Laboratory Diagnostic Approaches

Because many hospitalized patients are asymptomatic carriers, testing patients for C. difficle infection is only recommended if diarrheal stools are present.[12] Laboratory diagnosis is performed by detection of toxigenic C. difficile by culture, immunodetection of toxin, or cytotoxicity assay.[18]

Stool cultures on selective cycloserine-cefoxitin-fructose agar can yield to C. difficile isolation in 2-3 days. Unfortunately, culture is not specific for toxin-producing strains and is seldom used for clinical diagnosis.[5] However, isolated strains provide reliable bacteriological information for epidemiological purposes.

Cytotoxicity assay, which detects cytopathic effects of toxin B on mammalian cells, is the most sensitive and specific test available. It is considered the gold standard test to identify C. difficile toxins directly from the stools of infected patients. A filter-sterilized fecal suspension is added into a mammalian cell culture and monitored for the appearance of cytopathic effects caused by disruption of the cytoskeleton. In parallel, a control fecal filtrate supplemented with a specific antiserum neutralizing for clostridial cytotoxin is used to demonstrate the specificity of cytopathic changes. The drawback of this assay is that it is relatively slow (24-48 hr) and requires tissue culture expertise and facilities. Some authors suggest that performing cytotoxin assay directly on stools, as well as by culture followed by detection test for toxin produced in vitro, might improve the detection of patients harboring toxigenic strains.[18]

Enzyme immunoassays (EIAs) for toxin detection are now widely available from several manufacturers. These tests are rapid and simple to perform directly from stool samples. Most assays use monoclonal antibodies against toxin A. However, because some clinical isolates have been shown to produce only toxin B and still cause the disease, more recent EIAs combine the detection of both toxins A and B. However, none of the EIAs is as sensitive as the cytotoxicity assay.[19] This is why in the event that EIA testing is negative and diarrhea persists, it is recommended to repeat EIA testing on a second sample and even a third sample from patients suspected of infection by toxigenic C. difficile.[5]

Several sensitive and specific molecular diagnostic methods based on detection of toxin genes directly from feces have been proposed.[20-24] However, most of these tests targeted either only the gene for toxin A, or the gene for toxin B. Therefore strains belonging to toxinotypes altered or even deleted in the primer regions could be missed. In all cases, these assays where developed in classical PCR formats requiring time-consuming and contamination-generating post-PCR manipulations to identify the amplification products. For clinical laboratories, the potential risks of false-positive reactions due to carryover of previously amplified genetic materials render PCR assays relying on agarose gel electrophoresis impractical for analysis of amplicons. In addition, the PCR-inhibitory substances inherently present in feces specimens suppose an efficient sample preparation procedure to remove these inhibitors without having to sacrifice the sensitivity of the assay. Most of these procedures add complex technical steps that are time-consuming. One exception is the cleverly designed immunomagnetic preparation procedure of Wolfhagen et al.[24] However, it had not been tested on many specimens.

Recently, our team published the first real-time PCR assay for the detection of toxigenic C. difficile in feces.[25] This test targets both toxin genes and is a closed-tube assay. This means that amplification and detection of nucleic acids are performed in the same tube, therefore minimizing human intervention and reducing risk of contaminating laboratory surfaces with amplified material. The closed-tube assay format is a major improvement, which will permit a more general acceptance of PCR testing in clinical settings. Our test uses a rapid and simple sample preparation protocol requiring hands-on technician time of around 15 min, which is similar to EIA assays used for C. difficile. Target sequences for primers and probes were chosen by identifying conserved regions within tcdA and tcdB gene sequences available in GenBank. The tcdA and tcdB amplicons are 158 and 101 bp, respectively. The two molecular beacon probes were labeled with different fluorophores, hence allowing multiplex PCR and simultaneous detection of the two toxin genes. The use of two target genes increases assay robustness by maximizing the likelihood of detecting at least one toxin gene if the other presents variations in the primers or probe region. The 45-cycle PCR reaction took 45 min and the fluorescent signal was acquired in real time at each cycle on a Smart Cycler (Cepheid, CA) apparatus. Total time to response was less than 1 hr from reception of samples into the laboratory. The detection limit was 5 x 104 CFU/ g of stool. When compared to cytotoxicity testing, the real-time PCR assay demonstrated a specificity and a positive predictive value of 100%, a sensitivity of 97%, and a negative predictive value of 96%. Further validation of the test in a broader clinical trial is, of course, required. However, such specific, sensitive, and especially rapid identification and detection of toxigenic C. difficile may improve the management of patients and facilitate a more rational use of antibiotics.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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