The rapid serologic test of claim 1 wherein the site of tuberculosis is extrapulmonary.

4. The rapid serologic test of claim 1 wherein the test is also conducted in the presence of a selective inhibitor of bacterial GS.

5. The rapid serologic test of claim 4 wherein the selective inhibitor of bacterial GS is selected from L-Methionine-S,R-Sulfoximine or alpha-ethyl-D,L- Methionine-S,R-Sulfoximine.

6. The rapid serologic test of claim 1 wherein the test solution contains monoclonal or polyclonal antibodies specific to M. tuberculosis GS.

7. The rapid serologic test of claim 1 wherein Gamma-glutamylhydroxamate is assayed by its reaction with FeCU at acidic pH to form an orange-brown colored gamma-glutamylhydroxamate-Fe³⁺ complex, the absorbance of which is measured spectrophotometrically at about 540nm.

8. The rapid serologic test of claim 1 wherein a drop of the patient's serum is brought into contact with a GS active substrate, the presence of M. tuberculosis GS being evidenced by a visible color change of the substrate.

9. The rapid serologic test of claim 1 wherein a drop of the patient's serum is analyzed quantitatively for the presence of M. tuberculosis GS.

10. The rapid serologic test of claim 1 wherein the quantitative analysis is conducted using ELISA or latex agglutination.

11. The rapid serologic test of claim 1 wherein a first sample of patient sera is tested for Glutamine Synthetase activity and a second sample of patient sera is tested for Glutamine Synthetase activity wherein the test solution also includes a selective inhibitor of bacterial GS, and GS activity in the first sample but not in the second sample indicates the presence of active tuberculosis.

12. A rapid serologic test to distinguish between inactive and active tuberculosis in mammals comprising:

. obtaining a sample of patient serum and assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.

13. A rapid serologic test to distinguish between active tuberculosis in mammals and inactive tuberculosis, or previous exposure to BCG vaccination comprising: obtaining a sample of patient serum assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.

14. A process of culling animals with active tuberculosis from animals previously exposed to BCG vaccination or having inactive tuberculosis comprising: obtaining a sample of serum from the animal, assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase, and separating the animals with active tuberculosis from those animals with inactive disease or previously vaccinated for tuberculosis.

15. The rapid serologic test of claim 5 wherein L-Methionine-S,f?-Sulfoximine or alpha-ethyl-D,L-Methionine-S,R-Sulfoximine is present in a concentration of from about 0.2 μM to about 200 μM.

16. A rapid serologic test to monitor the success of therapy of tuberculosis in mammals comprising: obtaining a first sample of serum from a subject at the onset of therapy and subsequent samples at various times thereafter and assaying the samples for the level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase, wherein successful therapy is indicated by a lesser level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in a subsequent sample when compared to the first sample and unsuccessful therapy is indicated by an absence of a difference between the level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in successive samples when compared to the first sample or by a greater level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in a subsequent sample when compared to the first sample.

17. A rapid serologic test to monitor the presence or emergence of antibiotic resistant tuberculosis in mammals comprising: obtaining a first sample of serum from a subject at the onset of antibiotic therapy and subsequent serum samples at various times thereafter and assaying the samples for the level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase, a decline in the level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in subsequent samples when compared to the first sample indicating drug-sensitive tuberculosis and the absence of change in the level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in subsequent samples when compared to the first sample, or a greater level of bacterial Glutamine Synthetase activity or the level of M. tuberculosis Glutamine Synthetase in a subsequent sample when compared to the first sample, indicating drug-resistant tuberculosis.

18. A rapid serologic test to detect reactivation of tuberculosis in mammals with latent tuberculosis comprising: obtaining a first serum sample and periodically obtaining subsequent samples of serum from a subject, assaying each sample for bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase, the presence of bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase in any sample indicating reactivation of tuberculosis.

19. A rapid serologic test to distinguish active from inactive tuberculosis in a mammal with a positive tuberculin skin test or Quantiferon test for tuberculosis, comprising: obtaining a sample of serum from the subject, assaying the sample for bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase, the presence of bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase indicating active tuberculosis the absence of bacterial Glutamine Synthetase activity or M. tuberculosis Glutamine Synthetase indicating inactive tuberculosis.

20. A method of treating active tuberculosis comprising obtaining a sample of patient serum, assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase and if the assay shows the presence of bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase, treating the patient with a suitable anti-tuberculosis drug.

21. The method of claim 20 further including periodically during treatment with a suitable anti-tuberculosis drug obtaining a sample of patient serum, assaying that sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase and ceasing treatment with the anti-tuberculosis drug once the assay of the sample of patient serum no longer shows the presence of bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.

22. The method of claim 21 further including, after ceasing treatment, periodically obtaining serum from the patient and assaying the sample of patient serum for the presence of bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase to ascertain whether the patient has a re-occurrence of active tuberculosis.

23. A kit for determining the presence in a mammal of active tuberculosis comprising: apparatus suitable for obtaining a sample of blood from the mammal and separating a sample of serum therefrom and means for assaying the serum sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase.

24. A kit for treating active tuberculosis in a mammal comprising: apparatus suitable for obtaining a sample of blood from the mammal and separating a sample of serum therefrom, means for assaying the serum sample for bacterial Glutamine Synthetase activity or the presence of M. tuberculosis Glutamine Synthetase and and one or more doses of antibiotic suitable for treating active tuberculosis.