Clostridioides difficile infection, often referred to as C. diff, is a significant cause of infectious diarrhea, typically occurring after a person has taken antibiotics. The medication disrupts the normal balance of bacteria in the gut, allowing the C. diff organism to multiply and release toxins that damage the intestinal lining. When a patient experiences persistent, unexplained diarrhea—defined clinically as three or more unformed stools in 24 hours—a laboratory test is necessary to confirm the presence of the toxins or the bacteria itself and guide appropriate treatment.
Required Stool Quantity
The amount of stool needed for a C. diff test is small, but the quality and consistency of the sample are far more important than the volume. Most laboratories require a minimum volume in the range of 5 to 10 milliliters (mL) of diarrheal stool for testing. This quantity is roughly equivalent to two or three teaspoonfuls of liquid specimen, or a small scoopful of unformed stool.
The specimen must be unformed, meaning it takes the shape of the container it is held in, corresponding to Type 5, 6, or 7 on the Bristol Stool Chart. Formed or solid stool specimens are typically rejected because testing them often indicates colonization—the presence of the bacteria without active infection—rather than true disease. Insufficient quantity can lead to the sample being rejected or an inconclusive test result, necessitating a repeat collection.
Collection Methods and Best Practices
The process of collecting a stool sample must be performed carefully to ensure the specimen is clean and representative of the active infection. The first step involves passing the stool into a clean, dry receptacle that prevents contamination. Common collection tools include a special container, a bedpan, or a clean sheet of plastic wrap draped loosely under the toilet seat.
It is necessary to prevent the stool from mixing with urine or toilet water, as these contaminants can dilute the sample or interfere with the laboratory assays. If the stool is not entirely liquid, the collector should aim to select a portion that appears particularly watery, slimy, or bloody, as these areas are more likely to contain the highest concentration of the organism or its toxins. Using the provided collection spoon or a clean, disposable item, a small portion of the sample is then transferred into the sterile specimen cup.
After the sample is secured in the cup, it is important to screw the cap on tightly to prevent any leakage during handling and transport. Any external contamination on the cup itself can lead to rejection by the lab due to biohazard concerns. Immediately following the collection, proper hand hygiene using soap and water is necessary, especially since alcohol-based hand sanitizers are generally ineffective against C. diff spores.
Sample Integrity and Handling
Once the sample is collected, maintaining its integrity through proper handling is paramount, particularly because the C. diff toxin is unstable. The toxin can begin to degrade at room temperature in as little as two hours, which can lead to a false-negative result in toxin-based tests. Therefore, the specimen must be delivered to the laboratory as quickly as possible after collection.
If immediate transport is not feasible, the specimen must be stored correctly to preserve the toxin’s viability. Refrigeration at a temperature between 2 and 8 degrees Celsius is the standard recommendation for short-term storage. A refrigerated sample is stable for up to 24 hours, though some protocols allow for up to 48 hours. For longer-term storage, the specimen may need to be frozen at a much colder temperature, such as -20°C or -70°C.
Before submission, the container must be properly labeled with the patient’s full name, date of birth, and the exact date and time of collection. This labeling is a requirement for the laboratory to process the sample and link the result accurately to the patient’s medical record. A sample that is improperly sealed or labeled will often be rejected outright.
Understanding the Laboratory Tests
Sample quantity and handling are important due to the two primary methods laboratories use to test for C. diff. The first method involves Toxin Enzyme Immunoassays (EIA), which directly detect the toxins A and B produced by the bacteria. This test requires the toxins in the sample to be viable, which is why temperature and time are important for preventing toxin degradation.
The second common method is the Nucleic Acid Amplification Test (NAAT), often a Polymerase Chain Reaction (PCR) test. This test is highly sensitive and detects the genes that code for the toxins, meaning it searches for the bacteria’s DNA rather than the toxin protein itself. Because NAAT detects genetic material, the test is less affected by delays in transport or improper temperature, as DNA is much more stable than the toxin protein.
Many laboratories use a multi-step algorithm, often starting with a highly sensitive NAAT or a screen for the common C. diff enzyme Glutamate Dehydrogenase (GDH). While NAAT is excellent at detecting the presence of toxigenic bacteria, its high sensitivity sometimes detects colonization in patients who are not actively ill, leading to a potential over-diagnosis. The quality of the stool—specifically that it is diarrheal—remains the initial filter, ensuring the test is only performed when the patient is symptomatic and the result will be clinically meaningful.