Tuberculosis Methods

Abstract

The present invention relates to a method for diagnosing tuberculosis (TB) disease in a subject. The method involves mixing a mycobacteria-specific bacteriophage with a sample of peripheral blood mononuclear cells (PBMCs) from the subject, followed by determining the presence or absence of mycobacterial DNA sequences in DNA isolated from the mixture. The present invention provides a sensitive and specific test for the presence of early TB, i.e., asymptomatic infection that carries a high risk of developing active TB, or latent tuberculosis infection (LTBI), i.e., asymptomatic infection that is unlikely to develop into active TB in a subject.

Description

[Technical Field]

[0001] The present invention relates to methods for characterizing the state of tuberculosis (TB) in a subject so that individuals with early TB, asymptomatic or latent TB infection (LTBI) can be identified. The invention further relates to methods of treatment, methods for improving the prognosis of TB, and kits for use in assessing TB disease in a subject. [Background technology]

[0002] An estimated one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB). Globally, an estimated 10 million people developed TB disease in 2017. While TB cases occur in all countries and age groups, the highest prevalence is among adults in India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh, and South Africa. In the same year, there were an estimated 1.3 million deaths attributable to TB among HIV-negative people and an additional 300,000 deaths among HIV-positive people. TB is the leading cause of death from a single infectious pathogen in humans. In 2017, the proportion of people with TB who died from the disease was 16%, down from 23% in 2000, but far from the 10% rate needed to meet the World Health Organization (WHO) End TB Strategy 2020 target (Global Tuberculosis Report 2018, World Health Organization, https: / / www.who.int / tb / publications / global_report / en / ).

[0003] The traditional understanding of TB disease is dualistic. In one state, latent TB infection (LTBI) is defined as a state of persistent immune response to M. tuberculosi in the absence of clinically evident evidence of active TB disease, in which patients are asymptomatic and M. tuberculosi replication is low and controlled by the immune system. Latent TB infection provides M. tuberculosi with the ability to persist undetected in the population. Individuals can harbor latent TB infection for years without detection. Current estimates of the number of people worldwide with latent TB infection range from 1.7 billion to over 2 billion (23–27% of the world's population). In contrast, in active TB disease, patients are symptomatic due to pathology resulting from uncontrolled M. tuberculosi replication.

[0004] Only a fraction of individuals with LTBI develop active TB disease. The lifetime risk of developing active TB among infected individuals is 5–15%, with the highest risk occurring in the first 2 years after infection. Clinically latent infection appears to represent a continuum of outcomes, ranging from presumably cleared infection to asymptomatic disease without overt symptoms. Recent evidence supports the existence of a transitional state of human Mtb infection, termed early TB, characterized by clinically latent disease but a high risk of progression to active TB in the absence of further intervention, and a host blood transcriptional profile that overlaps with the disease (Cobelens F, et al., From latent to patent: rethinking prediction of tuberculosis. The Lancet Respiratory Medicine 2017;5(4):243-4; Zak DE, et al., A blood RNA signature for tuberculosis disease risk: a prospective cohort study. Lancet 2016;387:2312-22). Individuals with early TB have not yet developed elicited clinical symptoms, radiographic abnormalities, or microbiological evidence consistent with active TB.

[0005] Currently, treatment of LTBI is the primary therapeutic intervention available to prevent the development of active TB disease in people already infected with Mtb. While TB preventive treatment for LTBI is expanding, most people who would benefit from it still do not receive treatment. This is due in part to underdiagnosis of people with LTBI, which has multiple reasons, including asymptomatic or lack of symptoms, meaning people do not seek medical care, and the inability to accurately test for TB when people see a healthcare professional.

[0006] Therefore, there is a need for a rapid, simple and accurate test that allows for the detection of early TB early enough to allow effective intervention and treatment. It is against this background that the present invention has been developed. Summary of the Invention

[0007] In a first aspect of the present invention, 1. A method for diagnosing a disease in an asymptomatic human subject, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing mycobacteria-specific bacteriophage D29 with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; determining whether mycobacterial DNA sequences are present or absent in the DNA isolated from the mixture by one or more of: performing PCR on the DNA isolated from the mixture using forward and reverse primers specific for the mycobacterial DNA sequence, and / or performing appropriate sequencing to identify the mycobacterial DNA sequence, and optionally determining homology between the mycobacterial DNA sequence (if present) and known mycobacterial DNA sequences; optionally, performing a Mycobacterium-specific cellular immune (CMI) response test on the sample isolated from the subject to generate a positive or negative CMI response test result; the asymptomatic human subject is diagnosed with early tuberculosis (TB) if the mycobacterial DNA sequence, if present, is present, accompanied by either a positive or negative CMI response test result; Methods are provided in which an asymptomatic human subject is diagnosed with latent tuberculosis infection (LTBI) in the absence of mycobacterial DNA sequences and the presence of a positive CMI response test result.

[0008] In another aspect of the present invention, 1. A method for diagnosing early stage tuberculosis (TB) in a human subject, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing a mycobacteria-specific bacteriophage with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; determining whether one or more mycobacterial DNA sequences are present in the DNA isolated from the mixture; Methods are provided in which the presence of a mycobacterial DNA sequence indicates that the subject has early stage tuberculosis.

[0009] In one embodiment, the human subject is an asymptomatic human subject, i.e., the subject does not exhibit any symptoms of active tuberculosis disease.

[0010] In another aspect of the present invention, 1. A method for predicting whether a subject will develop or is at risk of having early stage tuberculosis (TB), comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing a mycobacteria-specific bacteriophage with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; and determining whether a mycobacterial DNA sequence is present in the DNA; Methods are provided in which the presence of a mycobacterial DNA sequence indicates that a subject is at risk of developing or having early-stage tuberculosis.

[0011] In another aspect of the present invention, 1. A method for predicting whether a subject is at risk for developing active tuberculosis (TB), comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing a mycobacteria-specific bacteriophage with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; and determining whether a mycobacterial DNA sequence is present in the DNA; Methods are provided in which the presence of a mycobacterial DNA sequence indicates that the subject is at risk for developing active TB.

[0012] In another aspect of the present invention, 1. A method for assessing the prognosis of tuberculosis (TB) disease in a subject, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing a mycobacteria-specific bacteriophage with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; and identifying the presence or absence of mycobacterial DNA sequences in the DNA isolated from the mixture; Methods are provided in which the presence of a mycobacterial DNA sequence indicates that the subject is at risk of having early stage TB and / or at risk of developing active TB disease.

[0013] In another aspect of the present invention, 1. A method for treating tuberculosis (TB) in a subject in need thereof, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; To identify the presence of early TB in a subject using a bacteriophage-mediated mycobacterial DNA release assay and, if early TB is identified, administering one or more anti-TB drugs or therapies to the subject.

[0014] Bacteriophage-mediated mycobacterial DNA release assay means any assay or method described herein whereby a mycobacterium-specific bacteriophage (i.e., a bacteriophage that selectively lyses mycobacteria, or a specific mycobacterium species or strain) is used to lyse mycobacteria in a sample to confirm the presence of mycobacterial DNA in the sample, and then the presence of mycobacterial DNA in the sample is confirmed by any suitable method, for example, PCR or next-generation sequencing using mycobacterium-specific primers.

[0015] In another aspect of the present invention, 1. A method for treating early stage tuberculosis (TB) in a subject in need thereof, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; mixing a mycobacteria-specific bacteriophage with a sample of PBMCs to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; Identifying the presence of mycobacterial sequences in the DNA; administering to the subject one or more anti-early TB drugs or therapies.

[0016] In another aspect of the present invention, 1. A method of improving prognosis of tuberculosis (TB) in a subject, comprising: Obtaining a sample of isolated peripheral blood mononuclear cells (PBMCs) from a subject; To identify the presence of early TB in a subject using a bacteriophage-mediated mycobacterial DNA release assay and, if early TB is identified, administering one or more anti-TB drugs or therapies to the subject.

[0017] Suitable anti-TB drugs or therapies according to any aspect of the present invention are known to those of skill in the art and can be selected based on clinical or other indications. Suitable anti-TB drugs or therapies include rifampicin, ethambutol hydrochloride, pyrazinamide, and isoniazid (including pyridoxine hydrochloride), which may be administered to a subject alone (one anti-TB drug or therapy) or in any suitable combination of two or more anti-TB drugs or therapies.

[0018] In one embodiment according to any aspect of the invention, a sample of peripheral blood mononuclear cells (PBMCs) may be isolated from a subject by any suitable method, for example, density centrifugation (e.g., Ficoll-Paque), isolation in cell preparation tubes (CPT™), isolation in SepMate™ tubes, or isolation using a hemagglutinating agent, for example, HetaSep™.

[0019] In another aspect of the present invention, Provided is the use of a bacteriophage to measure the presence of one or more mycobacterial biomarkers in a blood sample isolated from a human subject in the manufacture of a kit for assessing whether the human subject is at risk of developing or has early stage tuberculosis (TB) or active tuberculosis disease.

[0020] In one embodiment according to any aspect of the invention, determining whether one or more mycobacterial DNA sequences are present in the DNA may include performing a polymerase chain reaction using, for example, mycobacterium-specific primers.

[0021] In one embodiment according to any aspect of the invention, the one or more mycobacterial DNA sequences may include, for example, an IS6110 element and / or an IS900 element.

[0022] In one embodiment according to any aspect of the invention, the method may include sequencing one or more mycobacterial DNA sequences and, optionally, determining homology between the mycobacterial DNA sequences and known mycobacterial DNA sequences, which may include those identified from public sequence databases or from TB-infected individuals to whom the subject is known or suspected to have been exposed.

[0023] In one embodiment according to any aspect of the present invention, the subject may be a human subject. In one embodiment according to any aspect of the present invention, the subject may be a mammal, such as, but not limited to, a primate, ape, or human. In one embodiment according to any aspect of the present invention, the subject may be selected from at least one of an asymptomatic subject and / or a TB-contacted subject. In one embodiment according to any aspect of the present invention, the subject may be selected from at least one of an asymptomatic human subject and / or a TB-contacted human subject. An asymptomatic subject refers to any subject who does not exhibit any symptoms of active tuberculosis disease typically used to confirm a diagnosis. Such symptoms may be determined by clinical or radiological evaluation. Additionally, the subject may test negative by sputum smear microscopy and / or mycobacterial culture. A TB-contacted subject refers to any subject who has had contact with another individual known or suspected to have a tuberculosis infection. A TB-contacted subject may be identified by TB contact tracing.

[0024] In one embodiment according to any aspect of the present invention, the subject may be immunocompromised and / or immunosuppressed. Both immunodeficiency and immunosuppression may be considered to be immunodeficiency states. The subject's immunodeficiency may be caused by a disease that directly or indirectly causes immunosuppression. Examples of such diseases include certain cancers (e.g., anemia, lymphoma, multiple myeloma, bone marrow cancer, blood cell cancer), chronic infections, such as acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) infection, and various hormonal and metabolic disorders, including anemia, hypothyroidism, and hyperglycemia. In one embodiment, the subject may be infected with HIV. Immunosuppression in a subject can also be caused by malnutrition, aging, administration of immunosuppressive agents, drugs or immunosuppressants (e.g., chemotherapy, disease-modifying antirheumatic drugs (DMARDs)), immunosuppressive drugs (anti-rejection drugs) administered in connection with organ transplantation, steroids such as corticosteroids (e.g., glucocorticoids), exposure to environmental toxins, or abuse of alcohol, illicit drugs, or nicotine. In one embodiment, the subject is taking or has taken one or more immunosuppressive medications, particularly one or more medications selected from the group consisting of chemotherapy, DMARDs, anti-rejection drugs, and glucocorticoids.

[0025] In one embodiment according to any aspect of the invention, the subject may be a TB-exposed subject, and suitably the method is performed on a sample of PBMCs obtained from the subject within 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month after the subject has been in contact with an individual infected with TB.

[0026] In one embodiment according to any aspect of the invention, the bacteriophage may be (myco)bacteriophage D29 or TM4, preferably D29. Other bacteriophages suitable for lysing mycobacteria will be known to those of skill in the art, see, e.g., Hatfull GF. (2018) Mycobacteriophages. Microbiol Spectr. 2018;6(5):10.1128 / microbiolspec.GPP3-0026-2018.doi:10.1128 / microbiolspec.GPP3-0026-2018.

[0027] In one embodiment according to any aspect of the invention, incubating the mixture under conditions that allow for lysis of the mycobacteria may include incubation at about 37°C for about 6 hours, 5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour, or less, preferably about 3.5 hours.

[0028] In one embodiment according to any aspect of the invention, any CMI response test may include an interferon gamma release assay (IGRA) test. An example of a suitable IGRA test is the QuantiFERON-TB Gold Plus (QFT, Qiagen Inc.) assay. Other mycobacteria-specific CMI response tests will be known to those skilled in the art.

[0029] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0030] All documents mentioned herein are incorporated herein by reference in their entirety.

[0031] As used herein, "and / or" should be interpreted as a specific disclosure of each of two particular features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0032] Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0033] While the present invention has been described by way of example with reference to certain embodiments, those skilled in the art will further appreciate that it is not limited to the disclosed embodiments, and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

[0034] Unless otherwise specified, a process comprising steps may be performed in any suitable order. Thus, steps may be performed in any suitable order.

[0035] Sequence homology can be measured using known methods. For example, the UWGCG Package provides the BESTFIT program, which can be used to calculate homology (for example, used with default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or align sequences (usually with default settings). For example, they are described in Altschul SF (1993) J Mol Evol 36:290-300, Altschul, S,F et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0036] The BLAST algorithm performs a statistical analysis of the similarity between two sequences. See, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the minimum total probability in the comparison between a first sequence and a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, a sequence is considered to be similar to another sequence. [Brief explanation of the drawings]

[0037] [Figure 1]This graph shows the baseline results of a mycobacterium-specific cellular immune (CMI) response test (IGRA test: QuantiFERON-TB Gold Plus (QFT) Qiagen Inc.) assay) for a number of asymptomatic subjects who were determined to belong to Group 2 over the course of the study. All subjects included in Figure 1 had positive QFT test results at one or more time points during the study, and all subjects were asymptomatic at baseline. The Y-axis indicates the arbitrary participant number assigned to each subject, and the X-axis indicates the ratio of antigen-1 to mitogen (positive control antigen) determined using the QFT kit. Subjects assigned participant numbers 455, 493, and 494 tested positive at baseline by a bacteriophage-mediated mycobacterial DNA release assay (Actiphage™). [Figure 2] This graph shows the results of the QuantiFERON-TB Gold Plus (QFT) Qiagen Inc. assay at 3 months (approximately 8-12 weeks) for a number of asymptomatic subjects identified as belonging to Group 2 during the course of the study. All subjects included in Figure 2 had positive QFT test results at least at 3 months, and all subjects were asymptomatic at baseline and 3 months. The Y-axis indicates the arbitrary participant number assigned to each subject, and the X-axis indicates the ratio of antigen-1 to mitogen (positive control antigen) determined using the QFT kit. Subjects assigned participant numbers 455, 493, and 494 tested positive by QFT and a bacteriophage-mediated mycobacterial DNA release assay (Actiphage™) at 3 months. [Figure 3]This is an image of a gel showing PCR products generated using mycobacterium-specific primers designed to amplify a 123-bp region of the IS6110 element. Lanes 1 and 19: NEB 100-bp DNA ladder; lanes 2–15: subject samples prepared using the bacteriophage-mediated mycobacterial DNA release assay (Actiphage™) method. Lanes 3 and 10 represent samples obtained from participant 455 using PBMCs prepared by either Ficoll or Hetasep, respectively. Lane 16—PCR positive control (1 μL of BCG DNA); lane 17—blank; lane 18—PCR negative control (no template, water control). DETAILED DESCRIPTION OF THE INVENTION

[0038] Tuberculosis (TB) is the leading cause of human death from a single infectious pathogen. The causative bacterium, M. tuberculosis (Mtb), persists in the population by establishing asymptomatic latent infection (LTBI), a reservoir for future disease. It is estimated that 5–10% of the LTBI population will develop TB in the future, usually within 2 years of acquiring the infection. The pathobiological mechanisms underpinning progression to active TB and its severity are poorly understood.

[0039] The World Health Organization has identified an urgent need for a highly predictive test for the development of near-term TB disease. The ideal test would significantly improve the predictive value of the development of active TB among infected individuals over currently available tests for LTBI in order to achieve WHO disease treatment and eradication goals (Consensus Conference Report: Development of a Target Product Profile (TPP) and a framework for evaluation of a test for predicting progression from tuberculosis infection to active disease. Geneva: World Health Organization; 2017 (WHO / HTM / TB / 2017.18)).

[0040] Previously available diagnostic tests are not sensitive or specific enough to accurately identify all cases of LTBI. Furthermore, tests do not adequately predict whether an individual with LTBI will progress to active TB in the future (i.e., whether the individual has early TB). This leads to a large number of individuals requiring treatment to prevent a single case of active TB, which itself presents a further barrier to the expansion of programmatic management of TB.

[0041] Previously employed diagnostic tests for LTBI, such as the interferon-gamma (IFN-γ) release assay (IGRA) or tuberculin skin test (TST, e.g., Mantoux test), measure T cell responses to Mycobacterium tuberculosis and therefore provide indirect evidence of exposure to M. tuberculosis. Tests that rely on the patient's immune response, by definition, may not yield results until a measurable T cell response is induced, and such a response takes time to develop (at least 6 weeks after infection). The resulting diagnostic delay allows bacteria long time to replicate before diagnosis and therefore treatment can be administered, reducing patient prognosis and increasing the likelihood of community transmission. Additionally, this immune memory response is diminished in older, immunocompromised, or immunosuppressed patients, thereby leading to inaccurate (false-negative or indeterminate) test results. For example, subjects receiving immunosuppressive medications may have false-negative TST results (Agarwal S. et al., 2014, Steroids Decrease Prevalence of Positive Tuberculin Skin Test in Rheumatoid Arthritis: Implications on Anti-TNF Therapies. Interdisciplinary Perspectives on Infectious Diseases, 2014(5759):430134) or false-negative or indeterminate IGRA results (Belliere and Blancher, 2017, QuantiFERON test interpretation in patients receiving immunosuppressive agents: an alert. European Respiratory Journal Apr 2017,49(4)1602102; DOI:10.1183 / 13993003.02102-2016). Tests based on the immune response to Mtb may also incorrectly identify individuals whose immune system has effectively cleared the infection or who have previously been vaccinated against Mtb (false positives) as being at risk of developing active TB.Studies have shown that the positive predictive values ​​of the tuberculin skin test and interferon-gamma release assay for predicting active TB within 2 years are 1.5% and 2.7%, respectively (Consensus Conference Report: Development of a Target Product Profile (TPP) and a framework for evaluation of a test for predicting progression from tuberculosis infection to active disease. Geneva: World Health Organization; 2017 (WHO / HTM / TB / 2017.18)).

[0042] Furthermore, current testing methods have operational requirements that make them impractical or expensive for use in remote areas or countries where TB poses the greatest challenge. In addition, some TB diagnostic tests require the subject to produce sputum, which is generally not possible in asymptomatic individuals.

[0043] Initial TB Early TB describes a recently recognized classification of tuberculosis characterized by an individual being asymptomatic but at increased risk of progressing to active TB in the absence of further intervention compared with individuals with LTBI. Individuals with early TB have not yet developed elicited clinical symptoms, radiographic abnormalities, or microbiological evidence consistent with active TB (Drain, P et al. 2018. Incipient and Subclinical Tuberculosis: a Clinical Review of Early Stages and Progression of Infection. Clin Microbio Rev. 31(5):e00021-18). It is now widely accepted that the asymptomatic phase of early disease may linger while the disease progresses before clinical manifestations with active disease. This condition identifies early TB. Individuals with early TB may not progress to active disease for 12 months or more.

[0044] Early TB has been difficult to diagnose because there has been no satisfactory positive test for this condition that distinguishes it from latent TB (LTBI: asymptomatic TB that is unlikely to develop into active TB due to effective clearance or containment by the immune system). Traditionally, diagnosis of early TB relies on a positive test typically associated with LTBI, such as an IGRA, seen in combination with an initial absence of clinical or radiological symptoms that is subsequently replaced by the presence of one or more clinical or radiological symptoms, or on microbiological test results as active TB develops. This means that early TB is generally only diagnosable in hindsight, after active TB symptoms have already appeared. The inability to distinguish early TB from LTBI before symptoms of active TB disease appear can result in unnecessary treatment of individuals unlikely to develop active TB (i.e., individuals with LTBI but not TB) and / or delayed treatment of individuals with early TB, which increases the likelihood of infection and reduces the prognosis for these patients.

[0045] LTBI testing Interferon-gamma release assay The interferon-gamma (IFN-γ) release assay (IGRA) is an example of a cell-mediated immune (CMI) response test, a test used to diagnose several infectious diseases, particularly tuberculosis. The interferon-γ (IFN-γ) release assay relies on the fact that T lymphocytes release IFN-γ when exposed to specific antigens.

[0046] One example of an IFN-γ assay is the QuantiFERON-TB Gold Plus (QFT, Qiagen Inc.) assay, which quantifies the amount of IFN-γ produced in response to the ESAT-6 and CFP-10 antigens from Mycobacterium tuberculosis. Results are reported as ELISA absorbance or calculated IFN-γ levels in IU / ml.

[0047] Another example of an IGRA is the T-SPOT.TB assay (Oxford Immunotec), an enzyme-linked immunosorbent spot (ELISPOT) assay performed on peripheral blood mononuclear cells incubated with ESAT-6 and CFP-10 peptides. Results are reported as the number of IFN-γ-producing T cells. If the number of spots in the TB antigen well exceeds a certain threshold compared with the negative control well, the individual is considered positive for Mycobacterium tuberculosis infection.

[0048] Mantoux Tuberculin Skin Test The Mantoux test (tuberculin skin test, PPD test) is a screening tool for TB infection that relies on an immune memory response to intradermal injection of tuberculin, a glycerol extract derived from a culture of Mycobacterium tuberculosis. T cells sensitized by a previous infection are recruited to the skin site, where they release lymphokines and induce an induration (a discrete, thin bump in the skin) 6–10 mm in diameter after 48–72 hours.

[0049] Currently employed tests for LTBI for M. tuberculosis infection (e.g., interferon-γ release assay or tuberculin skin test) are imperfect as initial TB tests (ITT) for three main reasons. 1. The tests measure M. tuberculosis infection indirectly; that is, they measure the T cell response to M. tuberculosis. This is a delayed response after Mtb infection (which can take approximately 6 weeks to develop in humans). This means that at the time of a positive test in a recently infected individual, the bacteria has had time to replicate, thereby reducing the individual's progression and increasing the likelihood of transmission to others. Furthermore, these indirect tests cannot distinguish between vaccinated, previously infected, and currently infected individuals. 2. The test is insensitive to certain subgroups of the population. Individuals with weakened immune responses, such as aging individuals, immunocompromised individuals (e.g., those infected with HIV), or individuals taking immunosuppressive medications, have a reduced ability to mount a T cell response, making them less likely to produce a positive test result even if infected with M. tuberculosis. 3. LTBI testing does not adequately predict whether an individual will progress to active TB in the future.

[0050] The WHO expressed its expectations for a clinically useful initial TB test (ITT) consensus conference report: Development of a Target Product Profile (TPP) and a framework for evaluation of a test for predicting progression from tuberculosis infection to active disease. Geneva: World Health Organization; 2017 (WHO / HTM / TB / 2017.18). These report state that such tests should be considered rule-in tests, with negative results providing limited information, while positive results indicate that active TB will likely develop. ITT is ideally used to screen people recently exposed to MTB, such as contacts of infectious TB patients (TB contacts). TB contact tracing is a process used to halt the spread of TB in communities. It involves locating and notifying contacts of infected individuals (TB contacts) so that they can receive counseling, testing, and treatment as needed. An ideal ITT would have characteristics that would enable the scale-up of contact tracing strategies and facilitate mass test-and-treat campaigns.

[0051] An ideal early TB test has the following characteristics: Negative in individuals who may have symptoms of other (respiratory) illnesses but have never been exposed to TB, including individuals with other diagnoses. Negative in individuals infected with Mtb but without early TB. They may have persistent TB infection and a positive LTBI test (TST or IGRA), but will not develop TB disease within the next two years. Individuals who have been treated for LTBI must be negative. Tests positive in individuals who develop TB within a short period of time (e.g., 2 years) after the test is completed and who do not show any signs of re-exposure after the test is performed. - Tests positive in individuals with symptomatic TB. -Negative in individuals who have completed TB treatment and are considered cured. [Table 1]

[0052] The inventors' development of a method for detecting viable mycobacteria in body fluids using phage combined with DNA amplification (bacteriophage-mediated mycobacterial DNA release assay) has been previously described in WO2015 / 049516 and demonstrates the detection of low-grade M. bovis bacteremia (10 per ml) in the blood of infected cattle. 2 Its usefulness in demonstrating subcellular levels has been demonstrated (Swift BM et al., Evidence of Mycobacterium tuberculosis complex bacteremia in intradermal skin test positive cattle detected using phage-RPA. Virulence 2016;7(7):779-88).

[0053] A bacteriophage-mediated mycobacterial DNA release assay (Actiphage™) has now been employed to detect human Mtb infection. The results of a proof-of-concept study applying the highly sensitive bacteriophage-mediated mycobacterial DNA release assay, Actiphage™, to a well-characterized clinical cohort are described herein. The early TB detection method described herein offers numerous advantages over currently employed tests for LTBI, including high specificity (probability of a negative test result in the absence of disease) and high sensitivity (probability of a positive test result in the presence of disease) for patients likely to develop active TB disease, improved false-positive reporting, fast time-to-result (less than 24 hours), and no need for cold-chain transport. These are all highly advantageous attributes that follow the characteristics defined by the WHO for an ideal early TB test.

[0054] The early diagnostic potential of the disclosed methods in detecting human subjects with early TB, i.e., asymptomatic individuals at risk of developing active TB (including individuals who may have previously been identified as having LTBI), is described. It has been demonstrated that early human TB is associated with, and may be identified by, the detection of viable mycobacteria (e.g., Mtb bacteremia) in the blood during early infection.

[0055] Even during active TB, evidence of TB-associated Mtb bacteremia is unclear. Previous studies applying culture and nucleic acid amplification tests (NAATs) to blood samples from active TB patients have been disappointing (Shenai S. et al. Exploring alternative biomaterials for the diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB / RIF assay. J Clin Microbiol 2013;51(12):4161-6). Improved Mtb detection has been reported following analysis of higher blood volumes for more severe active TB, suggesting that these methods are limited by insufficient sensitivity. Because Mtb blood titers during active TB disease are expected to be significantly higher than those during the early, asymptomatic phase of TB infection (i.e., immediately after contact with / transmission from an infected individual), it cannot be expected that testing for viable Mtb in the blood will be an effective determinant of prognosis for either early TB or subsequent active TB disease. While not wishing to be bound by any particular theory, the inventors hypothesize that the relative inaccessibility of intracellular Mtb DNA within circulating PBMCs is a significant contributing factor to the insufficient sensitivity of other Mtb detection methods. The approach described herein overcomes this by phage-mediated lysis of intracellular Mtb, thereby efficiently releasing bacterial DNA for sensitive NAAT in a second step. The approach described herein offers the surprising advantage of effectively identifying even subjects in the early stages of TB infection, in whom circulating mycobacterial titers in the blood may be very low. The advantages of a rapid, low-cost assay that provides a positive microbiological diagnosis without the need for sputum are clear. [Example]

[0056] Example 1 HIV-seronegative adult patients (age 18 years) were recruited into four groups according to the following criteria (see Table 1 for detailed data). 1. Active TB disease (pulmonary TB, PTB) based on a positive Xpert-Ultra (Xpert MTB / RIF Ultra, Cepheid Inc) or Mtb culture from an airway sample in patients with supporting clinical and radiological disease features (n=15) 2. Asymptomatic recent PTB contacts identified through contact tracing with a positive QuantiFERON-TB Gold Plus ((QFT) Qiagen Inc) test and normal chest x-ray (n=18). 3. Non-TB acute respiratory disease control group - patients initially referred to the TB service with symptoms suggestive of PTB, but subsequently diagnosed with non-TB and confirmed negative microbiological tests for Mtb (n=5). 4. Healthy control group - asymptomatic and QFT-negative participants with no history of previous TB exposure (n=28)

[0057] All participants provided blood samples for the Actiphage™ bacteriophage-mediated mycobacterial DNA release assay (ACTI) to test for viable mycobacterial bacteremia at recruitment and underwent 12-month prospective clinical follow-up. Additionally, in Group 2, QFT and Actiphage™ testing was repeated 8–12 weeks later to capture IGRA (QFT) seroconversion events (participants who were QFT negative at the first time point but positive at the second time point were presumed to be due to delayed T cell responses / delayed IFN-γ detectability). Participants in Group 2 remained in the study if they were QFT positive at the second time point. None of these participants received chemoprophylaxis. Patients with active PTB (Group 1) were sampled before starting antituberculosis treatment.

[0058] The clinical and experimental teams were blinded to the results of the Actiphage™ bacteriophage-mediated mycobacterial DNA release assay testing and the study groups from which the samples originated until the end of the study. Ethics approval was provided by the local Research and Ethics Committee (REC15 / EM / 0109), and all participants provided written informed consent at the time of enrollment.

[0059] QFT and Xpert-Ultra tests were performed according to the manufacturer's instructions, and Mtb cultures were performed according to standard methods. A negative Mtb culture result indicated that no bacterial growth occurred after the 8-week incubation period.

[0060] QFT ELISA data for participants determined as asymptomatic recent PTB contact (group 2) are shown in Figures 1 and 2 (baseline and 3 months, respectively).

[0061] For the bacteriophage-mediated mycobacterial DNA release assay (Actiphage™) test, blood (5 ml) was collected in sodium heparin tubes (Sarstedt) and stored at room temperature until processing. PBMCs were isolated from 2 ml blood aliquots according to the manufacturer's instructions by one of two alternative methods: i. Ficoll-Paque Plus (GE Healthcare) using Leucosep tubes (Sigma) and ii. Hetasep (Stem Cell Technologies).

[0062] PBMCs were resuspended in 200 μl of Actiphage™ medium and samples were then transferred to Actiphage™ Rapid Tubes (PBD Biotech Ltd) and bacteriophage D29 was added (20 μl, approximately 10 7pfu). Samples were incubated at 37°C for 3.5 hours and then centrifuged (13,000 x g, 3 minutes, room temperature). The flow-through from the Rapid tubes, containing the released mycobacterial DNA, was further concentrated (Zymo DNA Clean and Concentrator-5, Zymo Research / Cambridge Bioscience), and Mtb DNA was detected using a PCR assay specific for the IS6110 element with forward and reverse primers 5'-CCTGCGAGCGTAGGCGTCGG-3' and 5'-CTCGTCCAGCGCCGCTTCGG-3', which produced a 123-bp PCR product (Eisenach et al., Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis. 1991 Nov;144(5):1160-3).

[0063] Sixty-six participants were recruited into the study (Table 1). Of the 15 participants with active PTB (Group 1), one had evidence of miliary tuberculosis with a single cerebral tuberculoma. The remainder of Group 1 had no radiological or clinical features of multiorgan failure. Of the 18 participants in Group 2, one had QFT seroconversion by serial testing. The remainder were QFT-positive at both time points, and all had normal chest x-rays reported by a chest radiologist. All five participants in the non-TB acute respiratory disease control group (Group 3) had PTB ruled out by bronchoscopy and were effectively treated with antibiotics for community-acquired pneumonia.

[0064] Table 1 shows that 11 (73%) of the 15 active PTB cohort (Group 1) and 3 of the 18 asymptomatic TB-contact participants (Group 2) had positive Actiphage™ bacteriophage-mediated mycobacterial DNA release assay test results (these were participants assigned participant numbers 455, 493, and 494). Four of the remaining Group 1 participants, 15 of the Group 2 participants, and all participants in both control groups (Groups 3 and 4) had negative Actiphage™ bacteriophage-mediated mycobacterial DNA release assay test results.

[0065] Figure 3 shows a positive PCR band of the expected size (123 bp) for one of the participants in group 2 (assigned participant number 455) using primers specific for the mycobacterial IS<em>6110 element.

[0066] All study participants were also tested for C-reactive protein (CRP) levels to determine the presence of inflammation. Additionally, all participants in Group 1, Group 3, and Actiphage-positive participants in Group 2 were also tested by sputum smear microscopy, by Xpert-Ultra testing, and by measuring the number of days to positive mycobacterial culture from airway samples.

[0067] In the PTB cohort (Group 1, active TB disease), a positive result on the Actiphage™ bacteriophage-mediated mycobacterial DNA release assay was associated with a positive sputum smear, elevated baseline CRP, and a shorter time to mycobacterial culture. In the asymptomatic TB contact group (Group 2), participants had a normal baseline CRP (similar to healthy controls) and were incapable of forming mycobacterial cultures at baseline (i.e., no bacterial growth was detected after 8 weeks of incubation). Of the three Actiphage™-positive participants identified at baseline in Group 2, two developed active culture-positive PTB disease 7 months later. The lack of clinical, radiological, and microbiological evidence of TB at presentation, coupled with the development of active disease at later time points, is consistent with a diagnosis of early TB. This demonstrates the utility of the bacteriophage-mediated mycobacterial DNA release assay in diagnosing early TB and its ability to distinguish between subjects with early TB and those with LTBI. Whole-genome sequence analysis of Mtb isolates from two cases in each group who developed active TB confirmed the bacterial origin from each index case. A third Actiphage™-positive subject in Group 2 demonstrated QFT seroconversion (i.e., a negative QFT result on initial testing followed by a positive result 3 months later) but did not develop active TB within the study timescale (12 months). After 12 months of follow-up, none of the Actiphage™-negative participants in Group 2 developed active TB. Thus, the bacteriophage-mediated mycobacterial DNA release assay did not appear to generate false-negative active TB results among the Group 2 cohort, demonstrating that it is an effective test for early TB. In addition, asymptomatic cases who test negative using the bacteriophage-mediated mycobacterial DNA release assay (Actiphage™) and positive on the QFT test can be considered to have latent TB infection (i.e., low risk of developing active TB disease) with greater confidence than by the QFT test alone.

[0068] For clinical diagnosis in symptomatic patients suspected of having active PTB at baseline (i.e., groups 1 and 3), the bacteriophage-mediated mycobacterial DNA release assay (Actiphage™ Rapid test) showed a sensitivity and specificity (95% CI) of 73.3% (48.1–89.1) and 100% (56.6–100), respectively.

[0069] As a clinical diagnosis for early TB (high risk of developing active TB) in patients asymptomatic at baseline (i.e., groups 2 and 4), the bacteriophage-mediated mycobacterial DNA release assay (Actiphage™ Rapid test) showed a sensitivity and specificity (95% CI) of 100.0% (15.8–100.0) and 97.3% (88.0–99.9), respectively.

[0070] When applied to the entire cohort (all groups) at baseline, the sensitivity and specificity (95% CI) for detecting active PTB were 73.3% (48.1–89.1) and 94.2% (84.1–98.4), respectively. [Table 2]

Claims

1. 1. A method for aiding in the diagnosis of disease in an asymptomatic human subject, comprising: combining mycobacteria-specific bacteriophage D29 with a sample of peripheral blood mononuclear cells (PBMCs) isolated from said subject to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; and performing PCR on the DNA isolated from the mixture using forward and reverse primers specific for Mycobacterium tuberculosis DNA sequences; and / or performing appropriate sequencing to identify the DNA sequence of said Mycobacterium tuberculosis; and If the Mycobacterium tuberculosis DNA sequence exists, determining the homology between the Mycobacterium tuberculosis DNA sequence and known Mycobacterium tuberculosis DNA sequences. determining whether Mycobacterium tuberculosis (Mtb) DNA sequences are present or absent in the DNA isolated from the mixture by Including, the presence of the Mycobacterium tuberculosis DNA sequence indicates that the asymptomatic human subject has early tuberculosis (early TB); method.

2. 2. The method of claim 1, wherein the Mycobacterium tuberculosis DNA sequence comprises an IS6110 element.

3. 3. The method of claim 1 or 2, wherein the asymptomatic human subject is a TB-infected subject.

4. 4. The method of claim 3, wherein the method is performed on a sample of PBMCs obtained from an asymptomatic human subject within 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month after the asymptomatic human subject came into contact with an individual infected with TB.

5. 5. The method of any one of claims 1-4, wherein incubating the mixture under conditions that allow for lysis of the mycobacteria comprises incubation at about 37°C for about 6 hours, 5 hours, 4 hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour, or less.

6. the method further comprising performing a mycobacterium-specific cellular immune (CMI) response test on the sample isolated from the subject to generate a positive or negative CMI response test result; the absence of the Mycobacterium tuberculosis DNA sequence and a positive CMI response test result indicates that the asymptomatic human subject has latent tuberculosis infection (LTBI). The method of claim 1.

7. 7. The method of claim 6, wherein the CMI response test comprises an interferon gamma release assay (IGRA) test.

8. A method for predicting whether a human subject is at risk of having early-stage tuberculosis (early TB), comprising: mixing a mycobacteria-specific bacteriophage with a sample of peripheral blood mononuclear cells (PBMCs) isolated from the subject to form a mixture; incubating the mixture under conditions that allow lysis of the mycobacteria; isolating DNA from the mixture; and performing appropriate sequencing to identify the DNA sequence of Mycobacterium tuberculosis; and If the Mycobacterium tuberculosis DNA sequence exists, determining the homology between the Mycobacterium tuberculosis DNA sequence and known Mycobacterium tuberculosis DNA sequences. determining whether Mycobacterium tuberculosis DNA sequences are present in said DNA by Including, The presence of said Mycobacterium tuberculosis DNA sequence indicates that said subject is at risk for having early-stage TB.

9. 9. A kit for use in the method of any one of claims 1 to 8, comprising a mycobacteria-specific bacteriophage for determining the presence of one or more Mycobacterium tuberculosis DNA sequences in a sample of peripheral blood mononuclear cells (PBMCs) isolated from the human subject.

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