College of American Pathologists
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  ‘One of the most challenging PCR
  tests to develop’





cap today

October 2005
Feature Story

A rapid polymerase chain reaction assay for direct detection of MRSA in nasal swabs “is one of the most challenging PCR tests to develop,” says Franklin Cockerill III, MD, chair of microbiology and director of bacteriology, Mayo Clinic. “You want a nucleic acid method that picks out MRSA from mixed infections with methicillin-sensitive S. aureus plus coagulase-negative staph [CNS] carrying the mecA gene, which could give you a false-positive result.”

A molecular route to solving this conundrum arose from basic research by Japanese bacteriologist Keiichi Hiramatsu, PhD, of Juntendo University in Tokyo, who discovered that the beta-lactam (methicillin) resistance gene mecA of S. aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec) (Ito T, et al. Antimicrob Agents Chemother. 2001; 45: 1323–1336). Dr. Hiramatsu initially described three predominant MRSA clones in the world with different types of SCCmec in their chromosome. However, all three mobile genetic elements are closely related: They all carry an identical mec gene as well as genes for recombinases that excise the mobile element from the chromosome of one bacterium and insert it into another. Since CNS carries genetically similar SCCmec mobile elements, a critical finding for devising an effective PCR assay was that all three mobile genetic elements in S. aureus insert into the same unique site (orfX) in the bacterium’s chromosome, a site that has not been reported for SCCmec insertion into the genome of CNS. Since the original publication by Dr. Hiramatsu’s group, five major types and several subtypes of SCCmec mobile elements in S. aureus have been reported.

In the Geneohm/IDI real-time PCR assay, as well as the in-house assay Dr. Cockerill’s research group is developing, PCR primers and probes are used that cover the S. aureus-unique insertion site for the SCCmec cassette as well as unique sequences in the SCCmec cassettes. Ironically, this approach does not detect the mecA per se. However, a positive result should indicate that a MRSA strain is present. “The potential risk of such an assay is twofold,” Dr. Cockerill says. “One, false-positives may occur. Although this is likely a rare occurrence, a recent report indicated that a nosocomial S. aureus strain contained SCCmec sequences but the mecA gene was not present (J Antimicrob Chemother. 2004;54: 229). Two, false-negatives may occur due to novel SCCmec types that are not covered by current assays.” But this is a screening test, he says, “so if you can cover 95 percent of possible MRSA strains, that would be pretty good.” He tested his assay against 462 MRSA isolates. “We only missed five out of 462 patients,” Dr. Cockerill says, for a 99 percent sensitivity. “I think that’s pretty darned good.”

—William Check, PhD