Immunoassays for differential detection of clostridium difficile

1. A method for measuring a level of multiple toxins of Clostridium difficile ( C. difficile ) in a stool sample suspected of containing a C. difficile strain and/or a toxin thereof, comprising:

(i) providing the stool sample suspected of containing a C. difficile strain and/or a toxin thereof,

(ii) measuring a level of toxin B of the C. difficile in the stool sample using an optical interrogation-based ELISA having a limit of detection of no greater than 20 pg/mL for toxin B in stool, and

(iii) measuring a level of toxin A of the C. difficile in the stool sample using an optical interrogation-based ELISA having a limit of detection of no greater than 10 pg/mL for toxin A in stool.

2. A method for treating Clostridium difficile ( C. difficile ) infection in a subject, comprising: selecting a subject having a diagnosis of C. difficile infection, the diagnosis a made by a method comprising:

(i) determining that the subject manifests a symptom associated with C. difficile infection; and (ii) determining a level of toxin A, toxin B, or both in a stool sample obtained from the subject, using an optical interrogation-based assay having a limit of detection of about: (a) no greater than 10 pg/mL for toxin A in stool, and (b) no greater than 20 pg/mL for toxin B in stool; and administering a suitable anti- C. difficile drug to the subject.

3. The method of claim 2 , wherein step (ii) is performed at a level of detection of about:

(a) 0.3 to 10 pg/mL for toxin A in stool, and

(b) 1 to 20 pg/mL for toxin B in stool.

4. The method of claim 3 , wherein the level of toxin B determined being higher than 20 pg/ml indicates that the subject has C. difficile infection.

5. The method of claim 3 , wherein the symptom is diarrhea.

6. The method of claim 3 , wherein step (ii) is performed by a differential detection method to determine whether the sample contains a highly virulent strain or a less virulent strain.

7. The method of claim 1 , wherein the stool sample is processed via dilution and filtration prior to step (ii) and step (iii).

8. The method of claim 3 , wherein the method on which the diagnosis is based further comprises detecting the presence of a nucleic acid that encodes toxin A, a nucleic acid that encodes toxin B, or both, in a sample obtained from the subject.

9. The method of claim 3 , wherein the stool sample is processed via dilution and filtration prior to step (ii).

10. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 10 pg/mL for toxin A in stool.

11. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 5 pg/mL for toxin A in stool.

12. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1 pg/mL for toxin A in stool.

13. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 0.45 pg/mL for toxin A in stool.

14. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 0.3 pg/mL for toxin A in stool.

15. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 20 pg/mL for toxin B in stool.

16. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 15 pg/mL for toxin B in stool.

17. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 5 pg/mL for toxin B in stool.

18. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1.5 pg/mL for toxin B in stool.

19. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1 pg/mL for toxin B in stool.

20. The method of claim 3 , wherein step (ii) is not performed using a cytotoxicity assay.

21. The method of claim 1 , wherein:

(a) step (ii) is performed at a limit of detection of about 1 to 20 pg/mL for toxin B in stool, and

(b) step (iii) is performed at a limit of detection of about 0.3 to 10 pg/mL for toxin A in stool.

22. The method of claim 21 , wherein step (ii) and step (iii) each comprises detecting a fluorescence signal.

23. The method of claim 3 , wherein step (ii) comprises detecting a fluorescence signal.

24. The method of claim 21 , wherein the measuring a level of toxin B of the C. difficile in the stool sample using an optical interrogation-based ELISA comprises:

(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin B molecules to the stool sample;

(b) associating at least some of the toxin B molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin B molecule and a statistically significant fraction of the capture objects do not associate with any toxin B molecules;

(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin B molecule in step (b); and

(d) determining a level of toxin B in the stool sample based at least in part on the measure indicative of a number determined in step (c).

25. The method of claim 21 , wherein the determining a level of toxin A of the C. difficile in the stool sample using an optical interrogation-based ELISA comprises:

(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin A molecules to the stool sample;

(b) associating at least some of the toxin A molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin A molecule and a statistically significant fraction of the capture objects do not associate with any toxin A molecules;

(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin A molecule in step (b); and

(d) determining a level of toxin A in the stool sample based at least in part on the measure indicative of a number determined in step (c).

26. The method of claim 3 , wherein step (ii) comprises

(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin A molecules and/or toxin B molecules to the stool sample;

(b) associating at least some of the toxin A molecules and/or toxin B molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin A molecule and/or toxin B molecule and a statistically significant fraction of the capture objects do not associate with any toxin A molecules or toxin B molecules;

(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin A molecule and/or a toxin B molecule in step (b); and

(d) determining a level of toxin A and/or a level of toxin B in the stool sample based at least in part on the measure indicative of a number determined in step (c).

27. The method of claim 3 , wherein the anti- C. difficile drug is an antibiotic drug.

28. The method of claim 27 , wherein the antibiotic drug comprises metronidazole, fidaxomicin, and/or vancomycin.

29. A method for treating Clostridium difficile ( C. difficile ) infection in a subject, comprising:

(i) determining that the subject manifests a symptom associated with C. difficile infection;

(ii) determining a level of toxin A, toxin B, or both in a stool sample obtained from the subject, using an optical interrogation-based assay having a limit of detection of about:

(a) no greater than 10 pg/mL for toxin A in stool, and

(b) no greater than 20 pg/mL for toxin B in stool;

(iii) determining based on the results of step (ii) that the subject has C. difficile infection; and

(iv) administering a suitable anti- C. difficile drug to the subject.

30. A method for treating Clostridium difficile ( C. difficile ) infection in a subject, comprising:

administering a suitable anti- C. difficile drug to a subject diagnosed with C. difficile infection, the diagnosis having been made based on a method comprising:

(i) determining that the subject manifests a symptom associated with C. difficile infection; and

(ii) determining a level of toxin A, toxin B, or both in a stool sample obtained from the subject, using an optical interrogation-based assay having a limit of detection of about:

(a) no greater than 10 pg/mL for toxin A in stool, and

(b) no greater than 20 pg/mL for toxin B in stool.