American Association for Clinical Chemistry
Better health through laboratory medicine
March 2008 Clinical Laboratory News: Molecular Testing for Respiratory Viruses

March 2008: Volume 34, Number 3    


Molecular Testing for Respiratory Viruses
FDA-Cleared Panels Expand Labs’ Choices
By Deborah Levenson
 

While respiratory viruses often aren’t a cause of serious illness, they trigger a significant number of hospitalizations and deaths in debilitated adults—including those with HIV and COPD or who are elderly or on chemotherapy—and in children lacking fully developed immune systems. In these patients, influenza and respiratory syncytial viruses (RSV)—among others—can quickly become life-threatening. The CDC estimates that influenza alone causes 200,000 hospitalizations and 36,000 deaths in the U.S. annually, and cites RSV as the most common cause of severe lower respiratory disease in infants and young children worldwide. In patients with symptoms of severe respiratory disease, quick and accurate diagnosis can lead to proper treatment and shorter hospital stays.

Traditional diagnostic methods, however, often leave much to be desired. Rapid testing via enzyme immunoassay (EIA) for one specific virus is easy and inexpensive, but not very sensitive, while the more complex direct fluorescent antibody (DFA) assays offer better sensitivity and the ability to test for more virus types. However, DFA requires staff with proper training and specialized equipment. Results from culture, once considered the gold standard, take at least a day or two, and often more. Molecular tests for single respiratory viruses generally offer the best sensitivity in about 3 hours, but they require costly instruments and properly trained staff. And until recently, labs that wanted to offer molecular tests for respiratory viruses had to develop their own tests and perform their own rigorous validation.

Now two FDA-cleared, multiplex molecular respiratory tests offer labs the ability to detect several viruses using just one assay and one sample, with less validation and ongoing QC than laboratory-developed molecular methods. In January 2008, FDA cleared both the xTAG Respiratory Viral Panel (RVP), marketed by Luminex Molecular Diagnostics (Toronto, Canada), and the ProFlu+ Assay, marketed by Prodesse, Inc. (Milwaukee, Wis.). ProFlu+ detects the three most common respiratory viruses, while xTAG RVP detects nine, including three influenza A subtypes.

These multiplex tests and laboratory-developed versions can identify dual infections and may be the wave of the future. “These tests have the potential to become standard of care over the next five years or so,” proclaimed Sheldon Campbell, MD, PhD, Associate Professor of Laboratory Medicine at Yale University School of Medicine in New Haven, Conn. But he and other lab directors emphasized that decisions about using multiplex molecular assays for respiratory viruses depend on the particular needs of individual labs and hospitals. Among the issues to consider are patient populations, staffing, workflow issues, and if the costs and work necessary to take on these tests will truly improve diagnosis of serious respiratory illnesses.

Very Different Assays

The cleared assays differ markedly in their scope, technology, and turnaround times. ProFlu+ is a real-time PCR, closed-tube test that simultaneously detects influenza A and B and RSV, which together cause the vast majority of serious respiratory disease. Using a sample from the nasopharynx, the test uses real-time technology to perform reverse transcription and PCR in one tube. The whole process, including extraction, takes about 3 hours by company estimates. Prodesse markets its kit at $5,250, which includes the necessary reagents for 100 reactions, according to Andy Shrago, Prodesse’s Vice President of Sales and Marketing. ProFlu+ is cleared for use with two automated nucleic acid extraction systems, and is the first in a series of real-time respiratory PCR assays for which the company intends to seek FDA clearance. These include tests for human metapneumovirus and parainfluenza, noted Steve Virsuri, PhD, Prodesse’s Chief Scientific Officer.

In contrast, the xTAG RVP assay detects many more viruses, but is more complex and time-consuming. The panel tests for and discriminates among influenza A H1, H3, and non-specific subtypes and also detects RSV A, RSV B, influenza B, parainfluenza 1, 2, and 3, metapneumovirus, rhinovirus, and adenovirus. The version of the test for the European market includes H5N1, commonly known as bird flu, but the FDA-cleared version does not, because “there were insufficient numbers of patient samples to meet the criteria for FDA submission,” according to Jeremy Bridge-Cook, PhD, Luminex’s Vice President. The test process includes amplification via PCR, and then analysis in a Luminex instrument. By company estimates, the entire process takes about 6.5 hours, including 1.5 hours of hands-on time. The kit comes with analysis software, but labs must purchase ancillary reagents from particular lots that have been vetted by Luminex’s quality control system, according to the product insert. Because the Luminex instrument is an open tube system, labs should perform pre-and post-PCR amplification in separate areas as a precaution against contamination, the insert adds.

Some labs that have used the xTAG RVP test question the company’s time estimate. Helen Fernandes, PhD, Associate Professor and Director, Molecular Diagnostics in the Department of Pathology at the University of Medicine and Dentistry of New Jersey in Newark, said, “It takes about seven to eight hours from start to finish, and much of that time the test requires undivided attention.”


Sensitivity and Specificity of xTAG RVP
Virus
Sensitivity
Specificity
Influenza A
96.4%
95.9%
Flu A Subtype H1
100%
100%
Flu A Subtype H3
91.7%
98.7%
Influenza B
91.5%
96.7%
RSV A
100%
98.4%
RSV B
100%
97.4%
Parainfluenza 1
100%
99.8%
Parainfluenza 2
100%
99.8%
Parainfluenza 3
84.2%
99.6%
Rhinovirus
100%
91.3%
Adenovirus
78.3%
100%
Metapneumovirus
96%
98.8%
Source: Luminex package insert for xTAG RVP kit reagents. Figures are based on testing of 544 prospectively collected specimens at four North American clinical laboratories during the 2005-6 flu season in a trial that compared xTAG with DFA and/or culture.

 A Role in Public Health

Bridge-Cook noted that his company’s target market includes molecular labs at medium-to-large hospitals with both inpatient and outpatient samples interested in the test’s comprehensiveness, greater sensitivity, and speed, as well as public labs looking for an additional tool for monitoring pandemic threats. While Bridge-Cook would not divulge the price of xTAG RVP, and CMS has not set reimbursement for the test, company conversations with the agency and other insurers have led him to expect reimbursement “in the $200 to $400 range” for the panel. He maintained that xTAG RVP is as cost-effective as labor-intensive DFA on a per-report basis.

In contrast to xTAG RVP’s dual clinical and public health roles, ProFlu+ is designed primarily for high-throughput clinical laboratories, according to Shrago. It includes only influenza A and B and RSV because they cause the vast majority of serious respiratory illness, and because antivirals are available to treat them, according to Visuri. While Prodesse’s first commercial product for respiratory viruses, Hexaplex, initially distinguished between RSV A and RSV B, “the company removed the discrimination because customers reported that it took more effort and was of little value to the clinician,” he said. “We didn’t include subtyping ability for influenza A or RSV in ProFlu+ because the typical clinical lab doesn’t care about them,” he added. “There’s no difference in treatment for either influenza A or RSV subtypes.”

Sensitivity and Specificity of ProFlu
Virus
Sensitivity
Specificity
Influenza A
100%
92.6%
Influenza B
97.8%
98.6%
RSV
89.5%
94.9%
Source: Prodesse package insert for ProFlu+. Figures are based on 891 samples involved in a prospective study at 3 U.S. clinical laboratories during the 2006-7 season in which the reference method was culture.

 

For labs interested in primarily influenza and RSV, the fact that ProFlu+ is closed tube makes it an attractive option, Virsuri and Shrago maintained. “With an open-tube test, you run the risk of contamination. In recent years, labs have tried to get away from open-tube systems,” Virsuri explained, adding that contamination can result in false positives and the need to re-test batches of samples.

Although there’s no treatment available for many of the viruses on the xTAG RVP, hospitals can benefit from the knowledge that a patient has them, Bridge-Cook pointed out. “The hospital can take infection control measures like isolating or cohorting, meaning the hospital wouldn’t place people with different infectious agents in the same room. If the test comes back negative for everything, the patient likely has a bacterial infection. In either case, early diagnosis leads to shorter length of stay and savings for the hospital,” he explained.

A Real Advance?

The FDA-cleared multiplex tests represent a step forward in automation, according to Kelly J. Henrickson, MD, Professor of Pediatrics and Microbiology at the Medical College of Wisconsin in Milwaukee and Director of the Midwest Respiratory Virus Program at its Children’s Research Institute and Children’s Hospital of Wisconsin. Henrickson, who invented the original Hexaplex technology and founded Prodesse, holds company stock but is otherwise no longer involved with the company. He has a number of grants from NIH and works closely with Nanogen (San Diego, Calif.) on a number of technologies intended to diagnose respiratory viruses. While Luminex has automated the detection process, “You still must do the purification and amplification first,” Henrickson explained.

He pointed out that it’s possible to use other, research-use-only multiplex tests to simultaneously test for several respiratory viruses. These include a research-use-only test from EraGen Biosciences (Madison, Wisc.), the MultiCode-PLx Respiratory Virus RUO Panel (RVP), a PCR test with 17 viral targets. Seegene (Rockville, Md.) will soon launch in the U.S. a research-use only PCR assay that can simultaneously detect 13 respiratory viruses and 5 pneumonial bacteria targets.

Worth the Money?

A decision to use either FDA-cleared test—or any new molecular test—comes down to speed and accuracy versus cost, plus labs’ particular workflow, staffing patterns, and patient population, according to Angela Caliendo, MD, PhD, Professor and Vice Chair of Pathology and Laboratory Medicine at Emory School of Medicine and Medical Director of Microbiology and Molecular Diagnostics at Emory University Hospital, both in Atlanta, Ga. “When you bring any test in-house you have to ask: Will it change clinical management? How often will we run it and what will be the actual turnaround time?” She is currently evaluating the ProFlu+, Luminex, and EraGen assays.

Caliendo, who is also an infectious disease clinician, is now using rapid culture and EIA for respiratory viruses. She noted that shell vial culture, which involves living cultured cells on a cover slip in a vial of media and fluorescent antibody staining of the virus particles, yields the majority of positive results in 24 to 48 hours and is quite specific. But both Caliendo and a review article by Henrickson note that molecular methods are generally 10-30% more sensitive (Pediatric Infectious Disease Journal 2004; 23 (1s) S6-S10). However, a study detailed in the ProFlu+ insert and summarized in a presentation available on Prodesse’s Web site that shows ProFlu+’s sensitivity exceeds this range for influenza A and especially for RSV, in comparison to shell vial culture.

Which Viral Targets Do Labs Need?

Another consideration is whether clinicians and labs want all the pathogens on the xTAG RVP or if the ability to identify only the most common ones included in ProFlu+ will suffice. “We struggle with this issue on a daily basis. Each physician and institution has its own opinion,” said Henrickson. But he added that most clinicians and labs would want influenza and RSV, and those serving large pediatric populations would probably also want parainfluenza 1, 2, and 3, human metapneumovirus, and possibly adenovirus. The picornaviruses—including rhinovirus and enterovirus— are more controversial because while they cause the vast majority of respiratory illness, they are generally milder upper respiratory infections, not the lower respiratory illnesses that usually hospitalize patients. “However, both rhinovirus and enterovirus can cause pneumonia in kids. But a lot of kids have these viruses and are asymptomatic. It’s very hard to say what a positive result for rhinovirus means, and it’s up to the clinician to decide,” he explained.

Other Considerations: Batching, Validation, and Follow-Up Testing

Caliendo, Campbell, and Fernandes predicted that most hospitals would batch both FDA-approved assays and run them once a day, making the differences in the time needed to run the tests and get results less important. Despite the complexities of xTAG RVP, Fernandes is considering using it or EraGen’s Multicode PLx Respiratory Virus Panel along with ProFlu+ in a respiratory virus testing algorithm she hopes to begin next year. The first step would be rapid EIA testing for flu and RSV from symptomatic patients, with results back in 30 to 60 minutes. “This saves on tech time, reagents, and money and it’s the simplest, cheapest, and quickest,” she explained. If the EIA comes back negative, she would reflex to ProFlu+ because it identifies the most common viruses. Caliendo would use either the EraGen or Luminex panel when EIA and ProFlu+ are both negative and testing is clinically indicated. But Caliendo added that she is also in the process of validating homebrew PCR tests for cases with EIA and ProFlu+ negative results.

Another consideration in these tests’ value is how quickly clinicians will act on results. “Regarding timing and making the test quick enough to be worth the money, there has to be good, fast communication with clinicians. That’s not a problem here, because the lab is right on campus,” Fernandes pointed out.

For Campbell, Luminex’s ability to detect different influenza A subtypes—especially nonspecific ones—is an attraction due to their role in public health. “The ASR methods don’t usually discriminate the weird types,” he noted, but with xMAP RVP “if you detect a flu that isn’t one of the common subtypes, it raises suspicion of something new.” He also likes having an FDA-cleared molecular assay for all of the influenza subtypes because it lessens the burden of validation. “You can’t validate a molecular home brew or ASR flu test unless it’s flu season, and each year there’s only a short window for doing that. You have to keep live specimens, both positive and negative, and the positive specimens are available for only a short time.”

Of note is the fact that the FDA approval letters for both xTAG and ProFlu+ suggest that labs do follow-up testing with other methods to confirm results. While Bridge-Cook says such language is standard for influenza tests, Caliendo called it “very concerning because it puts labs in a difficult spot,” adding, “Most labs can’t and won’t confirm with a different test. PCR is the most sensitive and specific method we have. Most labs can’t afford to do two of them.”

What About Reference Labs?

Some of the lab directors think these tests, particularly xTAG RVP, may be useful as reference tests, if results come back quickly enough. Two day turnaround isn’t enough, said Fernandes. “RNA viruses are not very stable. A lot of these respiratory diseases, like flu, are self-limiting. If a clinician is concerned about infection control, decisions have to be prompt.”

Henrickson said next morning or same- day results from a reference lab may be worthwhile. “Accomplishing that probably depends on timing and number of pickups by the reference lab. If it takes 24 hours to get the sample to their lab, the test may not be useful. If a regional lab does four pickups daily and you can get results in the morning, then that’s useful,” he explained.

While Shrago reports reference lab interest in ProFlu+, none have thus far become clients. ViraCor Laboratories, a specialty molecular diagnostic reference lab in Kansas City, Mo., is offering the xTAG RVP to its clients at a list price of $396, but does not offer ProFlu+. ViraCor offers results for all tests, including xTAG RVP, on the same day that it receives the samples, and offers couriers service in many situations, according to Steve Kleiboecker, PhD, ViraCor’s Chief Scientific Officer. The majority of the labs’ clients are transplants centers.

The primary reason to order the xTAG RVP from ViraCor is the test’s complex workload and open-tube system, Kleiboecker added. “The workflow requirements and complexity might dissuade a hospital from running the test in its own lab, where space is often at a premium. A standard molecular lab philosophy is that you want to establish unidirectional workflow. We built additional space to accommodate the multi-step process.”

Tests of the Future

Henrickson, among other researchers, is working on technology that would make simultaneous testing for multiple respiratory viruses far less complex. “What people want is a box you can stick a sample into and walk away. It would do purification, amplification, and detection. No one’s there right now. We have only components of that system,” he explained. Meanwhile, the Asian SARS epidemic and U.S. federal government concern about bioterrorism and pandemics has spurred academic, commercial, and military research on other technologies that may someday be useful in clinical settings. In his review article, Henrickson lists some emerging methods including capillary electrophoresis, with or without PCR, microarrays, and handheld reverse transcription PCR devices.