Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-29
2003-01-07
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007320, C435S007900, C435S007920, C435S007940, C435S007950, C435S026000, C435S973000, C436S518000, C436S528000, C436S531000, C436S532000, C436S808000, C436S811000
Reexamination Certificate
active
06503722
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of diagnostic methods and kits for detecting
Clostridium difficile.
The methods and kits provide rapid, sensitive, and accurate assays for the presence of toxigenic strains of
C. difficile
in an biological sample.
2. Background
Clostridium difficile,
an anaerobic organism, is the major causative agent of pseudomembranous colitis (PMC) in humans. PMC is characterized by diarrhea, a severe inflammation of the colonic mucosa, and formation of pseudomembranes that are composed of fibrin, mucus, necrotic epithelial cells, and leukocytes. The pseudomembrane can form a sheath over the entire colonic mucosa. In addition to causing PMC,
C. difficile
is believed to play a role in other less severe gastrointestinal illnesses; the organism is estimated to cause approximately 25% of reported cases of antibiotic-associated diarrhea. Brettle and Wallace (1984)
J. Infect.
8: 123-128; Gilligan et al. (1981)
J. Clin. Microbiol.
14: 26-31. Diarrhea affects approximately 25 million persons annually in the US alone, and causes almost 11,000 deaths. Peterson and Kelly (1993)
Lab. Diagnosis Infect. Dis.
7: 277-292.
C. difficile
-caused diseases are not limited to gastrointestinal illnesses, as the organism can cause abscesses, wound infections, osteomyelitis, urogenital tract infections, septicemia, peritonitis, and pleuritis. Lyerly et al. (1988)
Clin. Microbiol. Rev.
1: 1-18; Hafiz et al. (1975) Lancet 1: 420-421; Levett (1986)
J. Infect.
12: 253-263; Saginur et al. (1983)
J. Infect. Dis.
147: 1105. Antibiotics can predispose a host animal to PMC and other
C. difficile
-related illnesses, as the disturbance of the normal bacterial flora by the antibiotic disrupts the major barrier against colonization by pathogens, rendering the host animal susceptible to colonization by pathogens such as
C. difficile.
Hospitals and chronic care facilities are significant sources of
C difficile
infection, with one study finding that 21% of patients acquired
C. difficile
infection during hospitalization. McFarland et al. (1989)
N. Engl. J. Med.
320: 204.
The high frequency of
C. difficile
infection, coupled with the likelihood of a poor clinical outcome for cases that are not treated promptly, makes clear the need for rapid and accurate tests to detect
C. difficile
infection, determine whether any
C. difficile
present is toxigenic, and evaluate the effectiveness of treatment. Previously available methods for detecting
C. difficile
are much less than optimal for effective diagnosis and treatment of infection. One previously known method for detecting
C. difficile
infection is culture on agar media. The efficacy of this method is hampered by the significant variation in results that are obtained using different media, and the high rate of false positives (10-29%). Peterson and Kelly, supra. An additional disadvantage of this method is the lengthy culture period required before visible
C. difficile
colonies are discernable. A commercially available assay for
C. difficile
involves latex agglutination of an antigen that was eventually identified as
C. difficile
glutamate dehydrogenase. Lyerly et al. (1991)
J. Clin. Microbiol.
29: 2639; Lyerly et al. (1986)
J. Clin. Microbiol.
23: 622. However, this assay suffers from widely varying and insufficient sensitivity and specificity, with sensitivity ranging from 68% to 93% and specificity between 80% and 95%. Peterson and Kelly, supra. Moreover, previously available glutamate dehydrogenase assays were not thought to be useful for detection of toxigenic strains of
C. difficile
because glutamate dehydrogenase is produced by non-toxigenic strains of
C. difficile,
as well as toxigenic strains.
Non-toxigenic strains of
C. difficile
are generally considered clinically insignificant, while toxigenic strains can be lethal. Although distinguishing between toxigenic and non-toxigenic
C. difficile
strains is thus of great importance, previously known assays that were used in attempts to accomplish this goal were ineffective. One commonly used diagnostic method for detecting the presence of toxigenic
C. difficile
involves determining whether
C. difficile
is cytotoxic to susceptible cell lines. This cytotoxicity is the result of either or both of the two toxins produced by
C. difficile,
an enterotoxin designated toxin A and a cytotoxin designated toxin B, both of which are believed to be involved in the pathogenesis of PMC. The cytotoxicity assay has significant drawbacks for clinical use however, including the need to maintain tissue culture lines and the relatively low sensitivity of the assay. For example, Peterson and Kelly, supra., found that the sensitivity of cytotoxin detection alone ranged from 67% to 100%, and other researchers found sensitivity to be as low as 71%. Demlee et al. (1985)
J. Clin. Microbiol.
21: 323. Immunoassays for toxins A and/or B have also been used to detect
C. difficile
in samples, but these methods suffer from low sensitivity (63% to 88%). Peterson and Kelly, supra.
Thus, a need exists for assays to detect the presence of
C. difficile
in a sample that are rapid, sensitive, specific, and cost-effective. Assays to determine whether an infecting
C. difficile
strain is toxigenic are also needed. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods, compositions, and kits for the rapid detection of
C. difficile
in a test sample, in particular for detection of toxigenic
C. difficile
strains.
In a first embodiment, the methods involve detecting
C. difficile
toxin A or toxin B, and also detecting
C. difficile
glutamate dehydrogenase. The assay means for detecting the
C. difficile
antigens can be, for example, immunoassays. Sandwich assays provide a convenient, sensitive method for performing the methods. In one embodiment, the assay involves detection of
C. difficile
toxin A using a sandwich assay that employs an anchor moiety that is immobilized on a solid support and specifically binds to at least a first epitope of
C. difficile
toxin A, and one or more detection moieties, each of which is conjugated to a detectable label and specifically binds to at least a second epitope of
C. difficile
toxin A. The assay for detecting
C. difficile
glutamate dehydrogenase can also involve a sandwich assay in which glutamate dehydrogenase is bound to an immobilized anchor moiety that specifically binds to at least a first epitope of
C. difficile
glutamate dehydrogenase, after which bound glutamate dehydrogenase is detected using one or more detection moieties, each of which is conjugated to a detectable label and specifically binds to at least a second epitope of
C. difficile
glutamate dehydrogenase. The anchor moiety that specifically binds
C. difficile
toxin A and the anchor moiety that specifically binds
C. difficile
glutamate dehydrogenase can be immobilized in separate zones on a single solid support, on separate supports, or can both be present in a single zone.
The invention also provides devices for detecting the presence of toxigenic strains of
C. difficile
in a test sample. The devices include a porous member having an upper and a lower surface, being positioned in the device such that the test sample may be applied to the upper surface, wherein a plurality of anchor moieties that are capable of specifically binding to
C. difficile
toxin A are immobilized in a first zone of the porous member, and a plurality of anchor moieties that are capable of specifically binding to
C. difficile
glutamate dehydrogenase are immobilized in a second zone of the porous member. The devices also include a non-absorbent member having a textured surface with channels capable of forming a network of capillary channels when placed in communication beneath or around the porous member, said-capillary network being substantially parallel to the lower surface of the porous member. The test sample, alone or in combination with other fluids,
Baskar Padma
Biosite Diagnostics
Smith Lynette R. F.
Townsend and Townsend / and Crew LLP
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