Abstract:
The present disclosure relates to the filed of medical diagnostics, specifically directed towards the field of bacterial vaginosis diagnostic methods, systems and apparatus. Also included is a multiplexed platform test for the diagnosis of infectious vaginitis.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates generally to the field of medical diagnostics. More specifically, the present invention is directed toward the field of bacterial vaginosis diagnostic methods, systems and apparatus. 
       BACKGROUND 
       [0002]    The normal vaginal ecosystem is complex, predominated by  Lactobacilli. Lactobacillus  is estrogen dependent, maintaining a normal environmental pH of 4.0.  Lactobacillus  preserves mucin gel coating over the epithelium, produces lactic acid, H 2 0 2 , and bacteriocins (calprotectin), and maintains an innate immune response.  Lactobacillus  protects against HIV acquisition/transmission, prevents pro-inflammatory cytokines and reduces the risk of STIs (sexually transmitted infections). (Valore,  Am J Obstet Gynecol;  87: 561 (2002).) 
         [0003]    A change in normal bacterial flora, including the reduction of  Lactobacillus  allows other bacteria to multiply and produce toxins which affect the body&#39;s natural defenses and make re-colonization of healthy bacteria more difficult. Normal vaginal flora is commonly described in the literature. Specifically normal vaginal flora is described by: Fredricks, et al., Molecular identification of bacteria associated with bacterial vaginosis.  N. Engl. J. Med.  353:1899-1911. 2005; Hyman, et al., Microbes on the human vaginal epithelium.  PNAS  120:7952-7957, 2005; Verhelst, et al., Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between  Atopobium vaginae, Gardnerella vaginalis  and bacterial vaginosis.  BMC Microbiol.  4:16. 2004; Verhelst, et al., Comparison between Gram stain and culture for the characterization of vaginal microflora: definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora.  BMC Microbiol.  5:61. 2005; Zhou, et al., Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.  Microbiology  150: 2565-2573. 2004; May et al., The Identification of Vaginal  Lactobacillus  Species and the Demographic and Microbiologic Characteristics of Women Colonized by These Species.  The Journal of Infectious Diseases  180:1950-6. 1999; and Vasquez et al., Vaginal  Lactobacillus  Flora of Healthy Swedish Women.  Journal of Clinical Microbiology,  40 (8). 2746-2749. 2002. 
         [0004]    Infectious vaginitis accounts for more than ten million physician office visits each year, with approximately half of all adult women suffering at least one episode. The three most common forms of infectious vaginitis in decreasing incidence are bacterial vaginosis,  vulvovaginal candidiasis  and  trichomoniasis.  Because  vulvovaginal  infections can result in serious clinical sequelae, vulvovaginal symptoms and signs warrant careful evaluation and appropriate therapy. 
         [0005]    Bacterial vaginosis (BV) is a prevalent cause of vaginal infection and is associated with several serious health conditions. BV is caused by an imbalance of naturally occurring bacterial flora. BV results when the normal, predominantly  Lactobacillus,  vaginal flora shifts to one dominated by  Gardnerella vaginalis, Mycoplasma hominis,  and a variety of anaerobic organisms predominantly clostridial species. 
         [0006]    Fredricks, et al. have reported considerable bacterial diversity in subjects who had BV as compared to healthy subjects, including 35 bacterial phylotypes detected in samples from subjects with BV. (Fredricks, et al. “Molecular Identification of Bacteria Associated with Bacterial Vaginosis”  N. Engl. J. Med.  353;18 (2005).) Additionally, it was reported that extremely high levels of bacterial DNA from several rarely cultivated clostridial species were detected in the vaginal fluid of subjects with BV. Fielder, et al. “Changes in BV-Associated Bacterial concentrations with Vaginal  Metronicazole  Therapy” ISSTDR abstract (2007)). 
         [0007]    The pathogenesis of BV is as follows: the overgrowth of anaerobic microorganisms is accompanied by the production of proteolytic enzymes that act on vaginal peptides to release several biologic products, including polyamines, which volatize in the accompanying alkaline environment to elaborate foul-smelling trimethylamine. Polyamines facilitate the transudation of vaginal fluid and exfoliation of epithelial cells, creating a copious discharge. Clue cells are formed when such organisms are present in high numbers, adhere to exfoliated epithelial cells in the presence of an elevated pH. (Sobel, J D,  N Engl. J. Med.  337: 1896-1903 (1997).) 
         [0008]    Untreated BV may cause serious complications, such as increased susceptibility to sexually transmitted infections and may present other complications for pregnant women. BV has also been associated with an increase in the development of Pelvic Inflammatory Disease following surgical procedures such as a hysterectomy or an abortion. BV can be cured by antibiotics such as metronidazole and clindimicyn. Thus it is important to have an effective efficient method for diagnosis. 
         [0009]    Prior BV diagnostics focus on causative agents, either applying direct or indirect measures. These diagnostics include detection of  Gardnerella vaginalis  through a nucleic acid based test. Additionally, prior diagnosis of BV includes detection of elevated enzymes such as sialidase activity, an enzyme produced by bacterial pathogens associated with BV including  Gardnerella, Bacteroides, Prevotella  and  Mobilincus.    
         [0010]    All of the references cited in this disclosure are hereby incorporated in their entirety. 
       SUMMARY OF THE DISCLOSURE 
       [0011]    The present disclosure teaches a method for the diagnosis of BV through a quantitative determination of  Lactobacillus,  establishing a measure of clinically relevant  Lactobacillus  in healthy vaginas, and correlating a diminished amount of  Lactobacillus  with the diagnosis of BV. The present disclosure also teaches a method for detecting the occurrence or non-occurrence of bacterial vaginosis in a patient comprising the steps of collecting a biological sample from a patient, quantitatively measuring the presence of  Lactobacillus;  and correlating a diminished presence of  Lactobacillus  with the diagnosis of bacterial vaginosis. The disclosure further teaches using quantitative polymerase chain reaction to  Lactobacillus  in order to quantitate the amount of  Lactobacillus.  The disclosure further teaches using any analyte sandwich assay (i.e. lateral flow immunoassay, ELISA or direct DNA detection) to quantitate the amount of  Lactobacillus.  The disclosure further teaches a method for diagnosis of BV,  Candida  and  Trichomonas  utilizing lateral flow immunoassay to quantitate  Candida, Trichomonas  and  Lactobacillus  in a multiplexed assay. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0012]      FIG. 1  is a graphic representation of an inverse relationship between  Lactobacillus crispatus  and BV pathogens. 
           [0013]      FIG. 2  is a graphic representation of changes in BV flora following Metronidazole treatment. 
           [0014]      FIG. 3  is a flow chart representation of a syndromic approach to diagnosis of BV,  Candida  and  Trichomonas,  collectively called vaginosis. 
           [0015]      FIG. 4  is a flow chart representation of an additional step in the syndromic approach to diagnosis of BV,  Candida  and  Trichomonas,  collectively called vaginosis. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0016]      Lactobacillus crispatus,  is the predominant  Lactobacillus  species present in “healthy vaginas” as shown in the control subjects of Table 1. Table 1 includes the results of a study of the bacteria identified by broad-range 165 rDNA polymerase chain reaction in vaginal fluid from subjects with bacterial vaginosis. (Fredricks et al.  N Eng J Med  2005) There are a large and diverse number of bacterial species found in the vagina of subjects with BV.  FIG. 1  illustrates the existence of an inverse relationship between  Lactobacillus crispatus  and BV pathogens in subjects with BV. Thus the level of  Lactobacillus crispatus  significantly decreases with a diagnosis of BV. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Bacteria Identified by Broad-Range 16SrDNA Polymerase Chain Reaction in  
               
               
                 Vaginal fluid from Subjects with and without Bacterial Vaginosis. 
               
             
          
           
               
                   
                 Subjects with BV 
                   
                   
                   
                 Control Subjects without BV 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6  
                 7 
                 8 
                 9 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                 Percentage of Clones 
                   
                   
                   
                   
                   
               
               
                   
               
             
          
           
               
                 
                   Lactobacillus crispatus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 49 
                 74 
                 99 
                 48 
                 60 
                 89 
                 40 
                 100 
               
               
                 
                   L. jensenii 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 2 
                   
                 1 
                   
                   
               
               
                 
                   L. gallinarum 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   L. gasseri 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 9 
                   
                   
                 1 
                   
                   
                   
               
               
                 
                   L. vaginalis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 2 
                   
                   
                   
                   
               
               
                 
                   Staphylococcus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 2 
                   
                   
                   
                   
                   
                   
               
               
                 
                   epidermidis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   S. lugdunensis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
               
               
                 
                   Clostridium 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 3 
                   
                   
                   
               
               
                   perfringens  (96%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AB045286 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Ureaplasma parvum 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
                   
                   
               
               
                 
                   L. iners 
                 
                   
                   
                   
                 7 
                 22 
                 3 
                   
                 1 
                 5 
                 38 
                   
                 1 
                 46 
                 36 
                 10 
                 60 
                   
               
               
                 
                   Gardnerella vaginalis 
                 
                 35 
                 13 
                 2 
                 4 
                 28 
                 1 
                 25 
                 31 
                 39 
                   
                 14 
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured AB034121 
                   
                 43 
                 66 
                 34 
                   
                 36 
                 17 
                   
                 26 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (90.6%) (BVAB1) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured AF407407 
                 10 
                 4 
                   
                 10 
                 3 
                 5 
                 4 
                   
                 4 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (90.9%) (BVAB2) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured 
                 1 
                 1 
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 ULO278163 (92.9%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (BVAB3) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Atopobium vaginae 
                 
                 5 
                   
                 3 
                 21 
                 1 
                 3 
                 3 
                 11 
                 11 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Leptotrichia  amnionii 
                 6 
                   
                 2 
                 8 
                 1 
                 3 
                 10 
                 10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Megasphaera elsdenii 
                 
                 4 
                 10 
                 7 
                 1 
                 18 
                 3 
                 2 
                 13 
                 6 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (93.8%) AY038994 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   M. micronuciformis 
                 
                   
                   
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (94.6%) AF473833 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Eggerthella 
                 
                 2 
                 2 
                 2 
                 1 
                 4 
                 1 
                 3 
                 8 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   hongkongensis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (91.8%) AY288517 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Porphyromonas 
                 
                   
                   
                   
                 2 
                   
                 5 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   asaccharolytica 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Dialister  species (a) 
                 1 
                 1 
                 2 
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
               
               
                   Dialister  species (b) 
                   
                 1 
                 2 
                   
                   
                 2 
                 2 
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (94.8%) AF371693 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Sneathia sanguinegens 
                 
                 3 
                   
                 2 
                   
                 16 
                 9 
                 9 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Prevotella  genogroup 
                 21 
                 24 
                   
                 1 
                 7 
                 9 
                 12 
                 20 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Prevotella  genogroup 
                 7 
                   
                   
                 4 
                   
                 6 
                 7 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Prevotella  genogroup 
                   
                   
                 7 
                   
                   
                   
                   
                 3 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 3 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   P. bivia 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   P. buccalis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                 6 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   P. dentalis  (93.2%) 
                   
                   
                   
                   
                   
                 2 
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X81876 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   P. disiens 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   P. oulorum  (90.6%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 L16472 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   P. shahii  (90.7%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AB108825 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured 4C28d-23 
                 1 
                   
                 3 
                 1 
                   
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (91.2%) AB034149 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Candidate division 
                   
                   
                 2 
                 2 
                   
                 8 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 TM7 (93.7%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF125206 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Mobiluncus mulieris 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Peptoniphilus 
                 
                   
                   
                   
                   
                   
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   lacrimalis 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Peptoniphilus  species 
                   
                 1 
                   
                   
                   
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Peptostreptococcus 
                 
                 3 
                   
                   
                 2 
                   
                   
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   micros  (97.8%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF542231 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Gemella bergeriae 
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (95.8%) Y13365 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Aerococcus  species 
                   
                   
                   
                   
                   
                   
                   
                 1 
                 2 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   Anaerococcus 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   tetradius 
                 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured (89.8%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AF371910 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Uncultured (88.4%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 AJ400235 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   Veillonella  species 
                   
                   
                   
                   
                   
                   
                 1 
               
               
                   
               
             
          
         
       
     
         [0017]    As shown in  FIG. 2 , Polymerase Chain Reaction (PCR) was used to survey BV flora before and after metronidazole treatment. (Ferris et al.  J. of Clin. Microbiology,  2007). The species composition for patients prior to treatment was variable.  Lactobacillus  (in particular  Lactobacillus inners ) was prominent in all patients post-treatment.  Atopobium vaginae  concentrations were highest for patients who failed or responded incompletely to treatment and lowest for patients who were cured. Thus the results illustrated that post treatment,  Lactobacillus  predominate. Thus, a normal level of  Lactobacillus  present in the vaginal flora is keystone to a “healthy vagina”. 
         [0018]    The present disclosure teaches a method for the diagnosis of BV. Quantitative measure of  Lactobacillus  as a measure of a healthy vagina and an inverse correlation with BV yields a diagnostic test for BV. This method differs from previous diagnostics where the presence of the multitude of agents associated with BV, such as  Gardnerella,  indirect enzyme measures,  Amsel  criteria etc., was identified for diagnosis. The present disclosure teaches quantitatively measuring clinically relevant  Lactobacilli.  These  Lactobacilli  include, but are not limited to,  crispatus, jensenii,  and  gasseri,  either independently or in combination. Quantitative measurement can be achieved by a variety of methods known in the art. These methods include, but are not limited to: antibody/homologous cross reactive but specific reagents, quantitative polymerase chain reaction, and any “sandwich” assay—both antibody and nucleic acid based. Further included are direct antigen measurement and lateral flow immunoassay including utilizing SERS technology. The present disclosure teaches establishing a clinically relevant cut off value for the  Lactobacillus  measure from clinical trials and the use of statistical measures of relevance (ROC curves (receiver operator curves) as would be understood by one of skill in the art. 
         [0019]    Table 2 lists the analyte targets, antibodies for cytoplasmic proteins, antibodies for surface proteins and sequence information for applicable targets, including  Candida, Trichomonas  and  Lactobacillus.  Using the disclosed sequences, one with skill in the art can readily and without undue experimentation, develop antibodies to  Candida, Trichomonas  and  Lactobacillus.  As such, antibodies for  Candida  and  Trichomonas  are already known and listed in the table. Additionally, methods to develop antibodies using sequence derived antigens as described can be found at Strategic Diagnostics Inc, http://www.sdix.com/. In the case of  Lactobacillus,  the sequences disclosed and known are used to determine antigenic regions and will be further used to raise antibodies. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 Antibody for 
                   
               
               
                 Analyte 
                 Antibody for cytoplasmic 
                 surface  
                   
               
               
                 Targets 
                 protein 
                 protein 
                 Sequence information 
               
               
                   
               
             
             
               
                 
                   Candida 
                 
                 PA1-7206 Polyclonal, 
                 MA1-7009, 
                 The completed assembly #21 contains 15.845 Mb of 
               
               
                 
                   albicans 
                 
                 ABR—Affinity BioReagents 
                 Monoclonal, 
                 DNA, organized into the 8  C. albicans  chromosomes. 
               
               
                   
                 ab53891 Polyclonal, Abcam 
                 ABR—Affinity 
                 The gene and protein sequences or the sequence of part 
               
               
                   
                 Ab34211 monoclonal, Abcam 
                 BioReagents 
                 or all of a chromosome can be retrieved from the 
               
               
                   
                 1. monoclonal, 
                   
                   Candida  Genome 
               
               
                   
                 AbD serotec 
                   
                 Database.http://www.candidagenome.org/ 
               
               
                   
                 2. monoclonal, 
                   
                 * The genomic sequence of  Candida glabrata   
               
               
                   
                 AbD serotec 
                   
                 chromosomes A-M is available in Genebank. 
               
               
                   
                 3. monoclonal, 
                   
                 http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid= 
               
               
                   
                 AbD serotec 
                   
                 284593&amp;chr=A 
               
               
                   
                 1750-5007 Polyclonal, 
                   
                   
               
               
                   
                 AbD serotec 
                   
                   
               
               
                 
                   Trichomonas 
                 
                 9155-9529 monoclonal, 
                 C65101M 
                 The  Trichomonas vaginalis  G3 whole genome shotgun 
               
               
                 
                   vaginalis 
                 
                 AbD serotec 
                 monoclonal, 
                 (WGS) project has the acession# 
               
               
                   
                 The cell localization of the 
                 Meridian 
                 NZ_AAHC00000000. This version of the project(01) 
               
               
                   
                 immunogen for this antibody 
                 C65657M 
                 has the accession# NZ_AAHC01000000, and consists 
               
               
                   
                 is unknown. 
                 monoclonal, 
                 of sequences NZ_AAHC01000001- 
               
               
                   
                   
                 Meridian 
                 NZ_AAHC01075128. 
               
               
                   
                   
                 C65675M 
                 Length: 178,315,610 bp (unfinished). 
               
               
                   
                   
                 monoclonal, 
                   
               
               
                   
                   
                 Meridian 
                   
               
               
                   
                   
                 C65526M 
                   
               
               
                   
                   
                 monoclonal, 
                   
               
               
                   
                   
                 Meridian 
                   
               
               
                   
                   
                 PTV 65 
                   
               
               
                   
                   
                 polyclonal, 
                   
               
               
                   
                   
                 HyTest 
                   
               
               
                   
                   
                 Ab24317 
                   
               
               
                   
                   
                 Polyclonal, 
                   
               
               
                   
                   
                 abcam 
                   
               
               
                 
                   Lactobacillus 
                 
                   
                   
                 146 nucleotide sequences in CoreNucleotide Database, 
               
               
                 
                   crispatus 
                 
                   
                   
                 for example: gene for 16S rRNA, glyceraldehyde-3- 
               
               
                   
                   
                   
                 phosphate dehydrogenase, S-layer proteins and 60 kDa 
               
               
                   
                   
                   
                 chaperonin. 
               
               
                   
                   
                   
                 63 protein sequences, for example: phenolic acid 
               
               
                   
                   
                   
                 decarboxylase, alpha-enolase, transposase, truncated 
               
               
                   
                   
                   
                 PTS EIIC-like protein and surface layer protein. 
               
               
                 
                   Lactobacillus 
                 
                   
                   
                 37 nucleotide sequences in CoreNucleotide Database, 
               
               
                 
                   jensenii 
                 
                   
                   
                 for example: gene for 16S ribosomal RNA, rpoA gene 
               
               
                   
                   
                   
                 for RNA polymerase alpha subunit, pheS gene for 
               
               
                   
                   
                   
                 phenylalanyl-tRNA synthase alpha subunit, ptsH gene, 
               
               
                   
                   
                   
                 promoter region and 5′ UTR, rpsU gene, promoter 
               
               
                   
                   
                   
                 region and 5′ UTR, 60 kDa heat shock protein, recA 
               
               
                   
                   
                   
                 gene for recombinase A, mRNA for putative elongation 
               
               
                   
                   
                   
                 factor Tu, 60 kDA chaperonin and 16S-23S rRNA 
               
               
                   
                   
                   
                 intergenic spacer region and 23S rRNA gene 5&#39; flanking 
               
               
                   
                   
                   
                 region. 
               
               
                   
                   
                   
                 6 protein sequences, for example: RNA polymerase 
               
               
                   
                   
                   
                 alpha subunit, phenylalanyl-tRNA synthase alpha 
               
               
                   
                   
                   
                 subunit, 60 kDa heat shock protein, recombinase A, 
               
               
                   
                   
                   
                 putative elongation factor Tu and 60 kDa chaperonin. 
               
               
                 
                   Lactobacillus 
                 
                   
                   
                 261 nucleotide sequences in CoreNucleotide Database, 
               
               
                 
                   gasseri 
                 
                   
                   
                 for example: gene for 16S ribosomal RNA, 60 kDa 
               
               
                   
                   
                   
                 chaperonin, rpoA gene for RNA polymerase alpha 
               
               
                   
                   
                   
                 subunit, pheS gene for phenylalanyl-tRNA synthase 
               
               
                   
                   
                   
                 alpha subunit, putative complement factor, acidocin 
               
               
                   
                   
                   
                 LF221B, and putative immunity protein genes, recA 
               
               
                   
                   
                   
                 gene for recombinase A, pbgal gene for phospho-beta- 
               
               
                   
                   
                   
                 galactosidase, ATP synthase beta chain, aggregation 
               
               
                   
                   
                   
                 promoting factor, gassericin T gene region, Gassericin 
               
               
                   
                   
                   
                 A, phospho-beta-galactosidase and many sequences 
               
               
                   
                   
                   
                 from patents. 
               
               
                   
                   
                   
                 Genome sequences for  Lactobacillus gasseri  ATCC 
               
               
                   
                   
                   
                 33323, Length: 1,894,360 bp.3967 protein sequences, 
               
               
                   
                   
                   
                 for example: putative immunity protein, acidocin 
               
               
                   
                   
                   
                 LF221A, putative complemental factor, Lysin, Holin, 
               
               
                   
                   
                   
                 phospho-beta-galactosidase, gassericin K7 B, putative 
               
               
                   
                   
                   
                 ATP-dependent transport protein, aminopeptidase N, 
               
               
                   
                   
                   
                 putative branched-chain amino acid transporter, beta- 
               
               
                   
                   
                   
                 glucuronidase, putative regulatory protein, gassericin T, 
               
               
                   
                   
                   
                 Gassericin A, ATP synthase alpha subunit, RNA 
               
               
                   
                   
                   
                 polymerase alpha subunit, Ribosomal protein L34, 
               
               
                   
                   
                   
                 RNase P protein component, Preprotein translocase 
               
               
                   
                   
                   
                 subunit YidC, Predicted membrane protein, 
               
               
                   
                   
                   
                 transcriptional regulator, Aggregation promoting 
               
               
                   
                   
                   
                 factrelated surface protein and uncharacterized 
               
               
                   
                   
                   
                 conserved secreted or membrane protein 
               
               
                   
               
             
          
         
       
     
         [0020]    The present disclosure also incorporates by reference the technology disclosed for the use of SERS tags in assays including sandwich assays and lateral flow immunoassays (LFI). In particular, the following patents and pending applications are incorporated herein by reference; U.S. Pat. No. 6,514,767, Surface Enhanced Spectroscopy—Active Composite Nanoparticles; U.S. Pat. No. 7,192,778, Surface Enhanced Spectroscopy—Active Composite Nanoparticle; U.S. Patent App. Publication No. US-2005-0219509; Surface Enhanced Spectroscopy—Active Composite Nanoparticles; PCT Patent App. No. PCT/US07/61136 (unpublished), Lateral Flow Immunoassay with Encapsulated Detection Modality; PCT Patent Publication No. WO-2007/092941, SERS Nanotag Assays. 
         [0021]    SERS allows detection of molecules attached to the surface of a single Raman-enhancing nanoparticle. A Raman-enhancing metal that has associated or bound to it a Raman-active molecule(s) is referred to as a SERS-active nanoparticle. Such SERS-active nanoparticles can have utility as optical tags. For example, SERS-active nanoparticles can be used in immunoassays when conjugated to an antibody against a target molecule of interest. If the target of interest is immobilized on a solid support, then the interaction between a single target molecule and a single nanoparticle-bound antibody could be detected by searching for the Raman-active molecule&#39;s unique Raman spectrum. Furthermore, because a single Raman spectrum (from 100 to 3500 cm −1 ) can detect many different Raman-active molecules, different SERS-active nanoparticles can be used in multiplexed assay formats. In one embodiment, the disclosure provides for a single diagnostic for BV through the measurement of  Lactobacillus.  In an alternative embodiment, the disclosure provides for a multiplexed assay for BV,  Candida,  and  Trichomonas,  determining BV through the measurement of  Lactobacillus.    
         [0022]    The present disclosure also provides a lateral flow immunoassay (LFI) featuring encapsulated metal particles. Briefly, the encapsulated particles may use SERS nanotags as the detection modality. The use of encapsulated particles as a detection modality, in particular encapsulated SERS tags increases the sensitivity of an LFI prepared for visual reading and introduces the ability to obtain substantially more sensitive qualitative results or quantitative results through the analysis of a SERS spectrum read from an LFI prepared in accordance with the present embodiment. The use of SERS as detection modality also enhances the ability of an LFI device to be used for a multiplexed test. Other embodiments include LFI devices specifically configured to test vaginal samples, a reader for the detection and interpretation of a multiplexed assay and the hardware and software components used to implement the reader. 
         [0023]    Note that throughout this application various citations are provided. Each of these citations is specifically incorporated herein by reference in its entirety. 
       EXAMPLE 1 
       [0024]    In one embodiment,  Lactobacillus  sequence will be used to determine antigenic regions and use this to raise antibodies. These antibodies will be used for quantitative measurements of  Lactobacillus.    
       EXAMPLE 2 
       [0025]    In another embodiment, the sequences known for  Lactobacillus  are used to perform quantitative polymerase chain reaction for a determination of the amount of  Lactobacillus.    
       EXAMPLE 3 
       [0026]    In one embodiment, a quantitative measurement of clinically relevant  Lactobacilli  is performed. This may include  crispatus, jensenii, gasseri  either independently, or with antibody/homologous cross reactive but specific reagents. As is known by one with skill in the art, a clinically relevant cut off value for the  Lactobacillus  measure via clinical trials and the use of statistical measures of relevance (i.e. ROC curves) are developed. Once the clinically relevant cut off value for  Lactobacilli  is established, a diagnostic test is performed, wherein the  Lactobacilli  of a patient is measured, and a diagnosis of BV is established based on a low clinically relevant  Lactobacilli  value. 
       EXAMPLE 4 
       [0027]    In one embodiment, the disclosure teaches a syndromic approach to vaginitis diagnosis. The three most common forms of infectious vaginitis in decreasing incidence are bacterial vaginosis,  vulvovaginal candidiasis  and  trichomoniasis.  As shown in  FIG. 3 , incorporating the three measures in one test 300 results in a very powerful test.  Candida  and  Lactobacillus  measures  302 ( a ),  302 ( b ),  304 ( a ) and  304 ( b ), respectively, are quantitative while the  Trichomonas  would be a test based on the presence or absence of  Trichomonas    306 ( a ),  306 ( b ). As is known by one with skill in the art, a clinically relevant cut off value for the  Lactobacillus  measure via clinical trials and the use of statistical measures of relevance (i.e. ROC curves) are developed. Once the clinically relevant cut off value for  Lactobacilli  is established, a diagnostic test is performed, wherein the  Lactobacilli  of a patient is measured, and a diagnosis of BV is established based on a low clinically relevant  Lactobacilli  value. Similarly, for vulvovaginal  candidiasis,  clinical trials and the use of statistical measures of relevance (i.e. ROC curves) are developed. Once the clinically relevant cut off value is developed for  Candida,  diagnosis of vulvovaginal  candidiasis  is established based on a high clinically relevant  Candida  value.  Trichomoniasis  is diagnosed based on the presence of  Trichomonas.  In one embodiment a kit comprising the three tests based on LIF is envisioned using SERs tags for the relevant moieties. As shown in  FIG. 4 , an additional step  308  may selectively be implemented where analytes for causative agents to increase clinical sensitivity and specificity are added to the initial quantitative  lactobacillus  determination. 
         [0028]    While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.