By: Heather Williams

 

The Cyclospora outbreak is hitting Texas hard, with more cases in this outbreak than previously experienced.  Salmonella in Papayas is equally concerning, with the potential for the link to be established with more states and more brands. Oyster lovers everywhere are concerned about the latest Vibrio outbreak to hit Seattle. Even Chipotle is again wrapped up in a Norovirus outbreak. Whether you are concerned about these particular outbreaks or one that may occur in the future, here are a few things to help you understand the diagnosis process.

The Process

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 is time to see your doctor.  To diagnose your illness, the doctor will ask you for the unthinkable – a stool sample.

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, trained professionals 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.

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 without mechanical assistance.  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 takes between a few days to weeks to occur.  Infection occurs when the sporulated oocysts are ingested by another host.  Lab tests can identify both sporulated and unsporulated forms of the oocysts.

Microscopy

Microscopy is a test to visually identify the cells in the sample.  This can be performed by traditional wet mount, on a stained slide, or with UV fluorescence

Traditional wet mount microscopy is performed by placing the specimen on a slide and adding a few drops of a saltwater solution to suspend it. The technician scans over every part of the sample looking for cells of interest.  This requires knowledge of the potential cells the technician is looking for and a good eye.

Stained slides are a more permanent slide method.  This allows the technician the ability to archive the sample or submit for further consultation without losing the sample.  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 also similar to traditional wet mount microscopy in the way the sample is added to the slide, however it is analyzed by looking at the sample under a UV light.  Filters can be set at various wavelengths to identify organisms.  Organisms reactive differently from each other under UV light, so the type of organism can be identified based on its color with different filters.  For example, Cyclospora oocysts light up an intense blue color when observed with a UV excitation filter set at 330-365 nm.

Molecular Testing

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.

Prior to PCR testing, 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 method practiced that particular laboratory has approved for extracting DNA.

Once a clean DNA sample is obtained, it is analyzed against a panel of organisms expected to be in the sample.  Different reagents 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 in the test tube by providing the building blocks needed to replicate and tricking the DNA to replicate repeatedly 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 contents of 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

Flow Cytometry is a method of identification that is growing in popularity.  While the preparation of the sample takes about as long or slightly longer than microscopy analysis, the analysis time 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.

Flow cytometry is a new method of analyzing cells to “type” them.  Using flow cytometry, scientists are looking to identify the target cell, in this case Cyclospora oocysts, in the sample.  A control is used to compare each cell with what the target cell is supposed to look like.  This means that scientists test a known sample alongside the tested sample for comparison.

To begin, the stool sample will come to the lab 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 method your doctor requests, you should have your results with a few days.  Both you and your doctor can feel confident in the treatment options performed in response to knowing exactly which pathogen you have been infected with.

 

 

Sources:

http://jcm.asm.org/content/43/5/2375.full

https://www.cdc.gov/dpdx/diagnosticprocedures/stool/moleculardx.htm

http://www.abcam.com/protocols/introduction-to-flow-cytometry

https://www.cdc.gov/dpdx/diagnosticprocedures/stool/microexam.html