By: Heather Williams
It seems like every couple of weeks a new foodborne outbreak is hitting the news. Right now, Cyclospora is affecting Texas. In fact, there are more cases in this current outbreak than Texas has previously experienced for this pathogen. Whether you may be concerned about this particular outbreak, or one in the future, here are a few things to help you understand the process your doctor may use to diagnose your illness.
You’ve seen the news, your stomach hurts, you have diarrhea, and all other symptoms on the health alert. You also know you have consumed a food item on the watch list for the outbreak. If you are feeling really bad, it might be time to see your doctor. To diagnose your illness, the doctor will ask you for the unthinkable. A stool sample. I know. It isn’t something you were planning on doing this afternoon.
Your doctor will ask you to deposit your specimen in a cup. That is not necessarily an easy task, as we are not quite programmed to do this behavior. And don’t worry, the trained professionals that will be analyzing your sample have seen it all. The texture, color, etc, will be noted before anything is done with the sample in an initial examination, so just allow your business to “go with the flow” and don’t mess with it once it is in the cup.
But what happens after you provide your sample? Doctors could order a variety of tests to check for the pathogen. Several different technologies can be used to achieve the same final answer. The doctor could request a microscopic examination, PCR analysis, or flow cytometry to determine if your sample contains the pathogen. Your sample will make its way to a lab where it will be tested.
Preparing the Sample
After initial examination, your sample will be processed to isolate the cells and depending on the test request, isolate DNA from the sample. In the case of Cyclospora, scientists are generally looking for the transmission phase of the pathogen, the oocyst. The oocyst is a spherical cell around 8 to 10µm in diameter. This is very tiny and cannot be seen with normal eyesight. These oocysts are passed into the environment in the feces of infected individuals. To become infectious, they undergo sporulation, a process where the cell wall hardens and becomes hardier. This may take anywhere from a few days up to a few weeks to occur. Infection takes place when the sporulated oocysts are ingested by another host. Lab tests can identify both sporulated and unsporulated forms of the oocysts.
Microscopy is a test used to visually identify the cells in the sample. This can be performed in a couple of different ways. For example, by traditional wet mount, on a stained slide, or with UV fluorescence
Traditional wet mount microscopy is performed by placing the specimen on a glass slide and adding a few drops of a saline to suspend it. The technician scans over every part of the sample looking for cells of interest.
Stained slides are similar to traditional we mount, but are a more permanent slide method. This allows the technician to archive the sample or submit for further consultation without losing the exact sample he or she is looking at. The sample is added to the slide in the same way as a traditional wet mount microscopy, however instead of looking at the slide immediately, a dye and fixative is added. This varies from specimen to specimen depending on what the technician is expecting to see on the slide.
UV fluorescence microscopy is another method similar to traditional wet mount microscopy in that the sample is added to the slide with a few drops of saline solution, however it is analyzed by looking at the sample under a UV light. Filters can be set at various wavelengths to identify particular organisms. Organisms react differently from each other under specific wavelengths of UV light, so the type of organism can be identified based on its color under different filter parameters. For example, Cyclospora oocysts light up an intense blue color when observed with a UV excitation filter set at 330-365 nm.
Molecular testing such as Polymerase Chain Reaction (PCR) is another method to confirm the presence of the pathogen. This method analyzes the DNA found in the sample and can be a follow up to confirm diagnosis after traditional microscopy indicates presence of the pathogen.
Before the sample can be analyzed using PCR, DNA from the sample must be extracted. There are a variety of ways to extract DNA from stool samples. This can be performed with commercial DNA extraction kits, cell lysis and organic DNA cleaning, or any other approved method the laboratory has in place for extracting DNA.
Once a clean DNA sample is obtained, it is analyzed against a panel of organisms expected to be in the sample. Specific ingredients are added to the sample based on which organisms are being tested against. PCR works by prompting the DNA to make copies of itself over and over again so the scientist can see more of what is in the sample. This is done by providing all of the building blocks needed to make copies the DNA and tricking it into replicating over and over again by changing the temperature of the sample in cycles. From just a small amount of DNA, thousands of copies can be made, allowing the technician to analyze the DNA in the sample. This analysis can be done on traditional gel electrophoresis where the replicated DNA sample is added to an agarose gel, an electrical current is applied, and the bands of DNA separate according to size on the gel. The bands represent size of DNA sections and are compared to a standard for each organism being analyzed. It can also be done with fluorescent dyes in a Real-time DNA instrument, where fluorescent dye is added to the PCR mix and incorporated into the sample. The instrument analyzes the amount of fluorescence and the type of fluorescent dye that is associated with each type of organism tested to identify the presence and type of pathogen in the sample.
Flow Cytometry is a new method of identification that is growing in popularity for pathogen identification. While the preparation of the sample takes about as long or slightly longer than microscopy analysis, the analysis time per sample is much faster. With a more automated analysis, more samples can be tested quickly and technician level of expertise and fatigue are not factors in the outcome, as the instrument does all of the work.
Flow cytometry is a new method of analyzing cells to “type” them based on the characteristics of the cell. Scientists can use flow cytometry to identify the target cell, in the case Cyclospora, the scientist is looking for oocysts in the sample. A known positive sample is tested alongside the unknown sample as a control. This allows the scientist to compare to each cell with what the target cell is supposed to look like.
When the stool sample comes to the lab, it will be fixed in SAP. Cells in the sample are stained, often with a fluorescent dye, and introduced into a fluid to be put in the instrument. The cells flow into the cytometer and funnel into a single cell path. Beams of laser lights are aimed at each cell, sending data to a reader. The reader looks for fluorescence emitted from the stained cells, and the way the light scatters from the cells after it is hit. Each cell type often has a unique signature. The results of the samples are compared to the results of the control sample to determine if the pathogen is present in the sample.
Whatever test your doctor chooses to use to diagnose your illness, your results should be available in a few days. Both you and your doctor can feel confident in the treatment options performed by knowing exactly which pathogen you have been infected with.