November 2010
Feature Story

Michelle Merrigan

C. difficile testing is in a tough spot—caught between the convenient but relatively insensitive immunoassays and the sensitive but more expensive and labor-intensive real-time PCR assays. Now, a new assay approved by the FDA in July has the potential to bridge the gap.

The Illumigene assay from Meridian Bioscience uses a new type of gene amplification that makes it possible for any lab to perform the test, in less than one hour, without investing in RT-PCR instrumentation.

C. difficile culturing and subsequent cytotoxin testing is highly sensitive and the gold standard against which all new tests are judged. But the 24- to 48-hour turnaround time makes it antiquated in today’s hospital setting. The more manageable stool cytotoxin assay, or CTA, is sensitive, but slightly less so than culture.

More than 90 percent of laboratories in the U.S. use immunoassays for C. difficile detection. Their low sensitivity makes the positive predictive value about 0.75, and it only worsens with repetition (Peterson LR, Robicsek A. Ann Intern Med. 2009;151: 176–179). Many labs employ multistep algorithms, usually for the surface antigen glutamate dehydrogenase, or GDH, followed by further screening with CTA or culture (Fenner L, et al. J Clin Microbiol. 2008;46:328–330). But few like the algorithms because GDH is not a sensitive screen. Christine Biggs, SM(ASCP), microbiology supervisor at Chester County Hospital in West Chester, Pa., says she and her colleagues had consistent problems with their GDH screen before abandoning it.

“The GI physicians would call me and say, ‘This patient clinically has C. diff. Can you run the ToxAB assay anyway?’ And we would do that, and we had a number of consecutive positives on their patients.” That was enough for the physicians to say, “We don’t want you to use the [GDH] antigen anymore.”

Further, hospital regulations often stipulate that labs can’t offer only a bundled choice without offering the tests singly. Paul Lephart, PhD, associate technical director of microbiology at Detroit Medical Center University Laboratories, says: “It just makes it confusing for the physicians. It would be better for the physicians to have one choice rather than offering a good choice and a not so good choice and hoping they know the difference.”

Given these limitations, the industry has been moving toward single molecular tests for all samples that detect the presence of C. difficile DNA in stool (see CAP TODAY, May 2010). “Nationwide, people are going to some form of molecular testing for C. diff,” Biggs says. Real time-PCR tests for C. difficile are on the market now: BD’s GeneOhm, Gen-Probe Prodesse’s ProGastro Cd assay, and Cepheid’s Xpert.

The Illumigene C. difficile assay employs a different type of nucleic acid amplification from standard PCR called loop-mediated isothermal DNA amplification, or LAMP. In standard PCR, primers anneal to the single strands of a melted DNA template, and polymerase replicates the strands. Thermal cycling repeatedly melts the DNA and allows for exponential amplification. LAMP is performed at a constant temperature of about 63°C and relies on the strand-displacing activity of the DNA polymerase. In PCR, one product is made. In LAMP, the action of multiple primers results in the amplification of a “cauliflower” structure, a complex of stem-loops. The amplification is rapid—it can be done in under an hour.

The Illumigene C. difficile assay uses a set of six primers directed to the highly conserved toxin A gene sequence in the PaLoc pathogenicity locus of C. difficile. C. diff strains that produce toxin B and not toxin A (A–B+) have been reported and caused complications previously in C. difficile testing. However, A–B+ strains still carry a target sequence for the Illumigene assay that can be detected in all the A–B+ strains described so far.

In standard PCR, the amplified product is detected by gel electrophoresis, or by the interaction of fluorescent probes or dyes in the case of real-time PCR. In the LAMP amplification reaction, there is a buildup of magnesium pyrophosphate, which forms a white precipitate, leading to a turbid reaction solution. The accumulation of turbidity indicates a positive amplification.

Sample preparation takes 15 to 20 minutes and does not require a centrifugation step. The stool sample is collected on a brush, which is placed in a filtering apparatus with diluent and then vortexed. The sample is filtered through the apparatus cap by squeezing the tube, and the filtrate is transferred to a new extraction tube with reaction reagents. After heating at 95°C for 10 minutes, the sample is ready for application to two conjoined reaction tubes, one containing C. difficile-specific primers (the test chamber), the other containing S. aureus primers (the control chamber). Says Biggs: “I liked the internal control with each patient. I think that was a smart design.”

In the Illumigene assay, the amplification and detection are performed in one instrument, the Illumipro-10 incubator/reader. The machine allows up to 10 samples per run. Time to completion is about 40 minutes. Each kit contains 50 tests and comes with positive and negative controls.

Kirk Doing, PhD, director of clinical and molecular microbiology at Eastern Maine Healthcare Systems, says his laboratory did a 450-sample head-to-head comparison of Illumigene and the Prodesse ProGastro Cd assay, which it has used for more than a year. It found 98 percent agreement using toxigenic culture as the gold standard.

At Chester County Hospital, Biggs’ lab did a validation test with 68 specimens, comparing Illumigene with the lab’s algorithm of a GDH screen followed by the ToxAB immunoassay. The result, Biggs says: “A false-negative rate of approximately 10 percent in our current C. difficile testing algorithm.”

The Illumigene trial in Dr. Lephart’s laboratory found the same: The immunoassay was missing true positives in about 10 percent of the total samples.

With Illumigene, Dr. Doing says, the laboratory has to run an external extraction control with every run, so Illumigene cannot be used to test samples as they arrive in the lab. “It’s still a batch test. Only Cepheid offers true random testing capability,” he notes, adding that the need for the extraction control “adds significantly to the cost per reportable.”

One of the main advantages of Illumigene is not having to spare time and personnel from the molecular lab to do C. difficile testing. “It’s definitely advantageous to keep it in the main lab,” Biggs says. “My molecular room is in a different building. I don’t always have someone to pull out to do that testing,” particularly on second or third shift. Dr. Lephart, who says the molecular technologists at Detroit Medical Center University Laboratories “are busy doing HIV tests,” says C. diff testing with Illumigene is done in the virology lab, “because traditionally that’s where cytotoxicity testing was done.” There is also the time savings that comes with a more efficient workflow. Detroit Medical was using the ToxAB immunoassay with molecular confirmation, if necessary, and planning at CAP TODAY press time to transition all such testing to Illumigene.

Now that there are new options, hospitals may face a culture change over when C. difficile tests are ordered and how physicians respond. One disadvantage to low-sensitivity tests is that when doctors doubt results, they treat empirically. Alternatively, they order multiple tests, a practice many labs have rules against. To avoid the problem of physicians ordering repeats of tests for which the results are negative, Dr. Lephart says, “We are going to battle that with lots of physician education.” Dr. Doing says the laboratory at Eastern Maine accepts only one sample in a seven-day period: “We showed that with PCR testing, testing multiple samples is of no value to the patient.”

Molecular tests also face a change in how they are interpreted. A positive molecular test indicates only the presence of the organism, not of disease. “It’s a paradigm shift,” says Dr. Lephart. He and Dr. Doing say this will be dealt with on the leading end by implementing careful pre-test criteria.

Despite having difficulties with CPT codes and getting some payers to cover the Illumigene assay, Biggs says the Chester County Hospital laboratory will probably bring the test in, saying “It’s the right thing to do.”

“The pathologists are in favor of this method, and the GI doctors are salivating over this—to finally get good information.”