In the 78th Edward Jackson Memorial Lecture, presented during Friday’s Opening Session, Russell N. Van Gelder, MD, PhD, presented an overview of molecular diagnostics for ocular diseases.
Old options for a new century. In reflecting on Dr. Jackson’s legacy, Dr. Van Gelder wondered what Dr. Jackson would think if his ghost were to return to wander the halls of today’s annual meeting.
While some innovations would undoubtably carry a whiff of science fiction, other techniques would be “terribly familiar” to Dr. Jackson, Dr. Van Gelder said. For instance, when today’s clinicians manage a presenting corneal ulcer, they start by taking a sterile swab and spatula — and then scrape the infectious material, streak it on plates, and smear some of it on a microscope slide for a gram stain. “These techniques, which we still do today, date from the 1880s.”
Moreover, Dr. Van Gelder said, “Like Dr. Jackson, we would consider ourselves fortunate if anything actually grew from those cultures that might guide our therapy.” In addition, he noted, “Many of our strongest antibiotic options are well over 50 years old. It would seem that our technology in ocular infectious diseases has not kept up with other aspects of ophthalmic practice.”
Enter molecular diagnostics. “The detection of genetic material of the pathogen is a proxy for the presence of that organism in the tissue of interest and, thus, is evidence of infection,” Dr. Van Gelder said.
PCR—the enzymatic method for replicating DNA in a test tube — is the primary technique. PCR is “nanotechnology at its finest,” Dr. Van Gelder said, and “it immediately changed how we all did molecular biology” when it was introduced.
The original PCR technique had to be run separately for each suspected pathogen, and it yielded a “yes or no” answer. Subsequently, Dr. Van Gelder and his colleagues adopted and developed techniques that both enabled “multiplex querying for multiple pathogens simultaneously and utilized real-time quantitative methods to allow estimation of infectious load.”
One case in particular — involving a novel fungus that was not in the standard databases — changed the direction of Dr. Van Gelder’s research. In what he described as “a big save,” the patient was empirically treated with an antifungal drug and ultimately recovered 20/25 vision. The case prompted Dr. Van Gelder to wonder how many other culture-negative cases actually harbored novel organisms. Indeed, in one study that he coauthored, 14 of 46 corneal ulcer specimens assessed with PCR were found to contain previously unknown or unusual organisms.1
One drawback of PCR? “You have to know what you’re looking for,” Dr. Van Gelder said. Fortunately, more recent sequencing techniques have allowed for “shotgun metagenomics.” In this approach, researchers sequence all the DNA in a biopsy sample and then use computer algorithms to find the needle in the haystack—“that is, the foreign DNA that may be hidden in a great mass of human sequence.”
Looking ahead. It’s fun to speculate about the future, Dr. Van Gelder said, and one next-generation advance on the horizon is point-of-service sequencing. Dr. Van Gelder expects this to be possible using a handheld DNA sequencer. The sequencer, which is the size of a large USB dongle, is capable of generating a billion base pairs of DNA a day for less than $100 per sample. Dr. Van Gelder’s lab is now developing a real-time shotgun metagenomics platform for point-of-service diagnosis with the sequencer. “Ultimately, we’d like to be able to rapidly extract the DNA from a corneal sample and read out the causative organism within minutes.”
The researchers are quite close to this goal, Dr. Van Gelder said. He added that if Dr. Jackson were watching, “I suspect he would be pleased—and maybe even a little impressed.”
—Jean Shaw
1 Kim E et al. Am J Ophthalmol. 2008;146:714-723.
Financial disclosures: Dr. Van Gelder—NEI: S; Research to Prevent Blindness: S.
Disclosure key. C = Consultant/Advisor; E = Employee; L = Lecture Fees; O = Equity Owner; P = Patents/Royalty; S = Grant Support.