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Detection of toxigenic Clostridium difficile in stool specimens by the polymerase chain reaction
1Nosocomial Pathogens Laboratory Branch, Centers for Disease Control, Atlanta, Georgia.
Abstract:
Polymerase chain reaction (PCR) amplification of a segment of the toxin A gene was used to detect toxigenic Clostridium difficile directly from stool specimens of patients with antibiotic-associated diarrhea. Although PCR-inhibitory substances were recognized in DNA prepared from stool specimens, the inhibitory substances were eliminated by using an ion-exchange column after phenol-chloroform extraction. Eventually, 39 stool specimens were evaluated by PCR. PCR results for detection of toxigenic C. difficile were in complete agreement with cell culture assay results; all 12 PCR-positive stool specimens were positive by cytotoxin assay, and all 27 PCR-negative specimens were negative by cytotoxin assay. Toxigenic C. difficile was cultured from all PCR-positive specimens. These results suggest that PCR amplification may be an effective method for laboratory diagnosis of C. difficile-associated diarrhea and colitis.
Insights
Polymerase chain reaction (PCR) effectively detects toxigenic Clostridium difficile in stool samples. This method shows complete agreement with cell culture, offering a reliable diagnostic tool for C. difficile-associated diarrhea.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Antibiotic-associated diarrhea is frequently caused by Clostridium difficile.
- Accurate and rapid laboratory diagnosis is crucial for effective patient management.
- Traditional diagnostic methods can be time-consuming or lack sensitivity.
Purpose of the Study:
- To evaluate the utility of Polymerase Chain Reaction (PCR) for direct detection of toxigenic Clostridium difficile in stool specimens.
- To assess the performance of PCR compared to conventional cell culture and cytotoxin assays.
- To determine if PCR can overcome inhibitory substances present in stool samples.
Main Methods:
- DNA extraction from stool specimens followed by phenol-chloroform extraction.
- Purification using an ion-exchange column to remove PCR inhibitors.
- PCR amplification targeting the toxin A gene of Clostridium difficile.
- Comparison of PCR results with cell culture and cytotoxin assay findings in 39 stool specimens.
Main Results:
- PCR successfully detected toxigenic Clostridium difficile directly from stool.
- PCR results demonstrated complete agreement with cell culture and cytotoxin assay results.
- All 12 PCR-positive specimens were confirmed positive by cytotoxin assay and culture; all 27 PCR-negative specimens were negative.
Conclusions:
- PCR amplification is a sensitive and specific method for detecting toxigenic Clostridium difficile.
- The developed PCR method effectively eliminates inhibitory substances from stool DNA.
- PCR offers a potentially effective and rapid laboratory diagnostic approach for C. difficile-associated diarrhea and colitis.