Methods for diagnosing and monitoring treatment of bacterial vaginosis

1. A method for determining the presence or absence of Bacterial Vaginosis (BV) in a subject, the method comprising:

(a) providing a clinical sample from a subject suspected of having BV;

(b) performing an assay for the detection of Lactobacillus sp., G. vaginalis , and Eggerthella sp. in the sample; and

(c) determining the presence or absence of BV in the subject based on a combination of a detection status of either positive or negative for each of Lactobacillus sp., G. vaginalis status, and Eggerthella sp. determined from step (b),

wherein

(i) a negative detection status for both G. vaginalis and Eggerthella sp. indicates the absence of BV in the subject,

(ii) a positive detection status for both G. vaginalis and Eggerthella sp. indicates the presence of BV in the subject,

(iii) if the detection status of Lactobacillus sp. is positive, then a negative detection status for at least one of G. vaginalis and Eggerthella indicates the absence of BV in the subject, and

(iv) if the detection status of Lactobacillus sp. is negative, then a positive detection status for at least one of G. vaginalis and Eggerthella indicates the presence of BV in the subject;

wherein the presence of BV is indicated in the subject and the method further comprises administering a treatment regime for BV to the subject; and

wherein the assay for detection of Lactobacillus sp., G. vaginalis , and Eggerthella sp. is a multiplex amplification-based detection assay comprising

(1) contacting the sample with

first, second, and third Lactobacillus -specific amplification oligomers for amplifying a target region of a Lactobacillus sp. target nucleic acid, wherein (i) the first Lactobacillus -specific amplification oligomer comprises a first Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:7, (ii) the second Lactobacillus -specific amplification oligomer comprises a second Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:6, and (iii) the third Lactobacillus -specific amplification oligomer comprises a third Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:8;

first and second G. vaginalis -specific amplification oligomers for amplifying a target region of a G. vaginalis target nucleic acid, wherein (i) the first G. vaginalis -specific amplification oligomer comprises a first G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii) the second G. vaginalis -specific amplification oligomer comprises a second G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12; and

first and second Eggerthella -specific amplification oligomers for amplifying a target region of an Eggerthella sp. target nucleic acid, wherein (i) the first Eggerthella -specific amplification oligomer comprises a first Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-51 of SEQ ID NO:17 and (ii) the second Eggerthella -specific amplification oligomer comprises a second Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:16;

wherein each of the first Lactobacillus -specific amplification oligomer, the third Lactobacillus -specific amplification oligomer, the first G. vaginalis -specific amplification oligomer, and the first Eggerthella specific amplification oligomer is a promoter primer or promoter provider further comprising a T7 promoter sequence located 5′ to the respective target-hybridizing sequence;

(2) performing an in vitro nucleic acid amplification reaction, wherein any Lactobacillus sp., G. vaginalis , and Eggerthella sp. target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the Lactobacillus sp., G. vaginalis , and Eggerthella sp. target regions; and

(3) detecting the presence or absence of the one or more amplification products.

2. The method of claim 1 , wherein

the nucleotide sequence of the first Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:7;

the nucleotide sequence of the third Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:8;

the nucleotide sequence of the first G. vaginalis -specific amplification oligomer consists of the sequence of SEQ ID NO:13; and/or

the nucleotide sequence of the first Eggerthella -specific amplification oligomer consists of the sequence of SEQ ID NO:17.

3. The method of claim 1 , wherein the detecting step (3) comprises

contacting the one or more amplification products with a first Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, a first G. vaginalis -specific detection probe that specifically hybridizes to the G. vaginalis target region, and a first Eggerthella -specific detection probe that specifically hybridizes to the Eggerthella sp. target region, and

detecting the presence or absence of any target-hybridized Lactobacillus -specific, G. vaginalis -specific, and/or Eggerthella -specific detection probe.

4. The method of claim 3 , wherein

the first Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:9,

the first G. vaginalis -specific detection probe comprises a target-hybridizing sequence consisting of residues 1-19 of SEQ ID NO:14, and/or

the first Eggerthella -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:18;

optionally wherein the detecting step (3) further comprises contacting the one or more amplification products with a second Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, wherein the second Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of residues 6-21 of SEQ ID NO:10.

5. The method of claim 1 , wherein the method comprises the detection of no more than ten bacterial genera associated with BV.

6. The method of claim 1 , wherein the method does not include detection of bacterial genera associated with BV other than Lactobacillus, Gardnerella , and Eggerthella.

7. The method of claim 1 , wherein the treatment regime comprises the administration of an antibiotic drug selected from the group consisting of metronidazole, clindamycin, and tinidazole.

8. A method for monitoring treatment of Bacterial Vaginosis (BV) in a subject, the method comprising:

(a) providing a clinical sample from a subject previously diagnosed with BV and already undergoing a treatment regime for BV;

(b) performing an assay for the detection of Lactobacillus sp., G. vaginalis , and Eggerthella sp. in the sample; and

(c) determining the presence or absence of BV in the subject based on a combination of a detection status of either positive or negative for each of Lactobacillus sp., G. vaginalis , and Eggerthella sp. determined from step (b),

wherein

(i) a negative detection status for both G. vaginalis and Eggerthella sp. indicates the absence of BV in the subject,

(ii) a positive detection status for both G. vaginalis and Eggerthella sp. indicates the presence of BV in the subject,

(iii) if the detection status of Lactobacillus sp. is positive, then a negative detection status for at least one of G. vaginalis and Eggerthella indicates the absence of BV in the subject, and

(iv) if the detection status of Lactobacillus sp. is negative, then a positive detection status for at least one of G. vaginalis and Eggerthella indicates the presence of BV in the subject;

wherein the presence of BV is indicated in the subject and the method further comprises continuing treatment of BV in the subject; and

wherein the assay for detection of Lactobacillus sp., G. vaginalis , and Eggerthella sp. is a multiplex amplification-based detection assay comprising

(1) contacting the sample with

first, second, and third Lactobacillus -specific amplification oligomers for amplifying a target region of a Lactobacillus sp. target nucleic acid, wherein (i) the first Lactobacillus -specific amplification oligomer comprises a first Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:7, (ii) the second Lactobacillus -specific amplification oligomer comprises a second Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:6, and (iii) the third Lactobacillus -specific amplification oligomer comprises a third Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:8;

first and second G. vaginalis -specific amplification oligomers for amplifying a target region of a G. vaginalis target nucleic acid, wherein (i) the first G. vaginalis -specific amplification oligomer comprises a first G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii) the second G. vaginalis -specific amplification oligomer comprises a second G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12; and

first and second Eggerthella -specific amplification oligomers for amplifying a target region of an Eggerthella sp. target nucleic acid, wherein (i) the first Eggerthella -specific amplification oligomer comprises a first Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-51 of SEQ ID NO:17 and (ii) the second Eggerthella -specific amplification oligomer comprises a second Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:16;

wherein each of the first Lactobacillus -specific amplification oligomer, the third Lactobacillus -specific amplification oligomer, the first G. vaginalis -specific amplification oligomer, and the first Eggerthella specific amplification oligomer is a promoter primer or promoter provider further comprising a T7 promoter sequence located 5′ to the respective target-hybridizing sequence;

(2) performing an in vitro nucleic acid amplification reaction, wherein any Lactobacillus sp., G. vaginalis , and Eggerthella sp. target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the Lactobacillus sp., G. vaginalis , and Eggerthella sp. target regions; and

(3) detecting the presence or absence of the one or more amplification products.

9. The method of claim 8 , wherein

the nucleotide sequence of the first Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:7;

the nucleotide sequence of the third Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:8;

the nucleotide sequence of the first G. vaginalis -specific amplification oligomer consists of the sequence of SEQ ID NO:13; and/or

the nucleotide sequence of the first Eggerthella -specific amplification oligomer consists of the sequence of SEQ ID NO:17.

10. The method of claim 8 , wherein the detecting step (3) comprises

contacting the one or more amplification products with a first Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, a first G. vaginalis -specific detection probe that specifically hybridizes to the G. vaginalis target region, and a first Eggerthella -specific detection probe that specifically hybridizes to the Eggerthella sp. target region, and

detecting the presence or absence of any target-hybridized Lactobacillus -specific, G. vaginalis -specific, and/or Eggerthella -specific detection probe.

11. The method of claim 10 , wherein

the first Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:9,

the first G. vaginalis -specific detection probe comprises a target-hybridizing sequence consisting of residues 1-19 of SEQ ID NO:14, and/or the first Eggerthella -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:18;

optionally wherein the detecting step (3) further comprises contacting the one or more amplification products with a second Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, wherein the second Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of residues 6-21 of SEQ ID NO:10.

12. The method of claim 8 , wherein the method comprises the detection of no more than ten bacterial genera associated with BV.

13. The method of claim 8 , wherein the method does not include detection of bacterial genera associated with BV other than Lactobacillus, Gardnerella , and Eggerthella.

14. The method of claim 8 , wherein the continued treatment comprises administering the same treatment regime that the subject is undergoing prior to step (a).

15. The method of claim 8 , wherein the presence of BV in the subject indicates a need to change the treatment regime that the subject is undergoing prior to step (a) and the continued treatment comprises administering a different treatment regime.