PCR Becoming PC for Flu

The advantages of molecular technologies are becoming more accessible for clinical laboratories seeking respiratory and flu diagnostic solutions.

If a laboratory test is inaccurate, then it does not offer much value. But if the results are not produced within a quick enough time frame to offer help in the way of clinical decision-making, then the test still holds little value (from a physician and patient standpoint). Laboratory administrators are charged with finding a balance that avoids either extreme and actually delivers real clinical value. In the area of respiratory and flu testing, this duty can be particularly challenging.

There are a number of diagnostic options for influenza, including viral culture serology, rapid antigen testing, polymerase chain reaction (PCR), and immunofluorescence assays. Each has varying sensitivity and specificity influenced by the type of test used, the type of specimen tested, and, in some cases, the laboratory that runs the test.

Traditional methods utilize viral culture, which typically provides results within 3 to 10 days but offers high sensitivity and specificity. Rapid influenza tests can deliver results within 15 minutes, but according to the Centers for Disease Control and Prevention (CDC), most that can be performed in a physician’s office are approximately 50% to 70% sensitive for the detection of influenza and roughly greater than 90% specific. This leads to more false negative results than false positive, a discrepancy that increases during peak flu season.

“One of the biggest things we learned from the 2009 H1N1 pandemic was that the methods that were commonly used for influenza testing were inadequate,” says Jay M. Lieberman, MD, medical director of infectious diseases, Quest Diagnostics and Focus Diagnostics, Cypress, Calif. “Either they were not sensitive enough or you had to wait a day or two for results, which is not what you want when you are trying to treat patients.”

The answer, at least for some laboratories, has been to acquire PCR technologies that had been previously limited to reference laboratories, which had the resources to support these systems. However, manufacturers have advanced their technologies in this arena, making the methodology more accessible to a greater number of clinical labs.

“Molecular testing is going to become the test of choice for influenza and other respiratory viruses and will eventually replace lateral flow rapid tests, DFAs [direct fluorescent antibodies], and cultures because it combines the two things you most need: accuracy and speed,” Lieberman says.

Bringing the test closer to the patient will not only further improve the turnaround time, but can also have a positive impact on cost. “[Molecular diagnostic technology] may be a little more money up front, but over the next several years, labs will find they save money. A test may be cheap to do, but if it’s giving the wrong result, there’s a significant cost of the test that has to be factored in,” Lieberman says.

CRITERIA TO CONSIDER

When laboratories consider what type of system to use for respiratory and flu testing, cost is just one factor to look at. The primary consideration should be clinical need. “[Laboratory administrators should consider whether] the test will make a difference in the practice of medicine within their institution for the clients that order tests from the lab,” says Wade Stevenson, FilmArray product manager, Idaho Technology Inc, Salt Lake City. “Will the test actually be ordered?”

Turnaround time is a critical element impacting clinical value. “Can the results be delivered fast enough to be of value?” asks Stevenson, noting that if the reply is no, the test will not be ordered.

The answer to this question must also be tempered by another consideration: the quality of the data produced by the test. How sensitive is it? Can user variation impact the result? Can the physician trust the data? “If a test’s sensitivity drops to 30%, how meaningful is its result?” Stevenson asks.

From an operational standpoint, administrators and acquisition committees need to consider the capabilities of the laboratory and its technologists. What resources are required to run the system? What technical skills are needed? How user-friendly is the technology? “[Laboratory administrators need to ask] whether they can successfully implement and run the test,” Stevenson says.

Another question should focus on sample type. What types of specimens are required, and how will they be collected and transported? Specimen type has been shown to make a difference. The CDC notes that preferred samples include nasopharyngeal or nasal swabs and nasal washes or aspirates (determined by the test) taken within the first 4 days of illness.

And, of course, there is the issue of cost. Many institutions today no longer focus solely on the capital investment but consider the lifetime cost of the equipment, both direct costs (such as consumables and maintenance) and indirect costs (such as impact on patient care and outcomes). In addition, can the technology generate revenue? With laboratory tests, the question of reimbursement—whether it exists as well as the amount—is also an important consideration.

SWITCH TO SUBTYPING?

Another factor to consider when acquiring respiratory and influenza testing capabilities is the data that is produced: How much information can be determined from the test? Is it important to distinguish between types? Subtypes?

According to the CDC, there are more than 10 rapid influenza tests approved by the FDA. Some identify influenza A; some identify influenza A and B but do not distinguish between these two types; others detect and distinguish; none offer subtyping. Molecular diagnostic methods, which can provide subtyping information, offer similar variety, particularly as an increasing number of molecular diagnostic tests become available.

“Subtyping is a trend emerging because the different subtypes have different responses to the anti-influenza drugs used to treat the organisms,” Stevenson says. In addition, surveillance programs, both public and institutional, often require this data to compile an accurate picture of incidence. “Understanding what’s circulating can be very valuable,” Lieberman says.

Idaho Technology recently began commercializing its FilmArray Respiratory Panel, which tests for 15 viruses simultaneously, including subtypes (adenovirus, coronavirus HKU1, coronavirus NL63, influenza A, influenza A H1, influenza A H1 2009, influenza A H3, influenza B, metapneumovirus, parainfluenza 1, parainfluenza 2, parainfluenza 3, parainfluenza 4, respiratory syncytial virus or RSV, and rhinovirus/enterovirus). Cleared by the FDA in April, the test requires 2 minutes of hands-on time and 1 hour to run.

The system performs sample preparation, reverse transcription-PCR, PCR, and detection using freeze-dried reagents hydrated with a solution injected by the laboratory technologist. A result is then generated by the system software automatically using end point melting curve data.

Currently, the menu is restricted to viral conditions, but Idaho Technology plans to expand the panel to incorporate bacterial infections. “Oftentimes, the symptoms for a bacterial upper respiratory tract infection are identical to the symptoms for a viral upper respiratory tract infection, but the treatment is very different,” Stevenson says. “We’ve had a lot of people very interested in the bacterial targets that we want to add on our panel.”

ProAdeno™+ is the latest product to join the GEN-PROBE® PRODESSE® respiratory line of products from Gen-Probe Inc, San Diego, which features the ProFlu™+ assay, introduced in 2008 for the detection of influenza A&B and RSV. ProAdeno+ is a real-time PCR assay that detects, but does not differentiate, serotypes 1 to 51 in NP swab specimens and has a simple workflow that makes it easy to implement and validate. “ProAdeno+ offers laboratories and clinicians an important tool in the diagnosis of viral respiratory diseases,” says Steve Visuri, senior director of development for Prodesse Products.

MAD FOR MOLECULAR

The Plus series of molecular assays from Gen-Probe, which includes ProFlu+, Pro hMPV™+, ProParaflu™+, ProFAST™+, and ProAdeno+, is a modular panel of tests where a single sample can be quickly analyzed for a variety of pathogens. These tests offer scalability and flexibility that physicians and laboratorians need during the respiratory testing season. Having the ability to order only the tests appropriate for each patient, physicians can provide personalized testing.

The Simplexa Flu A/B & RSV test, developed and manufactured by Quest Diagnostics’ Focus Diagnostics business, does not require confirmation of negative results. The RT-PCR test was approved by the FDA to run on the 3M Integrated Cycler and uses nasal or nasopharyngeal swabs. The system carries a small footprint and features ease of use.

“We are trying to make it easier for labs that are now ready to take that first step into molecular testing as well as offer an alternative for labs that are already molecular but find their current platforms and technologies cumbersome, too time-consuming, etc,” Lieberman says.

Newer molecular technologies can help to save time and cost by reducing manual labor, speeding turnaround time, increasing result accuracy, improving physician confidence, and/or positively impacting patient outcome. “A false negative test leads to a patient management decision that’s not optimal, which can lead to an adverse patient outcome,” Lieberman says.

OVERCOMING THE OBSTACLES

Any negative result should be considered against the clinical characteristics, a directive that comes from the CDC (among other institutions). The organization also recommends negative results from rapid tests be confirmed using another methodology. Use of a rapid test technology can quickly screen out some positives (with definite benefit regarding treatment) and reduce the number of tests to be run on advanced technology. But the delay in diagnosis can sometimes be problematic.

“With influenza, we know therapy is most effective when started earlier, and so you would like an answer around the time you are seeing the patient and trying to make management decisions,” Lieberman says. Complicating matters with flu and respiratory diseases is the unknown.

A big challenge for everyone involved in diagnosing respiratory and influenza viruses is the constant evolution of viral agents. “New respiratory viruses come and go all the time, and this evolution is difficult to predict. We can only respond after the fact because we can only make a test for a virus that is known and fairly well characterized,” Stevenson says.

At the same time, tests for viruses that have become obsolete may still be on the market. “The FDA has no authority to revoke clearance, and there are some tests out there that are so old that viruses they were optimized on are not circulating among humans—at least not in a form easily detected by some of these older assays. So it’s a tough challenge because Mother Nature can throw a curveball any time she wants,” Stevenson says.

Ultimately, it is up to the laboratory to determine which test will best serve its patients, ordering physicians, and institution. Some combination of options may prove best, but more may find that PCR provides an accurate result in a time- and cost-efficient fashion—and provides the balance that offers clinical value.

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Renee Diiulio is a contributing writer for CLP.