Cell wall protein CwpV (CD0514) as a diagnostic marker for Clostridium difficile ribotype 027
Inventors
DAVIS, MANLI Y. • WILLIAMS, KRISTA A. • BROWNING, JOCELYN N. • Lyerly, David M.
Assignees
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Publication Number
US-9133527-B2
Publication Date
2015-09-15
Expiration Date
Abstract
Accurate and rapid differentiation of the outbreak strain ribotype 027 from other possible Clostridium difficile (C. difficile) strains, using stool samples, facilitates decision making for treatment options. Cell wall protein V (CwpV) contains a cell wall binding domain conserved among C. difficile strains and a variable domain which is antigenically different among C. difficile strains. In embodiments, antibodies against the 027-specific region in CwpV are used in diagnostic tests to detect ribotype 027 in culture or fecal samples.
Core Innovation
The described invention uses Clostridium difficile cell wall protein CwpV (CD0514) as a diagnostic antigen marker. In particular, it uses the ribotype 027-specific variable C-terminal region of CwpV to differentiate ribotype 027 from other C. difficile strains in culture or fecal samples.
Antibodies raised against the 027-specific CwpV region are used in immunoassays to detect the ribotype 027-specific CwpV antigen. The immunoassay formats referenced include ELISA and the lateral flow “QUIK CHEK,” with the assay results compared against PCR ribotyping as a gold standard.
The reported performance indicates that anti-CwpV ELISA detects ribotype 027 with sensitivity and specificity values on the order of ~86%. The lateral flow QUIK CHEK test is reported to show sensitivity and specificity values on the order of ~83% sensitivity and 100% specificity for toxigenic isolates, and no detection of non-027 strains in the reported comparisons.
Claims Coverage
The provided independent claim covers diagnosing a patient for a ribotype 027 C. difficile strain associated with severe C. difficile infection by detecting a ribotype 027-specific CwpV antigen in a fecal sample using an immunoassay. The independent-claim coverage is primarily focused on antigen detection with immunoassays, with dependent claim coverage refining the immunoassay format and antibody/detection configurations.
Diagnosis of ribotype 027 infection by ribotype 027-specific CwpV antigen immunoassay
Obtain a fecal sample from a patient, determine the patient has a C. difficile infection, and determine whether the patient has a ribotype 027 C. difficile strain infection by determining a presence of a ribotype 027-specific CwpV antigen in the fecal sample using an immunoassay against the CwpV antigen.
Immunoassay format selected from ELISA, immunochemical membrane reaction, and lateral flow
Determine the ribotype 027 C. difficile strain infection using the immunoassay in an immunoassay format selected from ELISA, an immunochemical membrane-based reaction, or a lateral flow assay.
Capturing antibody is monoclonal, polyclonal, or recombinant anti-ribotype 027-specific CwpV antibody
Define the immunoassay such that it uses monoclonal, polyclonal, or recombinant anti-ribotype 027-specific CwpV antibodies as capturing antibodies.
Detection antibody includes HRP-conjugated anti-ribotype 027-specific CwpV antibody
Use an HRP-conjugated polyclonal or monoclonal anti-ribotype 027-specific CwpV antibody as detection antibodies in the immunoassay.
Corroboration by determining ribotype 027-specific CwpV DNA fragment using nucleic acid amplification
Further determine the presence of a ribotype 027 C. difficile strain by identifying a ribotype 027-specific CwpV DNA fragment using nucleic acid amplification assays.
Dilute fecal sample prior to CwpV antigen immunoassay
Further determine the presence of a ribotype 027 C. difficile strain by diluting a fecal sample to make a diluted fecal sample and using the diluted sample in an immunoassay against the CwpV antigen.
The inventive coverage centers on detecting a ribotype 027-specific CwpV antigen in a fecal sample using an immunoassay, with refinements specifying immunoassay format, capturing antibody classes, HRP-conjugated detection antibodies, a possible nucleic-acid amplification determination of a ribotype 027-specific CwpV DNA fragment, and optionally diluting the fecal sample before immunoassay.
Stated Advantages
Improved diagnostic differentiation of ribotype 027 from other C. difficile strains using a ribotype 027-specific CwpV antigen marker.
Anti-CwpV ELISA reports ~86% sensitivity and specificity for detecting ribotype 027.
QUIK CHEK reports ~83% sensitivity and 100% specificity for toxigenic isolates and no detection of non-027 strains.
Documented Applications
Diagnosing a patient with a ribotype 027 C. difficile strain infection associated with severe C. difficile infection using fecal sample immunoassay detection of ribotype 027-specific CwpV antigen.
Use of immunoassays (ELISA and lateral flow “QUIK CHEK”) for detection in culture or fecal samples to differentiate ribotype 027 from other C. difficile strains.
Performance comparison of immunoassay detection results versus PCR ribotyping as a gold standard.
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