|"This method is not foolproof, but it's one way of knowing that you've at least got a single strain of an organism, which is absolutely essential for downstream studies. "
In the March issue of Emerging Infectious Diseases, CHORI clinical scientist Deborah Dean, MD, MPH, reports on a novel shotgun cell culture harvest assay she and her colleagues have developed which has the potential to revolutionize the field of Chlamydia research.
The leading cause of preventable blindness and bacterial sexually transmitted diseases in the world, Chlamydia trachomatis is a global epidemic. Over the last 20 years, Dr. Dean has dedicated herself to better understanding the obligate intracellular organism in the hopes of eventually developing a vaccine.
"Being able to precisely identify different strains and their impact on the human host will eventually allow us to develop a vaccine that's going to have maximum global impact," says Dr. Dean.
It's that precise identification, however, that's been vexing many an investigator, including Dr. Dean. Traditional assays to isolate individual Chlamydial strains from mixed infections have relied on the behavior of laboratory-adapted reference strains, in which cells infected with such strains form single, isolated plaques. Researchers like Dr. Dean have found, however, that samples of C trachomatis strains taken directly from human populations, behave in very different ways than laboratory reference strains.
"We desperately needed to develop a new way to segregate clonal populations, because if you aren't able to do this, you could unknowingly have a mixed population that is going to impact any results from downstream studies. It's absolutely essential to know what you're dealing with," says. Dr. Dean.
|"With our clinical isolates, we could find very few single, isolated plaques and that was for a distinct minority of clinical isolates - and that meant we couldn't use plaque assays, but needed to develop a new technique for isolating single strains of chlamydiae from each isolate."
Instead of using traditional plaque assays, Dr. Dean and her colleagues used microscopic assessment to identify infected cells, harvested those areas which appeared to show circumscribed infection, and stained what remained in the wells (where the cells had been infected) to determine if the the harvested cells represented an isolated infection, likely representing a single clonal population of a strain.
"Once we figured out the technique, we were off and running. But it took tons of trial and error over a couple of years," Dr. Dean says. "It was very simple, but it took a breakthrough to get there."
And it's a breakthrough of no small measure. With more and more evidence mounting that Chlamydial infections are rarely caused by single strains, being able to isolate purified samples is of the utmost importance. In fact, the results that Dr. Dean's shotgun assay have yielded underscore the necessity of the approach.
"With this central technique we started identifying strains we didn't even know existed," Dr. Dean says. "In this study alone we have a trachoma isolate taken from a patient in 1959 - over 50 years ago - and it ended up containing two different species, one of which had never been detected in the eyes of trachoma patients but is extremely important in regards to the different pathogens causing blinding eye disease.
Dr. Dean suspects that such results are just the tip of the iceberg, and the only way to unearth what's beneath is through being able to absolutely purify out the different strains from clinical samples. While the novel technique is expensive and time consuming, it represents a ground-breaking shift in research capabilities that will gain momentum from hereon out.
Thanks to Dr. Dean's efforts, researchers now have the capability of knowing with confidence exactly which strains they've isolated, so they can then identify exactly how those individual strains correlate with clinical populations - a critical step in ongoing vaccine development.
Tuesday, May 17, 2011 8:19 AM