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Sizing up ‘mega’ multiplex panels for respiratory viruses

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Ann Griswold, PhD

May 2013—During the flu season of 2012, patients crowded the emergency room at the University of North Carolina (UNC) Health Care’s Memorial Hospital. They presented with a cough. Congestion. Low-grade fever. In some cases, a sneeze. But in a matter of hours, their clinical pictures diverged: Some patients deteriorated, requiring hospitalization; others remained congested but stable.

Until fairly recently, the race to distinguish serious from benign pathogens depended almost exclusively on viral and bacterial culture. Despite the technological advances of recent years, viral culture continues to be the gold standard in terms of specificity, but it’s done at the often-prohibitive cost of time.

Amplification-based technologies for detecting multiple pathogens were born of the necessity for accuracy, speed, and a clear view of the pathologic big picture. To date, the FDA has cleared several multiplex panels for detecting multiple respiratory viruses, paving the way for clinicians to treat not just the single most obvious infection but sometimes co-infections that play a subtle—or not so subtle—role in the patient’s outcome. Of these panels, only a handful can simultaneously detect six or more respiratory viruses: Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP FAST. These four represent the rise of the “mega” multiplex panel for respiratory pathogen detection, and a study published recently may well be the first to compare them to one another (Popowitch EB, et al. J Clin Microbiol. 2013;51(5):1528–1533).

The authors analyzed 300 patient specimens—including 200 retrospective and 100 consecutive nasopharyngeal swabs—using all four multiplex platforms. Their findings yielded individual profiles of the hands-on time, time-to-result, ease of use, and relative cost of each assay. The overall conclusion: Each assay has its tradeoffs.

Dr. Melissa Miller (right) with Elena Popowitch, MHS, medical laboratory specialist at UNC Health Care and coauthor of the multiplex study, at last month’s Clinical Virology Symposium. “Multiplex is absolutely a move in the right direction,” Dr. Miller says. [Photo: Julie Fletcher]

“My position has always been that there’s a place for the majority of these assays,” says senior author of the study Melissa Miller, PhD, of the Department of Pathology and Laboratory Medicine, UNC School of Medicine, and director of the molecular microbiology laboratory, UNC Hospitals. “It’s a balancing act between workflow, expertise in the laboratory, the patient population you’re serving, and the sensitivity you need to achieve.”

At UNC’s Memorial Hospital, where a large volume of patients flock to the institution’s Center for Transplant Care, Dr. Miller notes a strong push from clinicians to detect adenovirus infections as reliably and efficiently as possible. Of the respiratory viruses that threaten transplant patients, the adenovirus is counted among the most severe. Early detection can alert clinicians to the need for additional blood or urine tests to prevent systemic infection.

For that reason, UNC’s molecular microbiology lab uses the Genmark eSensor RVP, which relies on voltammetry to detect adenoviruses C and B/E, along with influenza A (H1/2009, H1, H3), influenza B, metapneumovirus (MPV), parainfluenza (PIV 1, 2, 3), respiratory syncytial virus (RSV A/B), and rhinovirus (RhV). While the Genmark panel works well for UNC’s large transplant population, and returns results in 7.2 hours, the comparison by Dr. Miller and colleagues illustrates that the panel is time- and labor-intensive, and probably not necessary for every setting.

Of the four panels studied, the Genmark eSensor RVP is more or less matched in complexity by the Luminex xTAG RVPv1 and xTAG RVP FAST. While both of the Luminex systems rely on bead-based hybridization and detection, the RVP FAST delivers results in about 4.8 hours, about three hours before the RVPv1. “The RVP FAST attempted to lessen post-amplified manipulations, which I think we’ll begin to see with other companies that have multiplex respiratory viral tests,” Dr. Miller notes. “But because of that, they lost sensitivity to influenza A and B, among other viruses.”

She and colleagues found that the eSensor RVP had an overall sensitivity of 98.3 percent and an overall specificity of 99.2 percent. The xTAG RVPv1 and RVP FAST had overall sensitivities of 92.7 percent and 84.4 percent, respectively, and overall specificities of 99.8 percent and 99.9 percent.

“The three more complex systems provide a high level of sensitivity at the cost of a complicated workflow and the need for molecular expertise,” Dr. Miller says. “I’ve talked to other laboratories that don’t have a large transplant population, so it’s probably not as critical that they lose some adenovirus sensitivity. For their labs, it’s more helpful to use a simple workflow that doesn’t require as much molecular expertise.”

On the other side of the country, Seattle Children’s Hospital is home to one such lab. The core laboratory at Seattle Children’s faced the onslaught of the 2012 flu season armed with a very different multiplex assay, the FilmArray, which detects 15 viral agents from respiratory samples in under 1.2 hours. An expanded FilmArray has since been cleared by the FDA to detect 17 viruses and three bacterial agents of respiratory infection: Bordetella pertussis, Mycoplasma pneumoniae, and Chlamydophila pneumoniae.

The FilmArray is simple and straightforward: An automated system extracts and reverse transcribes nucleic acid, then performs nested PCR in a single pouch. The closed system eliminates the risk of laboratory contamination. This feature also distinguishes FilmArray from most other multiplex assays, which require a technician to pipette post-amplified material. Cross-contamination during that pipetting step can significantly compromise a test’s specificity.

Not surprisingly, the UNC study found that the FilmArray had an overall specificity of 100 percent. Though FilmArray offers slightly less sensitive detection of adenovirus and influenza B compared with the three other multiplex assays in the UNC study, lowering the overall sensitivity to 84.5 percent, Film­Array’s streamlined procedure allows for a quick turnaround in urgent situations.

“FilmArray is one of the easiest and quickest multiplex panels to use,” says Xuan Qin, PhD, division chief of the microbiology laboratory at Seattle Children’s Hospital and author of a recent study in the American Journal of Clinical Pathology that describes the experience of implementing the Film­Array in the hospital’s core laboratory (Xu M, et al. 2013;139:118–123).

“The system is so well designed, it’s not actually what you think of when you picture multiplex,” Dr. Qin says. “This is a two-step PCR: It auto-extracts nucleic acid from the specimens, goes through the first step of multiplex PCR amplification, and then [the sample is] sent to a film array of 102 cells, each for a specific PCR.” To account for viral polymorphisms and increase sensitivity, each organism is covered by more than one target, and each set is triplicated. “It’s a very effective design,” Dr. Qin says. “When more than one target is positive for the same species, your sensitivity and specificity improve—the same principle behind our homebrew pertussis PCR.” (Qin X, et al. J Clin Microbiol. 2007;45:506–511).

Before implementing FilmArray, Seattle Children’s Hospital sent every respiratory specimen to a reference lab for direct fluorescence assay testing, a process requiring multiple steps—and bringing with it opportunities for error, Dr. Qin recalls. “It involved logging into our system and then transporting the samples to another lab and logging into their system. So there were two additional handoffs involved before we could report the results.” The entire process took about seven hours, at minimum.

After surveying 10 clinical laboratories across the United States and analyzing the daily workload at Seattle Children’s Hospital, the group invested in three FilmArray modules. The team opted to incorporate the system into the hospital’s core lab to ensure round-the-clock service for the hospital’s emergency department and urgent care center. It was a new experience for the lab’s 35 general medical technologists. “The core labs typically do CBCs and blood tests,” Dr. Qin says. “This was the first implementation of a diagnostic test for infectious disease.”

Though the technologists in the core lab had limited knowledge of microbiology, training was fairly straightforward: Faculty members, including Dr. Qin and study coauthor Min Xu, MD, PhD, program director of the core lab at Seattle Children’s, educated each shift of technologists about the basics of sterile technique, the importance of handling one specimen at a time under a biological safety hood, and practices specific to Film­Array such as the need to avoid bubbles when injecting patient samples into the reagent pouch. The instructors posted photographs on the board in front of the FilmArray analyzer to illustrate how the pouch should look if it is sufficiently filled versus insufficiently filled. An independent observer documented the technologists’ competency prior to the study.

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