METHODS FOR MONITORING TUBERCULOSIS MEDICATION

Abstract

The present invention relates to methods of determining whether a subject has taken a dose of Tuberculosis medication through analysis of a sample of sweat obtained from the subject, in the form of a skin-print.

Claims

1. A method of determining whether a subject has taken a dose of Tuberculosis medication, the method comprising: analysing using mass spectrometry a sample of sweat obtained from the subject to determine the presence therein of Tuberculosis medication and/or a metabolite thereof; wherein the sample of sweat is obtained in the form of a skin-print.

2. The method of claim 1, wherein the skin-print is a fingerprint.

3. The method of any of the preceding claims, wherein the Tuberculosis medication comprises isoniazid, pyrazinamide, rifampicin and/or ethambutol.

4. The method of any of the preceding claims, wherein the Tuberculosis medication comprises isoniazid and/or pyrazinamide.

5. The method of any of the preceding claims, wherein the Tuberculosis medication comprises isoniazid.

6. The method of any of the preceding claims, wherein the dose of Tuberculosis medication comprises at least 300 mg isoniazid.

7. The method of any of the preceding claims, wherein determining the presence of Tuberculosis medication in the sample of sweat comprises checking for an M+H mass to charge ratio (m/z) peak between 138.06 and 138.08 using mass spectrometry, preferably between 138.06 and 138.07.

8. The method of any of the preceding claims, wherein the metabolite comprises acetylisoniazid.

9. The method of any of the preceding claims, wherein determining the presence of a metabolite of Tuberculosis medication in the sample of sweat comprises checking for an M+H mass to charge ratio (m/z) peak between 180.06 and 180.09 using mass spectrometry, preferably between 180.07 and 180.08.

10. The method of any of the preceding claims, wherein the Tuberculosis medication comprises pyrazinamide.

11. The method of any of the preceding claims, wherein determining the presence of Tuberculosis medication in the sample of sweat comprises checking for an M+H mass to charge ratio (m/z) peak between 124.04 and 124.06 using mass spectrometry, preferably between 124.05 and 124.06.

12. The method of any of the preceding claims, wherein the sample of sweat has a mass of 0.1 to 2 g.

13. The method of any of the preceding claims, wherein the sample of sweat has a volume of 5 to 50 nl.

14. The method of any of the preceding claims, wherein the sample of sweat comprises 0.1 to 2 ng of the Tuberculosis medication and/or the metabolite thereof.

15. The method of any of the preceding claims, wherein the mass spectrometry is liquid chromatography mass spectrometry.

16. The method of any of the preceding claims, wherein the mass spectrometry is paper spray mass spectrometry.

17. The method of any of the preceding claims, further comprising confirming the identity of the subject by checking the skin-print obtained from the subject.

18. The method of any of the preceding claims, further comprising analysing the sample of sweat to determine the presence therein of one or more markers which indicate that the level of Tuberculosis infection is declining or increasing.

19. The method of claim 18, wherein the one or more markers comprises one or more toxins.

20. A method of determining whether a subject has taken a dose of Tuberculosis medication, the method comprising: analysing using a lateral flow immunoassay a sample of sweat obtained from the subject to determine the presence therein of Tuberculosis medication and/or a metabolite thereof; wherein the sample of sweat is obtained in the form of a skin-print.

21. The method of claim 20, wherein the skin-print is a fingerprint.

22. The method of claim 20 or claim 21, further comprising confirming the identity of the subject by checking the skin-print obtained from the subject.

23. The method of any of claims 20 to 22, further comprising analysing the sample of sweat to determine the presence therein of one or more markers which indicate that the level of Tuberculosis infection is declining or increasing.

24. The method of claim 23, wherein the one or more markers comprises one or more toxins.

Description

[0081] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

[0082] FIG. 1: is a mass spectrum resulting from preparation of a blank piece of chromatography paper, focussed on the range where Isoniazid (if present) is observed.

[0083] FIG. 2: is a mass spectrum resulting from preparation of a piece of chromatography paper having a sample of sweat thereon, in the form of a fingerprint. The sample was obtained from a subject (TB2017-FP36) who had taken 300 mg of Isoniazid approximately 3 hours beforehand. The spectrum is focussed on the range where Isoniazid (if present) is observed.

[0084] FIG. 3: is a mass spectrum resulting from preparation of a blank piece of chromatography paper, focussed on the range where Acetylisoniazid (if present) is observed.

[0085] FIG. 4: is a mass spectrum resulting from preparation of a piece of chromatography paper having a sample of sweat thereon, in the form of a fingerprint. The sample was obtained from a subject (TB2017-FP36) who had taken 300 mg of Isoniazid approximately 3 hours beforehand. The spectrum is focussed on the range where Acetylisoniazid (if present) is observed.

[0086] FIG. 5: is a diagram showing relative amounts of Isoniazid and Acetylisoniazid present in fingerprint samples obtained from 16 subjects. The fingerprint samples were obtained without the subjects washing their hands.

[0087] FIG. 6: is a diagram showing relative amounts of Isoniazid and Acetylisoniazid present in fingerprint samples obtained from 16 subjects. The fingerprint samples were obtained after the subjects had washed their hands using soap and water.

[0088] FIG. 7: shows two mass spectra for two samples of fingerprint sweat, focussed on the range where Isoniazid (if present) is observed. The first sample was obtained from a subject who had taken Isoniazid 2 hours previously; the second sample was obtained from a subject who had taken Isoniazid 72 hours previously.

[0089] FIG. 8: shows two mass spectra for two samples of fingerprint sweat, focussed on the range where Acetylisoniazid (if present) is observed. The first sample was obtained from a subject who had taken Isoniazid 2 hours previously; the second sample was obtained from a subject who had taken Isoniazid 72 hours previously.

[0090] FIG. 9: shows two mass spectra for two samples of fingerprint sweat. The first sample was obtained from a subject who had taken pyrazinamide; the second sample was obtained from a subject who had not taken pyrazinamide.

[0091] The following non-limiting examples further illustrate the present invention.

EXAMPLES

[0092] Experimental Methods

[0093] Fingerprint Collection Method

[0094] All subjects from whom fingerprint samples were taken were human. Fingerprints were taken from the five fingers (i.e. the four fingers and the thumb) of each subject's right hand: [0095] without the hand having been washed; or [0096] after the hand has been washed with soap and water, and covered with a clean surgical glove for 10 minutes, without contacting any other surface.

[0097] Liquid Chromatography Mass Spectrometry Analysis of Samples

[0098] The chromatography paper with fingerprint samples thereon were placed in a 2 ml Eppendorf microcentrifuge tube, following which an extraction solution (1.5 ml of 10% dichloromethane in methanol) was added. The tube was then centrifuged for 2 min (at 9.5 centrifugal force). The solvent extract was evaporated to dryness under a stream of nitrogen at room temperature (20 C.) and reconstituted in 100 l mobile phase solution (95:5 water/acetonitrile+0.1% formic acid+iosotopically labelled internal standards) before being vortexed and transferred to a 300 l glass micro-insert vial, with 5 l being injected onto an LC-MS/MS system.

[0099] Chromatographic separation was performed on a Thermo Scientific Ultimate3000 UHPLC system equipped with a binary solvent manager, column manager and autosampler. The injection volume was 5 L. Separation was performed on a Kinetex XB-C.sub.18 column (1002.1 mm, 5 m) operated at 30 C. at a flow rate of 0.25 ml/min. Mobile phase comprises 95% H.sub.2O (0.1% formic acid) and 5% acetonitrile (ACN) (0.1% formic acid).

[0100] The prepared samples were introduced to a Thermo Orbitrap Q-Exactive Plus mass spectrometer using the standard ESI interface with a capillary temperature of 320 C. and spray voltage 3 kV. Positive mass spectra were acquired in full scan mode within a range of m/z 50-500 at a mass resolution of 70 000 at m/z 200.

Example 1

[0101] Fingerprint samples were obtained from 15 subjects who had taken 300 mg isoniazid approximately 3 hours beforehand and one subject (TB2017-FP019) who had taken 400 mg isoniazid, without their hands having been washed. Of the 15 subjects who had taken 300 mg isoniazid, one (TB2017-FP010) was resistant to isoniazid. TB2017-FP010's results were therefore discounted from the analysis.

[0102] This provided 75 fingerprint samples (one for each finger/thumb of the subjects' right hands). Each of the 75 fingerprint samples was analysed using liquid chromatography mass spectrometry, checking for the presence of Isoniazid and Acetylisoniazid (a metabolite of Isoniazid).

[0103] The presence of Isoniazid is evidenced by a peak at an m/z ratio of 138.0661, as shown in FIG. 2, which is the mass spectrum resulting from the analysis of a fingerprint sample from subject TB2017-FP36. The presence of Acetylisoniazid is evidenced by a peak at an m/z ratio of 180.0766, as shown in FIG. 4, which is the mass spectrum resulting from the analysis of a fingerprint sample from subject TB2017-FP36.

[0104] The results from analysis of the 75 samples are displayed in FIG. 5. The analysis found that Isoniazid was detected in 51 out of the 75 fingerprint samples (68%) and Acetylisoniazid was detected in 73 out of the 75 fingerprint samples (97%).

Example 2

[0105] Fingerprint samples were obtained from the same 16 subjects as Example 1, after they had washed their hands using soap and water. Again, TB2017-FP010's results were discounted from the analysis. This provided 75 fingerprint samples (one for each finger/thumb of the subjects' right hands). Each of the 75 fingerprint samples was analysed using liquid chromatography mass spectrometry, checking for the presence of Isoniazid and Acetylisoniazid. The results are displayed in FIG. 6. The analysis found that Isoniazid was detected in 40 out of the 75 fingerprint samples (53%) and Acetylisoniazid was detected in 72 out of the 75 fingerprint samples (96%).

Example 3

[0106] Fingerprint samples were obtained from 5 subjects who had finished a course of Isoniazid at least 72 hours previously.

[0107] 25 fingerprints were obtained from the subjects, without the subjects having washed their hands. These samples were analysed using mass spectrometry. Isoniazid was detected in 0 fingerprint samples. Acetylisoniazid was detected in 0 fingerprint samples.

[0108] The 5 subjects then washed their hands using soap and water. 25 fingerprints were then obtained from the subjects. These samples were analysed using mass spectrometry. Isoniazid was detected in 0 fingerprint samples. Acetylisoniazid was detected in 0 fingerprint samples.

Example 4

[0109] Fingerprint samples were obtained from 3 subjects who had never taken Isoniazid. 15 fingerprints were obtained from the subjects, without the subjects having washed their hands. These samples were analysed using mass spectrometry. Isoniazid was detected in 0 fingerprint samples. Acetylisoniazid was detected in 0 fingerprint samples.

[0110] The 3 subjects then washed their hands using soap and water. 15 fingerprints were then obtained from the subjects. These samples were analysed using mass spectrometry. Isoniazid was detected in 0 fingerprint samples. Acetylisoniazid was detected in 0 fingerprint samples.

Example 5

[0111] Two samples of fingerprint sweat were obtained from the same subject. The first sample was obtained 2 hours after the subject had taken Isoniazid. The second sample was obtained from the subject 72 hours after the subject had taken Isoniazid. The two samples were analysed using liquid chromatography mass spectrometry and the results analysed. The results are shown in FIG. 7. The presence of Isoniazid is evidenced by a peak at an m/z ratio of 138.0660. Analysis of the first sample, obtained 2 hours after the subject had taken Isoniazid, shows the presence of Isoniazid. In contrast, analysis of the second sample, obtained 72 hours after the subject had taken Isoniazid, does not show the presence of Isoniazid.

Example 6

[0112] Two samples of fingerprint sweat were obtained from the same subject. The first sample was obtained 2 hours after the subject had taken Isoniazid. The second sample was obtained from the subject 72 hours after the subject had taken Isoniazid. The two samples were analysed using liquid chromatography mass spectrometry and the results analysed.

[0113] The results are shown in FIG. 8. The presence of Acetylisoniazid is evidenced by a peak at an m/z ratio of 180.0766. Analysis of the first sample, obtained 2 hours after the subject had taken Isoniazid, shows the presence of Acetylisoniazid. In contrast, analysis of the second sample, obtained 72 hours after the subject had taken Isoniazid, does not show the presence of Acetylisoniazid.

Example 7

[0114] A fingerprint sample was obtained from a subject who had taken 1,000 mg pyrazinamide approximately 3 hours beforehand. The sample was taken after the subject had washed their hands. The fingerprint sample was analysed using liquid chromatography mass spectrometry, checking for the presence of pyrazinamide. The mass spectrometry analysis of the fingerprint sample is shown in FIG. 9. The presence of pyrazinamide is evidenced by a peak at an m/z ratio of 124.0505. FIG. 9 also shows a second mass spectrum which shows the mass spectrometry analysis of a sample obtained from a subject who had not taken pyrazinamide.