College of American Pathologists
Printable Version

  PCR assays gear up to rule H1N1 market


CAP Today




December 2009
Feature Story

Anne Paxton

One of the difficulties of grappling with the H1N1 influenza virus has been getting a fix on the numbers. Since the pandemic was first declared in April, how many cases of swine flu have occurred? How many cases have led to hospital admissions?

More than seven months into the pandemic, the nation doesn’t know for sure, and a key reason has been the limited availability of testing that can definitively diagnose H1N1.

Under guidelines of the Centers for Disease Control and Prevention, hospitals and clinics dealing with suspeted cases of H1N1, a subtype of influenza A, can diagnose the disease based on symptoms and observation alone. The CDC does not require a subtype test that would identify H1N1.

“The system is really overwhelmed,” says Stephen Nichols Jr., MD, director of microbiology at Cedars-Sinai Medical Center in Los Angeles. “There’s so much testing that could be done, it’s difficult to say who should get tested.” Another factor the CDC has found: the poor sensitivity of most currently available rapid tests when it comes to H1N1.

The World Health Organization, in its 2009 “European Guidance for Influenza Surveillance in Humans,” calls RT-PCR (reverse-transcriptase polymerase chain reaction) “the only rapid diagnostic test for which a positive result is accepted as confirmation” of H1N1. “Molecular testing is becoming the test of choice to diagnose influenza,” says Jay M. Lieberman, MD, medical director of infectious diseases at Quest Diagnostics.

Diagnostic testing companies are stepping up to the task. Quest was the first to launch its laboratory-developed PCR test for H1N1, within two weeks of the announcement last April of the H1N1 pandemic and publication of the virus’ genetic sequence. Only the CDC and public health laboratories were using those PCR tests starting in April. “But the demand outstripped their capacity,” Dr. Lieberman says. “The Food and Drug Administration approached us about our lab-developed test, resulting in our submitting data on the test’s performance and granting of emergency authorization to use it in the U.S.”

The FDA granted an emergency use authorization, or EUA, to Quest sub­sidiary Focus Diagnostics for its H1N1 PCR test in July. Two and a half months later, the FDA granted an EUA to Focus for its PCR-based Simplexa Influenza A H1N1 test kit for use on the 3M Integrated Cycler. Under the EUAs, the test is authorized only for the duration of the emergency, which current law has set to expire April 26, 2010, unless it is renewed.

“The first EUA allowed us to start selling test kits to CLIA high-complexity labs. We recognized there would be a lot of demand for the test and we needed to make it more available. The most recent EUA was for the next generation of the test, the Simplexa, specifically to be performed on the 3M instrument, and we just started selling that in October,” Dr. Lieberman explains. The 3M Integrated Cycler is a compact (12 inches ∞ 12 inches) molecular diagnostic testing system that can process up to 96 samples per run and provides results in 30 to 85 minutes, depending on the test’s parameters.

The CDC regards the test as allowing a useful division of labor. “The Focus Diagnostics test is available for clinicians to order,” says CDC medical epidemiologist Dan Jernigan, MD, MPH, of the National Center for Infectious and Respiratory Disease. “This allows the public health labs to remain focused on surveillance testing.”

Gen-Probe announced Oct. 29 that its Prodesse ProFlu-ST test was granted an EUA. And Roche Diagnostics said Nov. 16 that its RealTime ready influenza A/H1N1 detection set has an EUA.

Companies developing H1N1 PCR tests include, among others, GlaxoSmithKline, Seegene, DxNA, and Osmetech—all of which say they have requested or plan to request emergency use authorization for assays.

The volume of PCR H1N1 tests Quest has performed was high in May, the initial month, then went down. “But as the second wave of H1N1 cases started in August we saw a sharp increase,” Dr. Lieberman says.

Though Quest is not releasing volume data on the number of test kits sold, the H1N1 tests represent the type of esoteric testing that is driving revenue growth for the company. In 2000, about nine percent of its revenues were driven by gene-based and other esoteric tests. By 2008, that number had grown to 20 percent of its overall revenues. In November, the company received authorization to sell the test in 35 countries in Europe, and it is now doing the necessary clinical studies to submit a 510(k) device application to the FDA.

Based on models that extrapolate from intensive studies of small groups of people, the CDC estimates that 14 million to 34 million cases of 2009 H1N1 occurred in the U.S. between April and Oct. 17, with between 2,500 and 6,000 H1N1-related deaths in 2009.

Those are the most precise statistics available. “This model assumes that cases are being underdiagnosed based on data collected over the spring and summer demonstrating that patients were not being tested, and for those with rapid antigen tests, the sensitivity may be lower than previously expected,” says the CDC’s Dr. Jernigan.

“One of the major lessons we’ve learned,” Dr. Lieberman says, “is that influenza tests commonly performed in local labs don’t perform the way we’d like. At urgent care centers that are doing so-called rapid diagnostic tests, you get answers fairly quickly, but they miss one-third to one-half of cases.” Dr. Lieberman notes the evidence that the rapid tests may not perform as well against H1N1 as against seasonal flu viruses.

The CDC recently evaluated three popular rapid tests, BinaxNow made by Inverness Medical Innovations, Directigen EZ Flu A+B test made by Becton Dickinson, and QuickVue made by Quidel. The agency found that the tests detected seasonal flu (H3N2) in respiratory specimens 80 percent of the time but for H1N1 the numbers were only 40 percent to 69 percent. And the tests can report only that the sample is positive for flu—they don’t identify which strain is present.

The American Society for Microbiology has come out with a statement saying viral culture or a PCR-based assay is preferred. The ASM’s “Interim Algorithm for Guidance in Testing Patients with Respiratory Illness for Influenza A (Including Novel H1N1),” released Oct. 9, specifies, “Because of unacceptably low sensitivity and suboptimal positive predictive value, rapid influenza antigen EIA tests cannot be recommended as a primary screening test for influenza.” A direct fluorescent antibody test is acceptable, the algorithm notes, but all negative specimens should be followed up with a culture or PCR test.

“This is a major shift in recommendations,” Dr. Lieberman points out. “But since even rapid viral culture takes between 36 and 48 hours, we believe PCR will really become the gold standard because it is fairly rapid and more sensitive.” He also thinks that the 3M Integrated Cycler, with its small footprint making it about the size of a bread-maker, will help a lot of hospital labs that lack the capacity to do molecular testing offer high-quality PCR testing that is now being done in reference labs. “That’s one of the most important things that will come out of it,” he says.

Cedars-Sinai Medical Center was quick to purchase the Simplexa and the 3M Integrated Cycler as soon as the EUA was issued in October.

“We purchased the 3M Cycler to run the test and we’re using it to facilitate our diagnosis of H1N1,” Dr. Nichols says. “We test patients with flu-like symptoms in the ER and patients admitted to the hospital first by the direct fluorescent antibody test [DFA]. The testing volume has been 20 to 50 DFA tests a day. And for hospital admits, we confirm all negative DFA stains by PCR tests to decide who is and is not to be isolated. We’ve found many patients who are positive for H1N1.”

“We feel the test is sensitive and specific on the basis of comparison with our own homebrew assay, which we do not use clinically but which we developed to look at the sensitivity of any test we would bring in-house.”

“In years past, we were using fluorescence staining with a backup of culture,” says Margie Morgan, PhD, scientific director in the Department of Microbiology at Cedars-Sinai. “We did a three-day rapid spindown culture for confirmation. But we’re discovering that the fluorescent assay is not nearly as sensitive to this disease as PCR. This influenza season our laboratory is using PCR as our primary testing method for inpatients.”

The test works with three signals, Dr. Nichols explains. “There is an internal control, then there is a target on the matrix, that’s for influenza A, then a target to the H1 gene or antigen. So for H1N1 we’ll see two positive signals, one for influenza A and one for H1N1.”

The Simplexa’s speed and accuracy are particularly important in containing the spread of influenza, Dr. Nichols says. “Within our own system, epidemiologically, the PCR test is very, very useful. If the patient has influenza symptoms, we go ahead and treat without waiting for the diagnostic test results. Otherwise this is about isolation and epidemiology. We have patients who are employees who appear to have H1N1, and the faster we obtain that diagnosis, the better off the whole system will be.”

In the process, the medical center’s clinicians have found they were underdiagnosing H1N1. “If we suspected flu from the presentation in the ER, in many cases we would have had a negative DFA. But then with PCR, we discovered 10 percent of the negatives are H1N1 positive,” Dr. Nichols says.

Dr. Morgan believes other hospitals are missing cases as well. “I think the amount of disease in our region has been very much underestimated. I know a lot of hospitals do not have virology labs or molecular capability, and they’re only using a membrane EIA test and rapid test for diagnosis.”

Many are hesitant to take on a PCR test. “I’ve talked to a few smaller hospitals that were inquiring what we were doing at Cedars-Sinai, and when I discussed our plan with them, they thought it was beyond their scope. They often have high-complexity labs, but not necessarily high complexity in the molecular area.”

Other than underdiagnosis, “I don’t think we’ve had any surprises so far,” Dr. Morgan says. “Early in the season we didn’t know what to expect and we prepared for the worst to happen. And initially it was intense. We had a lot of people in the community who were ill, a lot coming to our ER, but our admits have been somewhat consistent and lower than expected. And we’ve been very blessed not to have had any deaths related to influenza.”

But a particular advantage of a rapid, accurate H1N1 test, Dr. Nichols says, is that Cedars-Sinai, a large tertiary center with 915 beds, would be ready in the event of a severe outbreak. “If we had an absolute emergency, if there were a breakout of disease in the community, we’d be in command of our own fate, as it were. We would understand who’s got the disease and who’s bringing it into the system or not.”

The Simplexa test kit, Quest reports, is priced at about $50 per test run, before labor, supplies, extractions, and other miscellaneous costs. The 3M Integrated Cycler is priced under $75,000. Cedars-Sinai has found the cost comparable to that of other molecular tests, Dr. Morgan says. “Because we can do these daily in our own medical center, we’re able to establish diagnoses and release patients from isolation faster, and we save the medical center a lot of money that way. In the long run, the cost of molecular testing is being offset by savings in our institution, and it’s far cheaper for us to perform it than to send it out.” Medicare reimbursement for a PCR test for flu can range up to $200.

The U.S. has been pushed a little harder than expected by the H1N1 virus, Quest’s Dr. Lieberman says, and a number of predictions have not come to pass. “A lot of planning has gone on for a pandemic, and it’s served us well in the U.S. But one assumption turned out to be incorrect: that the pandemic would start elsewhere in the world. We thought we’d have time to learn about the virus as it started to spread elsewhere and use that knowledge here. But North America was where it all began. So we’re learning as we go along.”

In late November, Quest announced that although influenza A specimens right now are almost invariably—99 percent of the time—positive for H1N1, the pandemic may, in fact, have already peaked. After Oct. 27, demand for the test declined after several weeks of growth, and the number of specimens testing positive for H1N1 virus dropped in all age groups, except for those 65 and older. In that cohort, the percentage testing positive tripled to 14 percent. Another exception: The decline is not happening in the Northeast, where the number of positive results suddenly doubled in the first two weeks of November.

“The bottom line,” Dr. Lieberman says, “is that the 2009 H1N1 pandemic is far from over.” In the meantime, “We’re proud of our ability to launch the first test, and we see it as a way of providing accurate test results closer to the patient, so clinicians have the best information to help them manage their patients.”

Anne Paxton is a writer in Seattle.