Abstract:
Accurate and fast detection of the presence of  Clostridium difficile  ( C. difficile ) disease is crucial for the proper treatment of patients with  C. difficile  infection. Present tests detecting the presence of  C. difficile  disease are fast and cost effective, but are not very sensitive. Using an ELISA including Cell Wall Protein 84 (Cwp84) increases the sensitivity of the ELISA. Cwp84 may be used alone or in combination with other markers to support a diagnosis of  C. difficile -associated disease.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/539,209 filed Sep. 26, 2011, which is entirely incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]      Clostridium difficile  ( C. difficile ) is the most common cause of nosocomial diarrhea and accounts for about 3 million cases of diarrhea annually in the United States. The known risk factors of  C. difficile  infection (CDI) are exposure to antibiotics, advanced age, and residence in hospitals or long-term care facilities. The symptoms of CDI range from mild diarrhea to pseudomembranous colitis and toxic megacolon. The average cost of treatment is about $10,000 per case. The mortality rate of  Clostridium difficile  associated disease (CDAD) increased from 5.7 deaths per million population in 1999 to 23.7 deaths per million population in 2004 due to the emergence of hypervirulent outbreak strains. Accurate diagnosis is crucial for prompt and proper treatment of patients with CDI. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0003]    Illustrative embodiments of the present invention are described in detail below with reference to the attached drawing figures, and wherein: 
           [0004]      FIG. 1  depicts a sequence of Cwp84 from  C. difficile  strain 630; 
           [0005]      FIG. 2  depicts a graphical representation of a response curve of recombinant Cwp84 on anti-Cwp84 ELISA; 
           [0006]      FIG. 3  depicts a tabular representation of a comparison between the number of positive (+) and negative (−) samples detected by anti-Cwp84 ELISA and ethanol shock culture method; and 
           [0007]      FIG. 4  depicts a tabular representation of a comparison between the number of positive (+) and negative (−) samples detected by an ELISA with both anti-GDH antibodies and anti-Cwp84 antibodies and an ELISA with anti-GDH antibodies alone. 
       
    
    
     DETAILED DESCRIPTION 
       [0008]    The present invention is directed to test methods for detecting  C. difficile  in patients based on the presence of marker proteins derived from  C. difficile . In particular, a new antigen marker, Cell Wall Protein 84 (Cwp84) may be used in immunoassays for the detection of  C. difficile . Anti-Cwp84 antibodies could be used alone or in combination with antibodies against other  C. difficile  proteins, such as GDH (glutamate dehydrogenase) and Toxins A and B (i.e., TcdA and TcdB, respectively), in immunoassays for highly sensitive detection of  C. difficile.    
         [0009]    Generally, primary diagnostic methods for the detection of  C. difficile  in stool samples include plating samples on a selective medium, cytotoxicity assay, immunoassay and molecular assays such as polymerase chain reaction (PCR) based and isothermal DNA amplification. The presence of  C. difficile  in stool samples can be detected by selective culturing methods or immunoassays targeting a common antigen present in all  C. difficile  strains. 
         [0010]    Presently, there are four major types of tests for detecting  C. difficile  in stool samples. The first, ethanol shock culture, includes treating fecal samples with ethanol followed by plating on cycloserine-cefoxitin-fructose agar (CCFA) and is considered the gold standard for its high sensitivity. However, this test may take up to 72 hours to obtain results and it does not differentiate between toxigenic and non-toxigenic strains of  C. difficile.    
         [0011]    Cytotoxicity test in tissue culture, the second type of test, can differentiate between toxigenic and nontoxigenic strains of  C. difficile  but the sensitivity is lower compared to PCR-based tests. Also, it takes up to 48 hours for a conclusion to be obtained using this test. 
         [0012]    The third, DNA amplification-based tests, targets the toxin genes A or B and is highly sensitive but costly. There is also a concern that the presence of toxin genes does not necessarily correlate with the presence of amounts of toxin proteins that are relevant to the disease. 
         [0013]    Lastly, antibody-based tests, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests, are rapid tests to detect the presence of Toxin A, Toxin B, and/or  C. difficile  common antigen glutamate dehydrogenase (GDH). GDH is an abundant metabolic enzyme which is highly conserved among  C. difficile  strains. The presence of GDH indicates the presence of  C. difficile  but does not differentiate between toxigenic and non-toxigenic strains. The immunoassays are rapid, cost effective, and can be used as point-of-care tests. Immunoassays detecting GDH are highly sensitive while immunoassays detecting toxins A and B show lower sensitivity compared to toxigenic culture method and molecular methods targeting toxin genes. Therefore, immunoassays based on  C. difficile  common antigens such as GDH serve as economical screening tests for the presence of  C. difficile  with follow-up testing used to confirm that TcdA and/or TcdB is present. One such approach is to use immunoassays as a screening test for the presence of common antigens such as GDH followed by more sensitive tests for the detection of toxins. 
         [0014]    The sensitivity of antibody-based tests is influenced by multiple factors, such as interfering substances, the stability of the antigen analyzed and the variation of the amount of antigen produced by different  C. difficile  strains. The incorporation of antibodies against additional antigen markers that show less, or at least distinct patterns of, variability would help overcome these obstacles 
         [0015]    Several  C. difficile  proteins were examined as potential stand-alone or additional antigens in immunoassays, based on the criteria of being abundant, conserved among all sequenced  C. difficile  strains, and  C. difficile  specific. Preference was given to cell surface antigens, as these proteins are exposed on the surface of the bacteria and are accessible to the antibodies used in the assay. From among those candidate proteins, Cwp84, a cell surface protease and possible colonization factor, was selected. 
         [0016]      C. difficile  cells process a surface layer (S-layer) outside of the peptidoglycan layer. The S-layer is present in both vegetative cells and  C. difficile  spores, and proteins within the S-layer mediate host-pathogen interactions. Several  C. difficile  surface proteins bind to gastrointestinal tissues and are potential colonization factors. These proteins include flagellar proteins FliC and FliD, the heat shock protein GroEL, the fibronectin binding protein Fbp68, surface layer protein SlpA, and Cwp66. 
         [0017]    Surface layer protein A (SlpA) is the major component of the surface layer of  C. difficile . SlpA has to be post-translationally processed into a high molecular weight domain and a low molecular weight domain to be fully functional. There are two cysteine proteases located in the surface layer of  C. difficile : CD2787 (Cwp84, NCBI accession number YP — 001089300) and CD1751 (Cwp13, NCBI accession number YP — 001088255). The absence of Cwp13 in a virulent  C. difficile  strain R20291 indicates that Cwp13 is not crucial for pathogenesis. In addition, the normal processing of SlpA in a cwp13 mutant confirms that Cwp13 is not involved in SlpA maturation. This leaves Cwp84 as the probable cysteine protease involved in the maturation of SlpA. The accumulation of unprocessed SlpA in cwp84 mutant strains further suggests that Cwp84 is responsible for the cleavage of SlpA. 
         [0018]    Besides processing SlpA, Cwp84 may also be more directly involved in colonization of the intestine by  C. difficile . The ability of  C. difficile  to express/release proteins that degrade host tissues contributes to its ability to colonize the intestinal epithelium. Cwp84 is not only associated with the S-layer but is also secreted. Cwp84 is active over a wide range of pH (3.5 to 8.0) with the optimum pH at 7.5 and is capable of degrading human fibronectin, laminin and vitronectin, proteins of the extracellular matrix. External protease activity has been observed in all  C. difficile  isolates examined and the protease activity can be neutralized by the cysteine protease specific inhibitor E64, suggesting that Cwp84, since it is the major cysteine protease of the S-layer, is responsible for this degradative activity. 
         [0019]    The transcription, translation and post-translational modifications of Cwp84 have been characterized. The gene encoding Cwp84 is transcribed as a monocistronic message in the early exponential phase of growth. The location and maturation of Cwp84 requires multiple proteins. The secretion of Cwp84 to the S-layer involves the ATPases SecA1 or SecA2. Cwp84 is expressed as a proenzyme and the maturation of Cwp84 requires post translational modification. The mature Cwp84 is about 47 kDa. The post-translational modification of Cwp84 includes the removal of the signal peptide (amino acids 1-32), the removal of amino acids 33-91 by Cwp13 and autoproteolysis. Sequence analysis of Cwp84 reveals a C-terminal anchoring domain and an N-terminal protease domain that is homologous to cysteine proteases in other bacteria. Cysteine at position 116 is critical for the enzymatic activity of Cwp84, and activity can be abolished by a Cysteine 116 to Alanine (C116A) mutation. Illustrated in  FIG. 1 , and provided as SEQ ID NO 1, is an amino acid sequence from a sequenced  C. difficile  strain 630. Amino acids 1 to 32 are predicted to be a leader signal peptide. In the present embodiment, a portion of the Cwp84 sequence is used. In additional embodiments, full length of the Cwp84 sequence may be used. 
         [0020]    In an embodiment of the present invention, a method of detecting  C. difficile  in stool samples using antibodies to Cwp84 is provided. Several characteristics of Cwp84 make it a good diagnostic marker. First of all, compared to other colonization factors, such as Cwp66 and SlpA, Cwp84 is more conserved among sequenced  C. difficile  strains, which makes it a better diagnostic marker for the detection of all  C. difficile  isolates. Secondly, Cwp84 is  C. difficile -specific. No protein with significant homology is found in other bacteria. Most interestingly, the expression of Cwp84 is up-regulated if  C difficile  is exposed to certain antibiotics. Antibiotic exposure is the most important risk factor for CDAD. The usage of broad-spectrum antibiotics, such as clindamycin, aminopenicillins, cephalosporins and fluoroquinolones disturbs the normal gut flora and allows subsequent  C. difficile  infection. The expression of Cwp84 is induced by ampicillin and clindamycin in many clinical isolates and by fluoroquinolones in moxifloxacin-resistant  C. difficile  isolates. Among colonization factors tested, Cwp84 is the most up-regulated upon antibiotic treatment. This makes Cwp84 a good diagnostic marker for antibiotic treatment-related CDAD. 
         [0021]    An embodiment of the present invention provides methods for using Cwp84, either independently or in combination with existing markers such as GDH, as a diagnostic marker for detecting CDI using fecal samples of humans. In this embodiment, humans present with symptoms common to  C. difficile  but have not yet been diagnosed. In additional embodiments, a person may be diagnosed with  C. difficile  infection upon identifying the presence of  C. difficile  in the fecal sample. 
         [0022]    The present invention is directed to detection methods for using Cwp84 as an indicator for the presence of  C. difficile  in bacterial cultures or feces. An immunoassay such as ELISA, which utilizes either monoclonal or polyclonal antibodies to Cwp84, can be used to indicate the presence or absence of  C. difficile . To produce antibodies against Cwp84, Cwp84 from  C. difficile  strain 630 was recombinantly expressed in  E. coli . Polyclonal and monoclonal antibodies against the recombinant mutant Cwp84 were generated in goats and mice, respectively. An ELISA was developed using polyclonal anti-Cwp84 antibodies as capturing antibodies and horseradish peroxidase (HRP)-conjugated monoclonal anti-Cwp84 antibodies as detection antibodies. 
         [0023]    In the ELISA, the fecal specimen is diluted 5-fold and added to a well containing the immobilized polyclonal antibodies. If the fecal specimen contains  C. difficile , the Cwp84 produced by  C. difficile  will bind to the antibodies during the incubation at 37° C. Following the incubation, monoclonal antibodies coupled to horseradish peroxidase (HRP conjugate) are added and allowed to bind to the captured Cwp84. Unbound conjugate is then washed away from the well and substrate (tetra-methyl-benzidene and hydrogen peroxide) is added for color development. Following the substrate incubation, 0.1 M sulfuric acid is added to quench the reaction and an optical density (OD) at 450 nM is measured using a spectrophotometer. An OD reading equal or above 0.080 indicates the presence of  C. difficile  and an OD less than 0.080 indicates the absence of  C. difficile  in the fecal specimen. 
         [0024]    This ELISA can also be used to distinguish bacterial cultures of  C. difficile  from cultures of other types of bacteria. Bacterial cultures can be diluted 20-fold in phosphate buffered saline (PBS) and added to a well coated with polyclonal antibodies against Cwp84. If the culture tested contains  C. difficile , the Cwp84 produced by  C. difficile  cells will bind to the immobilized polyclonal antibodies during the incubation at 37° C. Following the incubation, monoclonal antibodies conjugated with HRP are added as the detection antibodies. After the unbound detection antibodies are washed away, substance is added for color development followed by the quenching of the reaction by 0.1 M sulfuric acid. The OD at 450 nM is then measured using a spectrophotometer. An OD reading equal to or above 0.080 indicates the presence of  C. difficile  and an OD less than 0.080 indicates the absence of  C. difficile  in the bacterial culture. 
         [0025]    To study the detection limit of the ELISA using polyclonal anti-Cwp84 antibodies as capturing antibodies and monoclonal anti-Cwp84 antibodies conjugated to HRP as detection antibodies, Cwp84 was expressed in  E. coli , purified and diluted to concentrations shown in Table 1. A four parameter logistics curve fit method was used to generate the response curve shown in  FIG. 2 . Using OD 0.080 as a cutoff, 0.2 ng/mL Cwp84 can be detected by this ELISA. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Detection Limit of Recombinant Cwp84 on anti-Cwp84 ELISA 
               
             
          
           
               
                   
                 Concentration of Cwp84 (ng/mL) 
                 OD 450 nm/600 nm 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 0.003 
               
               
                   
                 0.2 
                 0.114 
               
               
                   
                 0.4 
                 0.268 
               
               
                   
                 0.8 
                 0.567 
               
               
                   
                 1.6 
                 1.207 
               
               
                   
                 3.2 
                 2.572 
               
               
                   
                 6.4 
                 3.363 
               
               
                   
                 12.8 
                 4.104 
               
               
                   
                   
               
             
          
         
       
     
       Example 1 
       [0026]    To investigate whether or not Cwp84 can be used as a marker to detect frequently seen strains of  C. difficile , twelve  C. difficile  strains of prevalent ribotypes were tested using the anti-Cwp84 ELISA. The PCR ribotyping method is relatively easy to perform, reproducible, and is the most discriminatory method to differentiate and identify  C. difficile  strains. 
       Results 
       [0027]    Twelve  C. difficile  strains of prevalent ribotypes were inoculated into Brain-heart infusion broth (BHI; Oxoid). Cultures were grown at 37° C. over night under anaerobic conditions. The  C. difficile  strains in BHI cultures were diluted 1:20 in PBS and tested on an ELISA using polyclonal anti-Cwp84 antibodies as capturing antibodies and monoclonal anti-Cwp84 antibodies-HRP conjugate as detection antibodies. The absorbance on dual wavelength (OD450/620 nm) is shown in Table 2. The cutoff of this ELISA was set to be 0.080. All the ribotypes produced strongly positive reactions on this ELISA, indicating that the Cwp84 ELISA can be used for detecting many common toxigenic and non-toxigenic strains of  C. difficile . 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Twelve Prevalent  C. difficile  Strains Produce 
               
               
                 Positive Reactions on anti-Cwp84 ELISA 
               
             
          
           
               
                   
                 Ribotype 
                 Toxigenic 
                 OD 450/620 nm 
                 Interpretation 
               
               
                   
                   
               
               
                   
                 001 
                 YES 
                 0.837 
                 + 
               
               
                   
                 002 
                 YES 
                 0.228 
                 + 
               
               
                   
                 003 
                 YES 
                 0.826 
                 + 
               
               
                   
                 005 
                 YES 
                 3.882 
                 + 
               
               
                   
                 009 
                 NO 
                 1.123 
                 + 
               
               
                   
                 010 
                 NO 
                 0.649 
                 + 
               
               
                   
                 014 
                 YES 
                 0.591 
                 + 
               
               
                   
                 015 
                 YES 
                 1.143 
                 + 
               
               
                   
                 027 
                 YES 
                 0.936 
                 + 
               
               
                   
                 039 
                 NO 
                 0.720 
                 + 
               
               
                   
                 053 
                 YES 
                 0.876 
                 + 
               
               
                   
                 106 
                 YES 
                 1.906 
                 + 
               
               
                   
                   
               
             
          
         
       
     
       Example 2 
       [0028]    To investigate whether or not other bacteria are cross-reactive on the anti-Cwp84 ELISA, fifty-six strains of bacteria that are commonly found in feces or closely related to  C. difficile  phylogenetically were grown under required conditions and tested on the anti-Cwp84 ELISA using polyclonal anti-Cwp84 antibodies as capturing antibodies and monoclonal anti-Cwp84 antibodies conjugated to HRP as detection antibodies. The absorbance using dual wavelength detection (OD 450/620 nm) is shown in Table 3. The cutoff of this ELISA was set to be 0.080. The negative reaction produced by all of the bacteria tested on this ELISA indicated no cross-reactivity on the anti-Cwp84 ELISA. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 No cross-reaction with anti-Cwp84 ELISA 
               
             
          
           
               
                   
                   
                 Optical 
                 Interpre- 
               
               
                 Bacterium 
                 Strain 
                 Density 
                 tation 
               
               
                   
               
               
                 
                   Aeromonas hydrophila 
                 
                 ATCC 35654 
                 0.001 
                 − 
               
               
                 
                   Bacillus cereus 
                 
                 ATCC 14579 
                 0.005 
                 − 
               
               
                 
                   Bacillus subtilis 
                 
                 ATCC 6051 
                 0.001 
                 − 
               
               
                 
                   Bacteroides fragilis 
                 
                 VPI 13785 
                 0.002 
                 − 
               
               
                 
                   Campylobacter coli 
                 
                 ATCC 43478 
                 0.003 
                 − 
               
               
                 
                   Campylobacter jejuni 
                 
                 ATCC 29428 
                 0.006 
                 − 
               
               
                 
                   Candida albicans 
                 
                 ATCC 10231 
                 0.001 
                 − 
               
               
                 
                   Citrobacter freundii 
                 
                 ATCC 8090 
                 0.009 
                 − 
               
               
                 
                   Clostridium bifermentans 
                 
                 ATCC 638 
                 0.005 
                 − 
               
               
                 
                   Clostridium clostridiforme 
                 
                 VPI 316 
                 0.001 
                 − 
               
               
                 
                   Clostridium butyricum 
                 
                 ATCC 8260 
                 0.004 
                 − 
               
               
                 
                   Clostridium glycolicum 
                 
                 VPI 2048 
                 0.001 
                 − 
               
               
                 
                   Clostridium innocuum 
                 
                 ATCC 14501 
                 0.004 
                 − 
               
               
                 
                   Clostridium ghonii 
                 
                 ATCC 25757 
                 0.002 
                 − 
               
               
                 
                   Clostridium haemolyticum 
                 
                 VPI 2041 
                 0.002 
                 − 
               
               
                 
                   Clostridium histolyticum 
                 
                 ATCC 19401 
                 0.002 
                 − 
               
               
                 
                   Clostridium novyi 
                 
                 VPI 5771 
                 0.002 
                 − 
               
               
                 
                   Clostridium perfringens 
                 
                 NCIB 10748 
                 0.002 
                 − 
               
               
                 
                   Clostridium septicum 
                 
                 VPI 3533 
                 0.002 
                 − 
               
               
                 
                   Clostridium sordellii 
                 
                 VPI 9048 
                 0.004 
                 − 
               
               
                 
                   Clostridium sordellii 
                 
                 VPI 7319 
                 0.001 
                 − 
               
               
                 
                   Clostridium sporogenes 
                 
                 VPI 9743 
                 0.001 
                 − 
               
               
                 
                   Enterobacter aerogenes 
                 
                 ATCC 13048 
                 0.002 
                 − 
               
               
                 
                   Enterobacter cloacae 
                 
                 ATCC 13047 
                 0.002 
                 − 
               
               
                 
                   Enterococcus faecalis 
                 
                 ATCC 19433 
                 0.003 
                 − 
               
               
                   Escherichia coli  EIEC 
                 ATCC 43893 
                 0.001 
                 − 
               
               
                 
                   Escherichia coli 
                 
                 ATCC 11775 
                 0.000 
                 − 
               
               
                   Escherichia coli  0157 H7 
                 ATCC 43895 
                 0.002 
                 − 
               
               
                   Escherichia coli  0157 H7 
                 ATCC 43888 
                 0.003 
                 − 
               
               
                 (non-tox) 
               
               
                   Escherichia coli  ETEC 
                 ATCC 35401 
                 0.005 
                 − 
               
               
                   Escherichia coli  EPEC 
                 ATCC 12014 
                 0.002 
                 − 
               
               
                 
                   Escherichia fergusonii 
                 
                 ATCC 35469 
                 0.005 
                 − 
               
               
                 
                   Escherichia hermannii 
                 
                 ATCC 33650 
                 0.003 
                 − 
               
               
                 
                   Gardnerella vaginalis 
                 
                 ATCC 14019 
                 0.002 
                 − 
               
               
                 
                   Helicobacter pylori 
                 
                 ATCC 43504 
                 0.003 
                 − 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 ATCC 9997 
                 0.003 
                 − 
               
               
                 
                   Peptostreptococcus anaerobius 
                 
                 ATCC 27337 
                 0.002 
                 − 
               
               
                 
                   Proteus vulgaris 
                 
                 ATCC 8427 
                 0.002 
                 − 
               
               
                 
                   Providencia stuartii 
                 
                 ATCC 33672 
                 0.003 
                 − 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 ATCC 10145 
                 0.001 
                 − 
               
               
                 
                   Pseudomonas fluorescens 
                 
                 ATCC 13525 
                 0.002 
                 − 
               
               
                 
                   Salmonella enterica 
                 
                 ATCC 49284 
                 0.004 
                 − 
               
               
                 serovar minnesota 
               
               
                 
                   Salmonella typhimurium 
                 
                 ATCC 14028 
                 0.002 
                 − 
               
               
                 
                   Serratia liquefacians 
                 
                 ATCC 27592 
                 0.002 
                 − 
               
               
                 
                   Shigella dysenteriae 
                 
                 ATCC 13313 
                 0.005 
                 − 
               
               
                 
                   Shigella flexneri 
                 
                 ATCC 29903 
                 0.002 
                 − 
               
               
                 
                   Shigella sonnei 
                 
                 ATCC 11060 
                 0.002 
                 − 
               
               
                 
                   Staphylococcus aureus 
                 
                 ATCC 6358 
                 0.002 
                 − 
               
               
                 
                   Staphylococcus aureus 
                 
                 ATCC 12598 
                 0.003 
                 − 
               
               
                 (Cowans) 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 ATCC 27626 
                 0.002 
                 − 
               
               
                 
                   Vibrio cholerae 
                 
                 ATCC 14035 
                 0.012 
                 − 
               
               
                 
                   Vibrio parahaemolyticus 
                 
                 ATCC 17802 
                 0.006 
                 − 
               
               
                 
                   Stenotrophomonas maltophilia 
                 
                 ATCC 13637 
                 0.002 
                 − 
               
               
                 
                   Yersinia enterocolitica 
                 
                 ATCC 23715 
                 0.002 
                 − 
               
               
                   
               
             
          
         
       
     
       Example 3 
       [0029]    To investigate whether Cwp84 can be used as a diagnostic marker to detect  C. difficile  in fecal samples, 64 clinical fecal samples were tested using the anti-Cwp84 ELISA described above. The sensitivity and specificity of the anti-Cwp84 ELISA for the detection of  C. difficile  were calculated using an ethanol shock culture method as the gold standard. 
       Results 
       [0030]    The anti-Cwp84 ELISA detected twenty-nine (29) positive samples out of the thirty-four (34) positive samples identified by the ethanol shock culture method. All thirty (30) samples that tested negative by anti-Cwp84 ELISA were true negative samples that failed to grow using the ethanol shock culture method. Thus, the calculated sensitivity and specificity of the anti-Cwp84 ELISA were 85% and 100%, respectively. 
         [0031]      FIG. 3  illustrates the results of anti-Cwp84 ELISA on 64 human fecal samples compared to the results of ethanol shock culture method. Anti-Cwp84 ELISA detected the presence of  C. difficile  in 29 of the 34 positive samples determined by ethanol shock culture method. 
       Example 4 
       [0032]    To investigate whether Cwp84 can be used in combination with GDH as common antigens to indicate the presence of  C. difficile  in fecal samples, an ELISA was developed using a combination of both anti-Cwp84 polyclonal antibodies and anti-GDH antibodies as capturing antibodies and HRP conjugated anti-Cwp84 monoclonal antibodies and HRP conjugated anti-GDH monoclonal antibodies as detection antibodies. Forty-five clinical samples that had been determined to be positive for  C. difficile  by the ethanol shock culture method, but that showed either weak or negative results on a standard anti-GDH ELISA, were selected and tested on the ELISA containing antibodies against both Cwp84 and GDH. 
       Results 
       [0033]    Five of the 25 samples that were negative by single antibody anti-GDH ELISA were positive on the ELISA utilizing both anti-GDH and anti-Cwp84 antibodies. The combination of anti-Cwp84 antibodies with anti-GDH antibodies in the ELISA increased the sensitivity of the assay. The addition of anti-Cwp84 antibodies as both capturing antibodies and detection antibodies helped to detect the presence of  C. difficile  in stool samples that was missed in ELISAs utilizing anti-GDH antibodies alone. 
         [0034]      FIG. 4  illustrates the results of this example. Forty-five fecal samples determined to be  C. difficile  positive by the ethanol shock culture method, but that were weak or negative on the anti-GDH ELISA, were tested using the ELISA with both anti-GDH antibodies and anti-Cwp84 antibodies. The combination of both anti-GDH and anti-Cwp84 antibodies detected  C. difficile  in 5 additional samples that had tested negative by ELISA with anti-GDH antibodies only. 
         [0035]    In summary, the present invention provides Cwp84 as an additional diagnostic marker for detecting  C. difficile  in stool samples in addition to the existing diagnostic markers GDH, Toxin A, and Toxin B. The examples and embodiments of the present invention have been described in relation to particular embodiments which are intended in all respects to be illustrative rather than restrictive. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its scope. 
         [0036]    Embodiments of the present invention also provide a device to test a fecal sample obtained from a person and use Cwp84 as an indicator of  C. difficile . Broadly speaking, the device of the invention comprises any configuration of components that permit practice of the method of the invention. More specifically, the device of the invention comprises any configuration of components that permit a sample containing, or suspected of containing, a substance of interest to be retained in a pre-defined area or region of the device, where the area or region comprises a specific binding pair member that is specific, either directly or indirectly, for the substance. 
         [0037]    In its most basic form, the device of the invention comprises (1) a receiving portion to receive a fecal sample from a person and (2) a testing portion to detect the presence of Cwp84 of the fecal sample, where the testing portion comprises a specific binding pair member that is specific for Cwp84. Any additional components that permit the practice of the method of the invention may also be included in the device. 
         [0038]    From the foregoing, it will be seen that this invention is one well adapted to attain all the ends and objects herein above set forth together with other advantages which are obvious and which are inherent to the method. It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.