In detecting infections, are two rounds of polymerase chainreaction (PCR) better than one?

Molecular biologist Gregg Y. Lipschik, who heads a researchteam at the National Institutes of Health and colleagues atCopenhagen's Hvidovre Hospital, reported in The Lancet that"nested PCR could become the most cost-effective method ofdiagnosing P. carinii pneumonia."

In "nested" PCR, Lipschik explains, a DNA sequence amplifiedby PCR then goes through a second, more precise amplificationof a smaller segment, with different gene primers.

Today, AIDS patients suspected of harboring the deadlypneumonia germ must swallow a flexible fiberoptic hose thatsamples the lining of their bronchial tubes, or else must coughup pathogen-laden phlegm. Detecting P. carinii by theseconventional methods is quick, Lipschik told BioWorld, but maynot catch all possible cases and may lead to false-positivediagnoses.

The NIH group sampled 71 specimens of sputum and 113 ofbronchial-lining cells in a blinded study comparing nested PCRwith conventional staining. Of the 71 patients, 17 haddocumented P. carinii pneumonia, and nested PCR pinpointedall of them. Staining turned up only nine, or 53 percent. PCRcorrectly identified 50 of 54 negatives -- 93 percent.

But Lipschik also identified two shortcomings of nested PCR."For one thing, it now takes us 24 to 48 hours to do a detectionprocedure from start to finish, while staining results areavailable in two or three hours. Second, not many laboratoriesor hospitals are sophisticated enough to perform the PCR tests,while in Scandinavia they are almost commercial."

He added, "Routine clinical use of PCR-based diagnosis mustawait improvements in convenience and processing time."

Those betterments are here now, stated John J. Sninsky, seniorresearch director at Roche Molecular Systems of Alameda, Calif.Roche purchased the rights to PCR development and marketingfrom Cetus Corp.

"There is no need to do two amplifications," Sninsky toldBioWorld. "On the newest optimized machines andthermocyclers, one round takes only one and one-half hours;nesting would take three." He adds that "PCR by definitiondetects single molecules, so nesting raises the worry of morefalse positives."

But the Roche expert finds the NIH/Copenhagen study "anextremely valuable addition to detection of the array ofopportunistic infections that threaten HIV-infectedindividuals."

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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