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E W FEATURE 6 7 sensitivity of all diagnostic tests," he said. "The use of a commercially available kit for nucleic acid extrac- tion and/or the PCR reaction itself can render a rather complicated process more standardized and easier to optimize." In ophthalmology, where samples are commonly lim- ited in quantity, he observed, PCR testing could produce a significantly higher yield than traditional cul- tures. Regis P. Kowalski, MS, (M)ASCP, research professor of ophthalmol- ogy, University of Pittsburgh, and executive director of the Charles T. Campbell Ophthalmic Microbiology Laboratory, explained that PCR kits rely on primers to work. "If there is a piece of pseudomonas DNA, for example, the primers would go in there, then using a machine, you would go through many cycles and keep on replicating the DNA until you got a lot of DNA," he said. This way it becomes possible to amplify a minute amount of DNA. Such kits are often directed at specific DNA. For example, a PCR kit to identify MRSA would look for the mecA gene, with primers directed specifically to amplify that, Mr. Kowalski said. Renda L. Hawwa, PhD, scientific affairs liaison, IntelligentMDx, Waltham, Mass., explained how the I MDx kit to identify herpes simplex virus (HSV) 1 and 2 works. "The assay utilizes PCR to generate ampli- fied product from the glycoprotein D and UL -30 genes of HSV-1 and HSV-2, respectively," Dr. Hawwa said. This kit, which is CE-marked, is not validated on corneal ulcers, she said. It is intended to be used on s ymptomatic patients such as those displaying a genital or oral lesion suspected to be HSV. She further explained how the kit works: "The assay includes probes that will bind to an amplified HSV product, if it is present. A fluo- rescent signal is then generated and the cycle that the signal is generated at will be inversely proportional to how much HSV is present in the sample." The sensitivity of PCR tests can also be an unexpected drawback, said Dr. Jeng. "The downside of this is that because they're testing for DNA, dead organisms or contami- nant organisms also have DNA," he said, adding that practitioners po- tentially may pick up something that is not growing and begin treat- ing the patient for the wrong thing. Still, for certain infections PCR testing holds particular promise, said Dr. Iovieno. "The patients who can benefit most from molecular diag- nostic techniques are the ones af- fected by corneal infections that are difficult to detect with common cultures such as Mycobacteria, or for whom a rapid diagnosis can result in a more favorable clinical outcome," h e said. For example, those with Acanthamoeba, he thinks, may do better if they are diagnosed more promptly. Mr. Kowalski agreed that for Acanthamoeba, PCR may be promis- ing. However, the jury is still out, he stressed, on whether it is better to culture this. He also thinks that PCR i s very important for varicella zoster virus (VZV) diagnosis and HSV. "For every patient that we're thinking VZV, we do PCR," Mr. Kowalski said. This, he explained, is hard to cul- ture. PCR is the better test for HSV, although this one does well in cul- ture. However, he stressed that for bacteria it is just not practical. "There are too many bacteria that can cause keratitis," Mr. Kowalski said, explaining that it would re- quire use of too many different primers looking to amplify different genes. He pointed out that it's expen- sive. This is not something that just any lab can do. "Everything needs to February 2011 Poll size: 249 EyeWorld Monthly Pulse EyeWorld Monthly Pulse is a reader survey on trends and patterns for the practicing ophthalmologist. Each month we send a four-question online survey covering different topics so our readers can see how they compare to our survey. If you would like to join the hundreds of physicians who take a minute a month to share their views, please send us an email and we will add your name. Email carly@eyeworld.org and put EW Pulse in the subject line—that's all it takes. Copyright EyeWorld 2014 HSV keratouveitis complicated by secondary Pseudomonas aeruginosa sclerokeratitis Source (all): Alfonso Iovieno, MD continued on page 68 February 2014 Corneal infections 56-68 Feature_EW February 2014-DL2_Layout 1 1/30/14 10:45 AM Page 67