WO2010070581A1

WO2010070581A1 - Method and device for diagnosing tuberculosis

Info

Publication number: WO2010070581A1
Application number: PCT/IB2009/055751
Authority: WO
Inventors: Keertan Unkha Jairam Dheda
Original assignee: University Of Cape Town
Priority date: 2008-12-15
Filing date: 2009-12-15
Publication date: 2010-06-24
Also published as: BRPI0922798A2; BRPI0922798B1; CN102246040A; CN102246040B; AP2011005772A0; ZA201104416B

Abstract

A rapid method for diagnosing tuberculosis (TB) in a subject is described. Antibodies are used to detect the presence of specific biomarkers, such as cytokine or chemokines, and in particular IFN-gamma or IP-10, in an unstimulated sample from the subject. An indicator is used to indicate binding of the antibodies to the biomarkers, indicating that the patient has active extrapulmonary tuberculosis. The method can be performed using a test strip containing the antibodies and indicator and onto which the sample is placed, using an ELISA assay, or using flow cytometry, a bio-analyser, a waveguide biosensor or surface enhanced Raman spectroscopy. Results can be available within a few minutes to a few hours, depending on the type of test device used. The method can detect extrapulmonary tuberculosis and can distinguish between active and latent tuberculosis. A kit including antibodies and a chromatographic indicator and instructions for use is also described.

Description

METHOD AND DEVICE FOR DIAGNOSING TUBERCULOSIS

BACKGROUND OF THE INVENTION

The invention relates to a method for the diagnosis of active tuberculosis (TB).

Tuberculosis is the world's greatest infectious killer of women of reproductive age and the leading cause of death among people with HIV/AIDS. It is caused by mycobacteria, usually Mycobacterium tuberculosis in humans, and a third of the world's population is thought to be infected with M. tuberculosis. Most infections in humans result in an asymptomatic, latent infection (LTBI), but some latent infections eventually progresses to active disease, which, if left untreated, kills more than half of its victims.

When the disease becomes active, 75-85% of the cases are pulmonary TB. In the other 15-25% of active cases, the infection moves from the lungs, causing extrapulmonary tuberculosis (EPTB). Extrapulmonary infection sites include the pleura (in tuberculosis pleurisy), the central nervous system and sub-arachnoid space (in meningitis), the lymphatic system peripheral lymph nodes, the genitourinary system (in urogenital tuberculosis), pericardial space, abdominal cavity, and bones and joints (in Pott's disease). Active TB is particularly common in Africa and Asia, and inflicts substantial mortality, morbidity and economic regression in affected communities.

A common method for diagnosing tuberculosis infection is the Tuberculin Skin Test (TST), which is a composite measure of cell mediated immunity in response to tuberculosis antigen (PPD) stimulation. However, it takes 2 to 3 days before the results can be obtained and frequently delivers false positive or false negative results. This test also does not distinguish latent infection from active disease, which is important in the clinical setting. More recently, two assays which detect the release of interferon gamma (IFN-γ) in response to mycobacterial-specific proteins have been developed and commercialised: QuantiFERON-TB Gold In-tube (ELISA assay) and T-SPOT.TB, (ELISPOT assay). These interferon-gamma release assays (IGRAs) are based on the ability of the M. tuberculosis antigens for early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to stimulate host lymphocyte production of interferon-gamma. Lymphocytes from a patient's blood are cultured with the antigens and incubated for 16 to 24 hours. If the patient has been exposed to tuberculosis before, T lymphocytes produce interferon gamma in response. Both tests are blood tests though one uses whole blood whilst the other uses periphral blood mononuclear cells. The distinction between the tests is that QuantiFERON-TB Gold quantifies the total amount of interferon gamma in the supernatent when whole blood is exposed to the antigens, whereas T- SPOT.TB counts the number of interferon gamma-producing activated T lymphocytes by enumerating the number of spots within an ELISPOT well. The tests are time-consuming (they take about 2 days from sample collection to acquisition of a result) and labour intensive, requiring the use of a laboratory and complex instruments. Moreover, they do not distinguish active tuberculosis from latent tuberculosis. Clinical evaluation and additional tests, such as culture of blood or fluid from the area suspected to be infected, are required to distinguish active tuberculosis from latent TB infection.

Extrapulmonary TB is difficult to diagnose. Culture is only helpful in a minority of cases. As only small numbers of the bacteria are usually present in a sample, culturing this slow-growing organism in the laboratory takes a relatively long time (about 4 to 6 weeks), and is not always successful. Delays of this length are unacceptable, as by the time patients are diagnosed with active TB, they may have died or incurred severe disabilities, such as brain damage in the case of TB meningitis.

The development of a rapid and inexpensive diagnostic test for active tuberculosis would thus be very valuable, particularly in the developing world. SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a method of diagnosing tuberculosis in a subject, the method comprising the steps of: (i) providing a fluid sample from the subject;

(ii) contacting the fluid sample with an antibody of a biomarker for tuberculosis, without stimulating the fluid sample with an antigen; and

(iii) detecting whether or not the antibody binds to a biomarker from the sample.

The binding of the antibody to a biomarker may be indicative that the subject has tuberculosis, and more particularly, active tuberculosis.

The tuberculosis may be extrapulmonary tuberculosis.

The biomarker may be a cytokine, such as IFN-gamma, or a chemokine, such as IP-10.

The antibody may be an anti-IFN gamma antibody or an anti-IP-10 antibody, or a combination thereof, thereby to detect whether IFN-gamma and/or IP-10 are present in the sample.

A capture antibody that is capable of binding to the anti-biomarker antibody or to the biomarker may also be used in the method.

The antibody or capture antibody may be a monoclonal antibody.

The antibody may be complexed to a particle, such as colloidal gold.

An indicator may be used to indicate the binding of the antibody to the biomarker. The indicator may be a chromatographic, fluorescent, optical, enzyme-linked or radio-labeled indicator. The method may be a point-of-care immunoassay and, for example, the antibody and indicator may be located on a test strip assay onto which the sample can be placed. Alternatively, or in addition to the strip-test assay, the method may be performed in a laboratory using an ELISA assay, flow cytometry, a bio-analyser, a waveguide biosensor or SERS.

Typically, the method may be performed within about 5 hours, more preferably within about 3 hours and even more preferably within about 10 minutes.

According to a second embodiment of the invention, there is provided a test device for diagnosing tuberculosis in an unstimulated fluid sample from a subject, the test device comprising:

(i) an antibody capable of binding to a tuberculosis biomarker; and (ii) an indicator which indicates when the antibody binds to the biomarker.

The test device may be a strip-test device onto which the fluid sample from the subject is placed or an ELISA assay plate with wells into which the fluid sample from a single subject can be placed. Alternatively, the test device may be configured for use in a flow cytometer, a bio-analyser, a waveguide biosensor or a SERS spectrometer.

According to a third embodiment of the invention, ther is provided a kit for diagnosing tuberculosis in an unstimulated sample from a subject, the kit comprising:

(i) an antibody which is capable of binding to a tuberculosis marker; and (ii) an indicator which indicates when the antibody has bound to the biomarker.

The kit may further include instructions for performing the method described above. The kit may also include a capture antibody. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows scatter-plots (left panel) and area under the ROC (right panel) of IP-10 levels in 74 patients with pleural TB versus non-TB controls. At a cut-point of 4035 pg/ml (solid line in left hand panel) the sensitivity was 100% and NPV was 100%. The area under the

ROC curve was 0.82.

Figure 2 shows scatter-plots (left panel) and area under the ROC (right panel) of unstimulated IFN-gamma, using pleural fluid from 74 consecutively recruited patients with TB and non-TB effusions. At a cut-point of 0.31 IU/I the sensitivity, specificity, PPV and NPV

(95% Cl) of unstimulated IFN-gamma, was 97% (85-99); 100%

(81-100); 100% (90-100) and 94% (73- 99), respectively. Accuracy was 98% (90-100) and area under the ROC was 0.99.

Figure 3 Unstimulated IFN-γ levels in cerebro-spinal fluid obtained from 140 TB meningitis suspects. These data show the excellent performance outcomes of unstimulated interferon-gamma as a TB diagnostic marker in the cerebrospinal fluid.

Figure 4 shows the principle of lateral flow test technology using dyed microspheres, which migrate along the strip and if the biomarker of interest is present, bind to a detection line, which changes colour (antibody 1 = antibody bound to microspheres on the left hand side of panels A and B; antibody 2 = antibody in the second column of antibodies from the left in panels A, B and C and binding the antigen in panel D; antibody 3 is the last column of antibodies (extreme far right) of panels A to D).

Figure 5 shows an example of a strip test apparatus that could be used in the invention. DETAILED DESCRIPTION OF THE INVENTION

The invention provides a rapid method for diagnosing tuberculosis (TB) in an unstimulated fluid sample from a subject. Unlike other commercially available methods, the present method can distinguish between active and latent tuberculosis, which is important for determining treatment of the subject. The method can also detect extrapulmonary tuberculosis. Very importantly, the method also provide results within a matter of minutes, unlike other tests, which need to incubate the sample for more than 12 hours.

Active tuberculosis is the disease state where the biological fluid or tissue is smear microscopy or culture positive for M. tuberculosis, or M. tuberculosis is detectable by a nucleic acid amplification test, in a subject with clinical and radiological features of active disease; constitutional symptoms are often present. Latent tuberculosis infection is where potentially viable M. tuberculosis is present in human tissues but the individual is asymptomatic, and without clinico- radiological features of active disease.

As mentioned earlier, the available tests for detecting tuberculosis have poor sensitivity and are time consuming and labour intensive. Rapid, accurate diagnosis of disease is important in the clinical setting. It is also beneficial to have a diagnostic method that can be used easily and at the point-of-care, such as in a consulting room, hospital ward or home.

The present invention utilises antibodies to detect the presence of specific biomarkers, such as cytokine or chemokines, and in particular interferon-gamma or IP-10, in an unstimulated sample from a patient (i.e. the sample is not stimulated (or processed) with antigens). Generally, a chromatographic indicator will change colour on binding of the antibodies to the biomarkers, indicating that the patient has active extrapulmonary tuberculosis. However, the biomarker of interest can also be detected by other optical, molecular biological, microfluidic or nano technologies, or the like (e.g. SERS (surface enhanced Raman spectroscopy) or waveguide biosensors (such as a slot-waveguide biosensor). The biomarker is detected in the biological fluid itself and overnight antigen stimulation, as required with other methods, is not needed.

The sample can be fluid from the pleural space, the pericardial space, abdominal cavity or sub-arachnoid space, or plasma or serum from a blood sample, saliva or urine. More particularly, the sample can be fluid from pleural effusion. Unlike the commercially available tests, the sample is preferably not a blood sample.

The sample can be applied to a strip similar to the type commonly used in pregnancy tests and other urine analysis tests, the strip including biomarker antibodies and a chromatographic indicator for detecting binding of interferon- gamma and or IP-10 to the antibodies.

The strip can include antibodies for IFN-gamma or IP-10, or both interferon- gamma and IP-10. Including both of these biomarkers will make the test less likely to return a false positive or false negative result, as interferon-gamma is a good rule-in marker and IP-10 is a good rule-out marker.

Results from this strip-test can be available within a few minutes (such as 2-3 minutes) of applying the sample to the strip.

Alternatively or in addition, an immunoassay (e.g. ELISA), flow cytometry or a bio-analyser may be used. A kit including at least antibodies, a chromatographic indicator and instructions for use could also be provided.

While this test strip will be sufficient for some type of extrapulmonary TB, e.g. pleural TB, in some difficult-to-diagnose types of tuberculosis, such as TB meningitis, the strip test described above can be combined with an immunoassay, such as an ELISA assay, to ensure that an accurate diagnosis is made.

Commercially available IFN-gamma ELISA assay plates are available with the appropriate antibodies and a suitable indicating means. The plates have 96 wells and are capable of testing 24 samples at a time. As each plate costs between ~ US$ 300 and 500, using a plate to test for only one sample at a time is very expensive in a developing world setting, and thus not particularly suited to the present invention.

For the present invention, IFN-gamma ELISA assay plates for conducting an assay of a single sample will be provided. For example, the assay plate could have only 8 wells (4 standards, 1 positive control, 1 negative control, 2 sample wells). Results of this assay could be available within two to three hours.

Several novel tools and biomarkers for diagnosing TB pleural effusions were evaluated by the applicant, including T cell assays, LAM antigen detection, PCR, IP-10, IFN-gamma, ADA, smear microscopy, culture and pleural biopsy. Preliminary proof of principle case-control studies have suggested that T cell assays may be useful. However, when evaluted in a clinical setting they performed poorly. IP-10 was promising but still missed 1 in 5 TB subjects and over-diagnosed 1 in 5; by contrast at a higher cut-point IP-10 had a 100% negative predictive value (indicating that it is an excellent rule out test for TB pleural effusion (Figure 1)). However, the most accurate discriminator was unstimulated IFN-gamma, which had a sensitivity and specificty approaching 100% (Figure 2). IFN-gamma is thus a very accurate marker for TB pleural effusion.

The invention is further described by the following example. This example, however, is not to be construed as limiting in any way either the spirit or scope of the invention.

Example

Figure 3 shows the unstimulated IFN-γ levels in cerebro-spinal fluid obtained from 140 TB meningitis suspects. The data indicates that when used in conjunction with other readily and rapidly available test results (e.g. Gram stain and cryptococcal latex agglutination tests), unstimulated IFN-γ has a sensitivity and specificity approaching 95%.

Figure 4 provides a summary outline of how a suitable test would work. A strip test (Figure 5) is provided having a nitrocellulase membrane carrier with a colloidal gold conjugate solution (a chromatographic indicator) complexed to anti- IFN-gamma antibodies, and possibly also anti-IP-10 antibodies, and a band of capture antibodies, enclosed in a plastic casing.

A small amount of a sample from a patient, for example fluid aspirated from a specific body compartment, such as the pleural space, is placed onto the test strip.

If IFN-gamma cytokines (and possibly also IP-10 biomarkers/chemokines) are present in the unstimulated sample from the patient, they will bind to anti-IFN- gamma and anti-IP-10 antibodies, respectively, on the nitrocellulase membrane, and these complexes will migrate towards the band of capture antibodies. This would work similarly for other biomarkers. Upon binding, the chromatographic indicator will change colour, signifying a positive test for tuberculosis.

Thus, the applicant has developed a method for diagnosing tuberculosis which is able to provide a result considerably faster than current diagnosis methods. The method is also cost-effective and able to distinguish between latent and active tuberculosis. Moreover, it is a point-of-care test, and it does not necessarily have to be performed by a person with technical skill or in a laboratory.

Claims

CLAIMS:

1. A method of diagnosing tuberculosis in a subject, the method comprising the steps of:

(i) contacting a fluid sample from the subject with an antibody of a biomarker for tuberculosis, without stimulating the fluid sample with an antigen; and

(ii) detecting whether or not the antibody binds to a biomarker from the sample.

2. A method according to claim 1 , wherein the binding of the antibody to a biomarker is indicative that the subject has tuberculosis.

3. A method according to claim 1 or 2, wherein the tuberculosis is extrapulmonary tuberculosis.

4. A method according to any one of claims 1 to 3, wherein the tuberculosis is active tuberculosis.

5. A method according to any one of claims 1 to 4, wherein the biomarker is a cytokine.

6. A method according to claim 5, wherein the cytokine is IFN-gamma.

7. A method according to claim 6, wherein the antibody is anti-IFN-gamma antibody.

8. A method according to any one of claims 1 to 4, wherein the biomarker is a chemokine.

9. A method according to claim 8, wherein the chemokine is IP-10.

10. A method according to claim 9, wherein the antibody is an anti-IP-10 antibody.

11. A method according to any one of claims 1 to 10, wherein the method detects whether IFN-gamma and IP-10 are present in the sample.

12. A method according to claim 11 , wherein anti-IFN-gamma and anti-IP-10 antibodies are used.

13. A method according to any one of claims 1 to 12, which further comprises the use of a capture antibody that is capable of binding to the biomarker or to the anti-biomarker antibody.

14. A method according to any one of claims 1 to 13, wherein the antibody or capture antibody is a monoclonal antibody.

15. A method according to any one of claims 1 to 14, wherein an indicator is used to indicate the binding of the antibody to the biomarker.

16. A method according to claim 15, wherein the indicator is a chromatographic, optical, fluorescent or radio-labeled indicator.

17. A method according to claims 15 or 16, wherein the indicator is a colloidal gold indicator, and the antibody is bound to a gold particle.

18. A method according to any one of claims 1 to 17, wherein the method is a point-of-care method.

19. A method according to any one of claims 1 to 18, wherein binding of the antibody to the biomarker is detected within 5 hours.

20. A method according to any one of claims 1 to 19, wherein binding of the antibody to the biomarker is detected within 3 hours.

21. A method according to any one of claims 1 to 20, wherein binding of the antibody to the biomarker is detected within 10 minutes.

22. A method according to any one of claims 15 to 21 , wherein the antibody and indicator are located on a test strip assay onto which the sample is placed.

23. A method according to any one of claims 15 to 21 , wherein the antibody and indicator are located in an ELISA assay plate.

24. A method according to any one of claims 1 to 21 , wherein the method is performed using a flow cytometer, bio-analyser, waveguide biosensor or surface enhanced Raman spectroscopy.

25. A test device for diagnosing tuberculosis in an unstimulated fluid sample from a subject, the test device comprising:

26. A test device according to claim 25, which is able to detect active tuberculosis in a subject.

27. A test device according to claim 25 or 26, which is able to detect extrapulmonary tuberculosis is a subject.

28. A test device according to any one of claims 25 to 27, wherein the antibody is an anti-IFN-gamma antibody.

29. A test device according to any one of claims 25 to 27, wherein the antibody is an anti-IP-10 antibody.

30. A test device according to any one of claims 25 to 29, which includes an anti-IFN-gamma antibody and an anti-IP-10 antibody.

31. A test device according to any one of claims 25 to 30, which further comprises a capture antibody.

32. A test device according to claim 31 , wherein the capture antibody is capable of binding to the biomarker or to the biomarker antibody.

33. A test device according to any one of claims 25 to 32, wherein the biomarker antibody and/or capture antibody is a monoclonal antibody.

34. A test device according to any one of claims 25 to 33, wherein the indicator is a chromatographic, optical, fluorescent or radio-labeled indicator.

35. A test device according to any one of claims 25 to 34, wherein the indicator is a colloidal gold indicator and the antibody is bound to a gold particle.

36. A test device according to any one of claims 25 to 35, which is a point-of- care device.

37. A test device according to any one of claims 25 to 36, which is a strip-test device onto which the fluid sample from the subject is placed.

38. A test device according to any one of claims 25 to 36, which is an ELISA assay plate with wells for testing a fluid sample from a single subject.

39. A test device according to any one of claims 25 to 35, which is configured for use in a flow cytometer, bio-analyser, waveguide biosensor or surface enhanced Raman spectroscopy.

40. A kit for diagnosing tuberculosis in an unstimulated sample from a subject, the kit comprising:

(i) an antibody which is capable of binding to a tuberculosis marker; and (H) an indicator which indicates when the antibody has bound to the biomarker.

41. A kit according to claim 40, which further includes instructions for performing the method of any of claims 1 to 24.

42. A kit according to claim 40 or 41 , which is for diagnosing extrapulmonary tuberculosis.

43. A kit according to any one of claims 40 to 42, which is for diagnosing active tuberculosis.

44. A kit according to any one of claims 40 to 43, wherein the antibody is an anti-IFN-gamma antibody.

45. A kit according to any one of claims 40 to 43, wherein the antibody is an anti-IP-10 antibody.

46. A kit according to any one of claims 40 to 45, which includes both anti- IFN-gamma and IP-10 antibodies.

47. A kit according to any one of claims 40 to 46, which further includes a capture antibody.