Two sides of the coin on
  respiratory viruses

May 2005
Cover Story

William Check, PhD

Ready for a pop quiz? When testing for viral respiratory pathogens, the best method to use is which of the following: rapid culture, rapid immunoassay antigen detection kits, immunofluorescent antibody methods such as direct fluorescent antibody, or nucleic acid amplification-based molecular tests?

If you’re thinking its all of the above, give yourself a pat on the back.

Certainly there is a prevalent perception that molecular methods based on nucleic acid amplification, such as PCR and NASBA, are the ideal technologies for viral diagnosis. Speaking at a session on rapid detection of respiratory pathogens at last year’s meeting of the Association for Molecular Pathology, Kelly Henrickson, MD, associate professor of pediatrics and microbiology and director of the respiratory virus molecular diagnostics laboratory at the Medical College of Wisconsin, Milwaukee, was explicit about this position: "I am going to try to convince you that molecular methods are the new gold standard for sensitivity and specificity."

But does that mean molecular methods are best in all situations? Angela Caliendo, MD, PhD, vice chair of pathology and laboratory medicine at Emory University School of Medicine, offered a different perspective in her talk at the same AMP session: When choosing rapid tests for respiratory viruses, "there is no answer that is perfect for everyone." Later she told CAP TODAY, "I would recommend that you ask what test best fits your clinical situation based on your patient population—inpatient versus outpatient, pediatric versus adult, immunocompromised versus immunocompetent—your desired turnaround time, and your resources and financial situation. If a particular test does not allow you to impact clinical outcome or clinical management, maybe that test is not for you."

"Dr. Caliendo and I mostly agree," Dr. Henrickson told CAP TODAY. To say that molecular tests may not be the best option in every clinical setting, he says, "doesn’t contradict that molecular is the gold standard for sensitivity and specificity. There are many places where more rapid, cheaper, and less sensitive and specific tests are preferable, such as in the outpatient setting." However, he maintains that molecular methods are best for all hospitalized patients and for all seriously ill patients, such as those with cancer, where it is critical to identify viral pathogens accurately.

Marie Louise Landry, MD, professor of laboratory medicine at Yale University School of Medicine and director of the clinical virology laboratory at Yale-New Haven Hospital, has evaluated many methods for detecting viral respiratory pathogens. "Certainly the greatest increase in interest right now is in molecular testing," she says. Whether you choose a molecular test for detecting viral respiratory pathogens, however, depends on your clinical setting and capabilities. "I know that some virology laboratories are starting to replace conventional culture with molecular tests for respiratory viruses in hospitalized patients. That approach has a lot to say for it," says Dr. Landry, who does not use molecular methods now as her primary testing modality.

"If a molecular assay is as sensitive as culture or better, and if it gives you an answer in one day or less, there can be a benefit." But, she adds, "there is a cost to be paid, both in development time and for equipment." She notes also that molecular testing can be difficult to troubleshoot because these tests are generally in-house assays or analyte-specific reagents.

In viral diagnostics generally, molecular testing has moved quickly to replace traditional testing, says David Hillyard, MD, associate professor of pathology at the University of Utah and medical director for molecular infectious disease testing at ARUP Laboratories, who organized and chaired the AMP session. "These tests have demonstrated increased speed and sensitivity, and for some viral pathogens, such as HIV and hepatitis C virus, molecular is essentially the only practical method. Many people have assumed that what has been true of other viral scenarios—replacement of traditional methods by molecular—would also be true automatically for viral respiratory pathogens," Dr. Hillyard says. "But testing for viral respiratory pathogens is more complicated."

The field is very nuanced and rapidly changing, Dr. Hillyard says, and realizing there are many different clinical scenarios is important. "What is indicated for RSV [respiratory syncytial virus] testing may not be true for influenza or human metapneumovirus. Different approaches are taken for pediatric versus adult populations, inpatient versus clinic patients, and the demands on small versus large laboratories vary considerably." Analyzing the value of molecular testing in all these settings is complex. For this reason, Dr. Hillyard chose two individuals—Drs. Caliendo and Henrickson—experienced in viral testing "to showcase two contrasting perspectives" at the AMP session.

One undisputed fact is that viral respiratory infections are a major source of illness, with an estimated 1.5 billion upper respiratory infections (colds, conjunctivitis, pharyngitis, otitis media, sinusitis) and 16 million lower respiratory infections (pneumonia, bronchiolitis, bronchitis, croup) in the U.S. annually. Each year 430,000 children and 208,000 adults are hospitalized with viral lower respiratory infections. At higher risk for severe viral LRI are elderly, immunocompromised people, infants, and children, but the majority of those who get serious LRI have no known risk factor. Influenza causes about 36,000 deaths in adults each year and RSV an additional 17,000 deaths. (Corresponding figures for children are about 150 and 500.)

The most common viral respiratory pathogens (in decreasing order of hospitalizations) are RSV A and B, influenza A and B, human parainfluenza virus (HPIV)1-3, human metapneumovirus, adenovirus, and rhinovirus. Because these viruses cause similar-appearing clinical illness, Dr. Henrickson says, "You can’t diagnose which virus is causing a patient’s illness by the clinical presentation." And though several viruses show clear seasonality, it’s not possible to make a viral identification by the time of year.

Detection of viral respiratory pathogens can be used for cohorting (to guide accurate isolation procedures), for surveillance (to determine which viruses are circulating in a community at a given time), and to affect patient management—to reduce unnecessary antibiotic use, promote appropriate antiviral use, and reduce unnecessary diagnostic studies. The clinical benefits of rapid diagnosis of respiratory viruses have been demonstrated (Woo PC, et al. J Clin Microbiol. 1997;35:1579-1581; Barenfanger J, et al. J Clin Microbiol. 2000;38:2824-2828).

Dr. Caliendo reviewed relevant data about traditional tests. (She told CAP TODAY, "I was in a role with which I am less familiar—not advocating for molecular testing.") Children shed much higher titers of many viruses, so a test may have much lower sensitivity in adults than in children. Sampling methods are not all equal. Using 14-day cultures for influenza, one group found an 80 percent positivity rate with nasal aspirates, 90 percent with sputum, 65 percent with nasopharyngeal swabs, and only 52 percent with throat swabs (Covalciuc KA, et al. J Clin Microbiol. 1999;37:3971-3974). For this reason, some viral laboratories will not accept throat swabs for testing.

Major testing methods include immunofluorescent antibody (IFA) methods, rapid immunoassay kits, and conventional and rapid culture.

Dr. Landry tested a multiplex (seven-virus) direct fluorescent anti body (DFA) pool against culture. Using cytospin-prepared slides, she found the respiratory screen reagent equivalent to or better than culture for detecting all viruses except adenovirus (Landry ML, Ferguson D. J Clin Microbiol. 2000;38:708-711). In separate studies, she found that DFA and rapid immunoassay kits were closer in sensitivity for influenza detection in children, especially when nasopharyngeal aspirates were tested, but that DFA was much superior to ELISA for swab samples collected from adults (Landry ML, et al. J Clin Microbiol. 2000;38:429-430). She also found rapid immunoassay kits for influenza A and B to have low sensitivity, just over 50 percent, and to be inferior to DFA (Landry ML, Ferguson D. J Clin Microbiol. 2003;41:3407-3409; Landry ML, et al. J Clin Virol. 2004;31:113-115).

Though they have low sensitivity, immunoassay kits are rapid, Dr. Caliendonoted, yielding results in less than an hour, and are extremely simple to use. They may be appropriate for use in outpatient clinics and as point-of-care devices.

Dr. Caliendo called IFA "a workhorse in many labs." Its turnaround time can be almost stat, and it is possible to run several batches per day. Since the specimen is scored under a microscope, this is the only method in which the quality of the sample can be assessed—at least 25 ciliated columnar epithelial cells are required for an adequate sample. However, use of the fluorescence microscope also makes IFA a high-complexity test demanding trained personnel.

Conventional culture has a turnaround time of two to seven days or longer, so it can’t affect management decisions. Rapid culture using the shell vial method and commercial mixed cell cultures such as R-Mix is much faster. Dr. Caliendo finds that more than 95 percent of positives are detected at day one. "We set it up in the late afternoon and report out the next afternoon," she says.

Dr. Henrickson spoke for molecular testing, focusing on the Hexaplex assay, which he called "the oldest and most widely used" kit for detecting respiratory viruses. (Dr. Henrickson co-invented and co-developed Hexaplex, which is marketed by Prodesse, a company he founded but with which he currently has no management role.) Hexaplex is an RT-PCR assay using strepavidin plate technology; it detects RSV A and B, influenza A and B, and HPIV 1-3. A newer Hexaplex Plus kit also detects human metapneumovirus. In unpublished work by Dr. Henrickson and pediatrician John DeVincenzo, MD, of the University of Tennessee, Memphis, results from Hexaplex for RSV A were tightly correlated with a viral plaque assay.

Pooled results for several studies in which Hexaplex was compared with culture, EIA, or DFA showed sensitivity and specificity for Hexaplex from 97 percent to 100 percent. True specificity for the molecular test is probably close to 100 percent, Dr. Henrickson said, an anomaly that arises from the extremely high sensitivity of molecular tests. "If the molecular test is positive and the old method is negative, by definition we have to say that is a false-positive for molecular," he explained. However, when discrepant results are adjudicated with a second RT-PCR assay using primers specific for a different genomic region than that used in the Hexaplex assay, molecular "false-positives" almost always turn out to be true positives, according to Dr. Henrickson.

Working with Sue Kehl, PhD, of Children’s Hospital of Wisconsin, Dr. Henrickson evaluated Hexaplex on clinical laboratory samples. Of 456 samples that tested negative by EIAs for influenza A and RSV, 209 were positive by Hexaplex. Similar results were obtained with samples that were negative by DFA—133 of 502 were positive by Hexaplex (Kehl SC, et al. J Clin Microbiol. 2001;39:1696-1701).

Overall, RT-PCR assays are more sensitive and specific than traditional methods and are rapid and clinically useful, with a turnaround time of six hours, Dr. Henrickson said. (He summarized the advantages of this technology in Henrickson KJ. Pediatr Ann. 2005;34:24-31.) Remaining issues in molecular diagnostics are cost, reimbursement, reliable reagents, the need for open platforms so that new pathogens can be added easily, and the need for proficiency panels.

Dr. Caliendo has several thoughts about whether and when molecular tests are the best choice. "How will you be using the test?" she asks. If it is to cohort patients, current molecular methods are not yet rapid enough, though she predicts they will eventually meet that standard. "We still have to rely on EIA or DFA," she says. "If you are interested in surveillance, then any test will do, since the turnaround time is not so urgent."

Guiding clinical management of individual patients is a separate context. Testing affects clinical management when it is "as accurate as possible as quickly as possible," Dr. Caliendo says. In outpatient settings, molecular is not rapid enough and the faster turnaround time of EIA or DFA offsets their lower sensitivities.

For inpatients, laboratories must ask themselves how quickly they need a result to influence clinical care. "From my perspective," Dr. Caliendo says, "I would like the result that day or the next day at the most. We have found that rapid cultures are very successful. Molecular can also have a place here. If you do choose molecular, make sure you are in a situation in which you are going to get a result in the time frame you need it."

Molecular testing’s higher sensitivity has also raised new questions and new issues of which labs need to be aware, Dr. Caliendo notes. A positive result on a molecular test may be analytically true but of unclear clinical importance. For example, molecular tests can detect both viable and nonviable virus—do these have similar clinical significance? Also, people can shed adenovirus for days or weeks after they recover from the clinical illness—will detection of adenovirus always correlate with disease? Finally, following administration of live attenuated vaccine, recipients may shed the virus for up to three weeks, which may give positive test results.

Evaluating the role of molecular testing for respiratory pathogens, Dr. Hillyard notes that it can be rapid, but that there are rapid immunological methods "that can be done in 20 minutes to an hour, much more quickly than molecular."

"Molecular assays, especially for respiratory viruses, can be much more difficult to perform than some immunological assays," he says, since you are often looking for multiple pathogens. Real-time PCR assays are starting to emerge, with the potential for increased speed, but they must be run once or even twice a day to take advantage of their speed. Not all hospital laboratories can do this.

An important question, Dr. Hillyard says, "is how much the adoption of molecular testing is driven by a clear medical necessity." In patients with encephalitis, testing for enterovirus and herpes simplex should be done only by molecular methods. Though a case can be made for molecular testing for respiratory viruses, Dr. Hillyard says, "it is probably safe to say it is a work in progress."

The development of specific therapies could tip the balance in favor of molecular. "It’s not as though once you identify a respiratory virus you have a clear unique therapy, a drug that will be used only with that virus," Dr. Hillyard points out. "That could change dramatically as work proceeds on therapeutics for viral respiratory infections. A specific therapeutic ups the ante, and rapid molecular testing may naturally fall out of that equation."

In Dr. Landry’s weighing of molecular testing, cost is a major consideration. "You will need automated extractors," she says. "Doing a large volume of testing with manual extraction using conventional PCR is very labor-intensive." As real-time instruments enter the laboratory, they make things faster and simpler but increase the investment in equipment even further.

In addition, Dr. Landry says, "All the time in development and validation of molecular methods gives people pause in switching over. And in terms of charges, a single PCR charge is often $150 to $200 or more." If you test for all important respiratory viruses, charges multiply, unless they are multiplexed. "Is the cost justified by the benefit?" Dr. Landry asks. Like Dr. Hillyard, she says, "If we had clearly effective treatments for these viruses, a better case could be made."

Molecular tests have a place in reference laboratories, in Dr. Landry’s view. Samples are shipped long distances, with a decline in infectivity, so higher sensitivity is more important. And a reference laboratory is more likely to be able to afford the up-front investment in equipment. But why would a respiratory virus sample be sent to a reference laboratory? A tertiary care hospital would have a virology laboratory on site that could do rapid culture or DFA. For a hospital without a virology laboratory, sending a sample to a reference laboratory would not help with cohorting or patient management because of the time factor. "In some situations you may need to document a very serious infection," Dr. Landry says. "But the cost of a send-out PCR would be steep and the hospital has to ask, Does this impact patient care? What is the benefit to justify this cost?"

Drs. Caliendo, Henrickson, Landry, and Hillyard all work in different settings and use different strategies for diagnosing respiratory viruses.

"We have in recent years changed our approach to respiratory virus testing," Dr. Caliendo says. She now primarily uses rapid culture with R-Mix, which meets the need for a broad diagnostic tool. Emory University Hospital does not have many pediatric patients; it serves a large population of transplant patients. "When respiratory viruses are in the differential, we recommend rapid culture of nasopharyngeal aspirate or swab or a BAL [bronchoalveolar lavage]," Dr. Caliendo says. She also does DFA testing for RSV.

They are considering molecular. "It would really only be practical for us to run it once per day. Does that improve our turnaround time that much? And we would have to assess the cost and labor," Dr. Caliendo says. If she went to molecular, Dr. Caliendo would want a real-time assay. "Would one multiplex assay be adequate?" she asks. "Or would we need to run two or three assays to cover all pathogens?" Real-time PCR has a limited ability to multiplex because of the technical limitation on the number of dyes. Running multiple assays would increase the expense.

Dr. Hillyard has used Hexaplex for several years. "The current version of the assay shows good sensitivity," he says. His laboratory can get a result out the same day, with a turnaround time of about eight to 10 hours from receipt of the sample. They run it many times during the week. Still, since ARUP is a reference laboratory and samples come from a distance, would the sending physician get the result in time to influence patient care? "Even with electronic reporting, we are looking at a turnaround time of a couple of days," Dr. Hillyard says.

Dr. Landry uses different tests in different clinical circumstances. Her primary test, for patients who come to the emergency department when the virology laboratory is open, is "a very labor-intensive" multiplex cytospin-enhanced DFA test for RSV, influenza A and B, HPIV 1-3, and adenovirus that meets the sensitivity of culture. Negative specimens are cultured. "It requires a lot of effort on the part of the laboratory to turn around results in 1.5 to two hours," she says. The laboratory does the test continuously throughout the operating hours. Under these circumstances, DFA is a useful test for the ER. "They are trying to move patients through," Dr. Landry says. "To wait several hours for results is a long time for them. We have stayed with DFA because of the benefits. But we wonder: People get backed up, since the ER physicians don’t make decisions until they get the result, whereas rapid tests for influenza and RSV give results in 15 to 20 minutes." The problem is that rapid tests are much less sensitive than DFA done well, and Dr. Landry found some false-positive results in early evaluations of rapid tests.

It requires a major effort to do the DFA test well, Dr. Landry says. "A lot of laboratories do not do it well so they get poor results. We put in a lot of effort training our people. It is something you have to make a major commitment to."

To make this test available as much of the time as possible, virology is open seven days a week year round, from 7 AM to 8:30 PM Monday to Friday and for eight hours on Saturday and Sunday. Beginning in November, depending on virus activity, weekend hours increase to 12 hours a day. For two to three months in peak respiratory season, they are open 18 hours a day, seven days a week.

During peak season when the virology laboratory is closed, a rapid EIA influenza test is done in the chemistry laboratory. "We train and oversee that," Dr. Landry says.

Her laboratory does human metapneumovirus by real-time PCR. "We may bring other respiratory viruses on in PCR format," Dr. Landry says, "those that are difficult to culture, such as new coronaviruses." She may also add a PCR assay for rhinovirus, for which there is no rapid or DFA assay and which may be involved in more lower respiratory disease than previously thought. A molecular test for the dangerous avian influenza virus would also be helpful.

In a comparison study, Dr. Landry found a real-time PCR assay for influenza A to be almost as sensitive as her laboratory’s DFA method (Habib-Bein NF, et al. J Clin Microbiol. 2003;41:3597-3601). She chose to stay with DFA because she would be able to do the molecular assay only once per day, greatly extending turnaround time and making the test irrelevant for much of what rapid testing is used for—bed assignments, moving patients out of the ER, and deciding on the type of infection control. "We would lose the rapid turnaround times that we are providing now," she says. "We are not in a position to run molecular tests several times during the day. It would be too expensive and is not an option for us."

For 10 years Dr. Henrickson ran a virology service laboratory. Until last year he also ran a reference laboratory. (Currently he does only research.) During that time he recommended Hexaplex testing on all hospitalized children with moderate to severe lower respiratory infection of unknown etiology, all immunocompromised children or with chronic medical conditions with any LRI, and many adults in the same categories. He performed the assay once daily, six days per week. He collected specimens until 1 PM, then set up the assay and (depending on whether he had one or two shifts) ran it overnight and reported results in the morning or ran it straight through (about six hours) and reported results at 7 to 8 PM. Between 1996 and 2004, his lab tested about 1,500 respiratory samples per year from Children’s Hospital of Wisconsin, one of the busiest children’s hospitals in the country. The lab also tested many hundreds per year from the adult university hospital.

During his talk at the AMP meeting, Dr. Henrickson presented cost and reimbursement figures for standard format and real-time PCR in singleplex and multiplex mode. His conclusion: "You can make money doing molecular diagnostics, despite what people say."

But Dr. Caliendo showed different figures and draws a different conclusion, and says, "The issue of reimbursement is complex and different for reference labs compared with hospital-based clinical laboratories." For the latter, she says, "making money on molecular testing on inpatients, most of whom are covered by DRGs, can be difficult. For outpatients, whether the laboratory makes money depends on its payer mix, cost, and contracting agreements."

Even as laboratories struggle with PCR, yet more innovative formats are being developed. Dr. Henrickson previewed an 87-probe microarray from Metrigenix, a multiplex gene chip assay with a detection threshold of 12 copies from Nanogen, a microbead method to detect multiplex PCR products from Luminex, and a combination RT-PCR and capillary electrophoresis approach. Only for this last technology is there clinical data so far (Erdman DD, et al. J Clin Microbiol. 2003;41:4298-4303). Whatever methods come into clinical use, Dr. Henrickson said, "Multiplexing is how we must go."

What could tip the scales in favor of broader molecular testing? Again, the introduction of specific anti virals. Evidence that using existing molecular tools leads to better patient management or is cost-effective relative to traditional methods might also do it, in Dr. Hillyard’s view. Still another factor would be a technological breakthrough. "We need a rapid, inexpensive, robust technology that creates a test that is as simple to perform as an immunological test and is price competitive," Dr. Hillyard says. "There are candidate assays that people are taking a hard look at." These include rapid isothermal techniques, technology that minimizes or eliminates sample preparation, and major improvements in real-time technology.

In the meantime, laboratory directors have to make decisions about existing technology. Despite Dr. Landry’s reservations about current versions of molecular tests, she says it is important to become familiar with them.

"Where does that technology belong," she asks, "in the molecular lab or the virology lab? I think virology labs need to start getting molecular expertise because many tests are going in that direction."

William Check is a medical writer in Wilmette, Ill.