SYSTEMS AND METHODS TO ASSES MICROBIOMES AND TREATMENTS THEREOF

• US 20160040216A1
• Filed: 04/04/2014
• Published: 02/11/2016
• Est. Priority Date: 04/05/2013
• Status: Active Application

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Akins, Robert A., Sobel, Jack D.
• Granted Patent: US 10,253,377 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16H 10/40 for data related to laboratory analysis, e.g. patient specimen analysis
• CPC Class Codes: G16H 20/10 relating to drugs or medications, e.g. for ensuring correct administration to patients
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Systems and methods to assess the health of various microbiomes and to identity species therein are disclosed. Described assessments and identifications can inform treatment decisions if a microbiome is determined to have a less than optimal balance of bacterial species within it; the presence of one or more negative species; and/or the absence of one or more positive species.

Claims

1. A method of diagnosing, monitoring the treatment of and/or predicting success or failure of therapy of bacterial vaginosis (BV) in a subject comprising:

amplifying total bacterial DNA present in a vaginal sample from the subject; and

selectively amplifying non-Lactobacillus (LB) species DNA in the sample by amplifying total bacterial DNA while specifically blocking amplification of bacterial DNA from LB species,

wherein the relative abundance of non-LB species is indicative of the presence of BV.

2. A method of claim 1, wherein the vaginal sample is a vaginal lavage.

3. A method of claim 1, wherein the amplification of bacterial DNA is performed by polymerase chain reaction (PCR).

4. A method of claim 3, wherein the PCR is quantitative PCR (qPCR).

5. A method of claim 3, wherein the total bacterial DNA is amplified with at least one forward primer and at least one reverse primer selected from BU4F+, BU6R+, 8F+, 1501R1, 1501R2, 1492R, 27F, 338R, 341F, 806R, 338F, and 534R.

6. A method of claim 1, wherein amplification of LB species DNA is blocked by at least one forward blocking primer and at least one reverse blocking primer which partially overlap binding sites for sequences on the LB genome and wherein the blocking primer melting temperatures are at least 68°

C.

7. A method of claim 6, wherein the at least one forward blocking primer and at least one reverse blocking primer are selected from LBblocker3, LBB3p, LBblocker4, and LBB4p.

8. A method of claim 1, wherein the relative abundance of non-LB species is determined by calculating a ratio of non-LB species to LB species in the sample.

9. A method of claim 1, wherein the method is used for the diagnosis of BV and wherein if the ratio of non-LB species to LB species is more than 1:

99, the subject has BV.

10. A method of claim 1, wherein the method is used for monitoring the treatment of BV, and wherein if the ratio of non-LB species to LB species is more than 1:

99, the treatment is ineffective and a new treatment is initiated in the subject.

11. A method of claim 1, wherein the method is used for monitoring the treatment of BV, and wherein if the ratio of non-LB species to LB species is 1:

99 or less, the treatment is effective and the prescribed course of treatment is completed in the subject.

12. A method of claim 1, wherein the method further comprises the step of determining the identity of non-LB species in the sample.

13. A method of claim 12, further comprising determining an appropriate therapy based on the identity of the non-LB species.

14. A method of claim 12, further comprising predicting the success or failure of a specific therapy of BV.

15. A method of claim 1, further comprising determining the identity of non-LB species in the sample.

16. A method of claim 15, wherein the determining comprises performing a melting curve analysis on resulting amplicons to determine the identity of non-LB species.

17. A method of claim 15, wherein the determining comprises performing a melting curve analysis on resulting amplicons to categorize classes of vaginal bacteria.

45. A method of claim 1 or 32 further comprising calculating a Δ

Cq score and if the Δ

Cq is below 3, diagnosing the subject with BV;

predicting the recurrence of BV in the subject; and

/or directing continued treatment of the subject for BV.

46. A method of claim 1 or 32 further comprising calculating a Δ

Cq score and if the Δ

Cq is 3 or above, determining that the subject is free from BV;

predicting the non-recurrence of BV; and

/or directing the cessation of treatment of the subject for BV.

47. A method of claim 1 or 32 further comprising calculating a Δ

Cq score and performing a non-LB melt curve analysis to generate a combined score.

48. A method of claim 47 wherein a Δ

Cq score of 3 or above in combination with an absence of melt curves within a defined temperature threshold direct (i) a determination that the subject is free from BV;

(ii) a prediction of non-recurrence of BV; and

/or (iii) direction of the cessation of treatment of the subject for BV.

49. A method of claim 47 wherein a Δ

Cq score of below 3 or the presence of melt curves within a defined temperature threshold direct (i) diagnosis of the subject with BV;

(ii) prediction of the recurrence of BV in the subject; and

/or (iii) direction of continued treatment of the subject for BV.

50. A method of claim 48 or 49 wherein the defined temperature threshold is ±

10°

C. from the Tm of LB.

51. A method of claim 48 or 49 wherein the defined temperature threshold is ±

5°

C. from the Tm of LB.

52. A method of claim 48 or 49 wherein the defined temperature threshold is ±

2.5°

C. from the Tm of LB.

18. A kit for assessing health of a vaginal microbiome comprising:

a first set of primers comprising forward primers and reverse primers that support amplification of bacterial DNA present in the vaginal microbiome;

a second set of primers comprising at least one forward blocking primer and at least one reverse blocking primer that block amplification of LB species DNA; and

instructions for obtaining a vaginal sample, amplifying the total bacterial DNA in the sample by PCR using the first primer set, amplifying the non-LB DNA in the sample using the second blocking primer set, and calculating the relative abundance of non-LB species in the sample.

19. A kit of claim 18, wherein the vaginal sample is a vaginal lavage.

20. A kit of claim 18, wherein the amplification of bacterial DNA is performed by PCR.

21. A kit of claim 20, wherein the PCR is quantitative PCR (qPCR).

22. A kit of claim 20, wherein the at least one first forward primer and at least one first reverse primer are selected from BU4F+, BU6R+, 8F+, 1501R1, 1501R2, 1492R, 27F, 338R, 341F, 806R, 338F, and 534R.

23. A kit of claim 18, wherein the blocking primers partially overlap binding sites for sequences on the LB genome and wherein the blocking primer melting temperatures are at least 68°

C.

24. A kit of claim 23, wherein the blocking primers are selected from LBblocker3, LBB3p, LBblocker4, and LBB4p.

25. A kit of claim 18, wherein the relative abundance of non-LB species is determined by calculating a ratio of non-LB species to LB species in the sample.

26. A kit of claim 18, wherein the kit is useful for the diagnosis of BV and wherein if the ratio of non-LB species to LB species is more than 1:

99, the subject has BV.

27. A kit of claim 18, wherein the kit is useful for monitoring the treatment of BV, and wherein if the ratio of non-LB species to LB species is more than 1:

99, the treatment is ineffective and a new treatment is initiated in the subject.

28. A kit of claim 18, wherein the kit is useful for monitoring the treatment of BV, and wherein if the ratio of non-LB species to LB species is 1:

99 or less, the treatment is effective and the prescribed course of treatment is completed in the subject.

29. A kit of claim 18, wherein the kit is useful for choosing an appropriate therapy for BV.

30. A kit of claim 18, wherein the kit is useful to predict the failure of a specific therapy of BV.

31. A kit of claim 18, wherein the kit further comprises reagents and instructions for conducting melting curve analyses on resultant PCR amplicons.

53. A kit of claim 18 further comprising instructions to calculate a Δ

Cq score and if the Δ

Cq is below 3, diagnosing the subject with BV;

predicting the recurrence of BV in the subject; and

/or directing continued treatment of the subject for BV.

54. A kit of claim 18 further comprising instructions to calculate a Δ

Cq score and if the Δ

Cq is 3 or above, determining that the subject is free from BV;

predicting the non-recurrence of BV; and

/or directing the cessation of treatment of the subject for BV.

55. A kit of claim 18 further comprising instructions to calculate a Δ

Cq score and instructions to perform a non-LB melt curve analysis to generate a combined score.

56. A kit of claim 55 wherein a Δ

Cq score of 3 or above in combination with an absence of melt curves within a defined temperature threshold direct (i) a determination that the subject is free from BV;

(ii) a prediction of non-recurrence of BV; and

/or (iii) direction of the cessation of treatment of the subject for BV.

57. A kit of claim 55 wherein a Δ

Cq score of below 3 or the presence of melt curves within a defined temperature threshold direct (i) diagnosis of the subject with BV;

(ii) prediction of the recurrence of BV in the subject; and

/or (iii) direction of continued treatment of the subject for BV.

58. A kit of claim 56 or 57 wherein the defined temperature threshold is ±

10°

C. from the Tm of LB.

59. A kit of claim 56 or 57 wherein the defined temperature threshold is ±

5°

C. from the Tm of LB.

60. A kit of claim 56 or 57 wherein the defined temperature threshold is ±

2.5°

C. from the Tm of LB.

32. A method of characterizing health of a vaginal microbiome from a sample of a subject comprising:

amplifying total bacterial DNA present in the sample; and

selectively amplifying non-LB species DNA in the sample by amplifying total bacterial DNA while specifically blocking amplification of DNA from one or more LB species,

wherein the relative abundance of non-LB species is indicative of the health of the vaginal microbiome.

33. A method of claim 32, wherein the amplification of bacterial DNA is performed by PCR.

34. A method of claim 33, wherein the PCR is qPCR.

35. A method of claim 32, wherein the total bacterial DNA is amplified with at least one forward primer and at least one reverse primer broadly reactive with bacterial DNA.

36. A method of claim 32, wherein amplification of the LB bacterial DNA is blocked by at least one forward blocking primer and at least one reverse blocking primer which specifically bind to unique sequences on the LB bacterial genome and wherein the blocking primer melting temperatures are at least 68°

C.

37. A method of claim 32, wherein the relative abundance of non-LB species is determined by calculating a ratio of non-LB species to dominant species in the sample.

38. A method of claim 32, wherein the method is used for the diagnosis of a disease or disorder in the subject, and wherein if the level of non-LB species is higher than a pre-determined threshold level specific for the sample was obtained, the subject has a disease or disorder of the vagina.

39. A method of claim 32, wherein the method is used for monitoring the treatment of a disease or disorder in the subject, and wherein if the level of non-LB species is higher than a pre-determined threshold level specific for the vagina, the treatment is ineffective and a new treatment is initiated in the subject.

40. A method of claim 32, wherein the method is used for monitoring the treatment of a disease or disorder, and wherein if the level of non-LB species is lower than a pre-determined threshold level specific for the vagina, the treatment is effective and the prescribed course of treatment is completed in the subject.

41. A method of claim 32, wherein the method further comprises determining the identity of non-LB species in the sample.

42. A method of claim 41, further comprising choosing a therapy based on the identity of non-LB species in the sample.

43. A method of claim 32, wherein the method is used to predict the failure of a specific therapy for a disease or disorder.

44. A method of claim 43, wherein the method further comprises the step of determining the identity of non-LB species in the sample.