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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 |
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