Identifying respiratory pathogens: Pneumonia panel studied against standard of care

Sherrie Rice

December 2020—In an evaluation performed at Washington University in St. Louis and published recently, BioFire’s FilmArray pneumonia panel was found to have strong agreement with standard-of-care methods in identifying viral and bacterial targets in 200 lower respiratory tract specimens (Webber DM, et al. J Clin Microbiol. 2020;58[7]:e00343–20). It was also found to have strong agreement with the BioFire upper respiratory panel for common targets, making it unnecessary to perform both.

In comparison to standard-of-care methods, it has the potential to detect more Staphylococcus aureus and Haemophilus influenzae and to detect more antimicrobial resistance, particularly at low organism concentrations or in mixed cultures.

Those are the findings reported in a CAP TODAY webinar (captodayonline.com) on the implementation of multiplex PCR assays for pneumonia, made possible by a special educational grant from BioFire.

“Pneumonia is a clinical and diagnostic challenge because the etiologic agents can be variable, the conventional diagnostics are slow, and the long window of diagnostic uncertainty combined with the increase in antibiotic resistance can lead to overly broad spec­trum empiric antimicrobial therapy,” Carey-Ann Burnham, PhD, D(ABMM), professor of pathology and immunology at Washington University and medical director of the Barnes-Jewish clinical microbiology laboratory, said in the Sept. 15 webinar.

“Interpretation of our diagnostic tests can be challenging,” she said, “because many of the bacteria that cause pneumonia are also common colonizers of the respiratory tract, and health care providers may need to order multiple diagnostic tests and laboratories may need to have available many methods for a comprehensive patient evaluation.”

The methods used today to diagnose bacterial pneumonia are culture based. In the laboratory at Barnes-Jewish Hospital, Dr. Burnham and Neil Anderson, MD, ABP-MM, D(ABMM), use qualitative culture for sputum and quantitative respiratory culture for other lower respiratory tract specimens, such as tracheal aspirate or bronchoalveolar lavage.

“We have multiple pieces of media with variable lengths of incubation. There is much interpretation required from our technologists,” said Dr. Anderson, assistant professor of pathology and immunology at Washington University and medical director of the Barnes-Jewish molecular infectious diseases laboratory, who co-presented and asked, “Is there a better way?”

The pneumonia panel and the BioFire upper respiratory pathogen panel have some targets in common, but “what’s new about the pneumonia panel are the 15 bacterial targets that are semiquantitative in nature,” Dr. Anderson said. Also new are the antimicrobial resistance determinants, both for Gram-positive organisms (such as mecA/C) and the multiple Gram-negative antimicrobial resistance determinants.

“This is a new and a different panel compared with what we have seen in the past,” he said, “and one of the things that’s new about it is the semiquantitative nature of reporting.” Bacterial organism abundance is estimated based on real-time PCR relative to an internal standard. Quantitation is reported in 1-log bins, ranging from 10⁴ to ≥10⁷ copies/mL.

Dr. Burnham, Dr. Anderson, and colleagues evaluated the pneumonia panel’s performance in comparison with standard-of-care testing. “We took 200 consecutively available lower respiratory specimens, collected from July 2018 to November 2019, with an entire flu season in that interval,” Dr. Anderson said. All patients had aerobic culture performed on their specimen and, depending on what the provider thought was necessary, a BioFire upper respiratory panel (RP2), a PCR test for other viral targets, and any other additional infectious disease workup as needed—all performed as part of standard-of-care testing.

They ran the BioFire pneumonia panel on the 200 specimens and compared the results of the panel with what was resulted from standard-of-care testing.

The average age of the patients was about 60, with a slight male predominance. “Some were immunocompetent, some were immunocompromised, some were in the ICU, some were in the ED. We had a good representation of different patients in our hospital,” Dr. Anderson said. Of the 200 specimens, 59 were bronchoalveolar lavage, 11 bronchial wash, 54 sputum, and 76 tracheal aspirate.

A viral or bacterial target was detected in 117 of the 200 specimens (58.5 percent), with more than one pathogen detected in 43 percent of the positive specimens. “So not only are many of these specimens positive, but many of the positives have multiple organisms detected.”

“The real question,” Dr. Anderson added, “is how this stacks up against the standard-of-care testing.” For the viral targets, there were nine instances of viral infections detected by standard-of-care testing for pathogens not on the pneumonia panel: five HSV and four CMV. “It is important to keep in mind that the panel doesn’t necessarily cover everything,” he said.

There were also nine instances of extra viral infections detected in which standard-of-care testing was not ordered. “These were cases in which the provider may have not been thinking about a viral etiology. But by running this broad panel, we were able to show positivity for a virus that may not have been suspected.” The clinical relevance of that detection depends on the situation, he noted, but in some of these cases, the finding may have been important.

When the target was tested for by standard-of-care methods and it was also in the pneumonia panel, they typically agreed.

For the bacterial targets, the story is similar, Dr. Anderson said. There were eight instances of bacterial detections by the standard of care for organisms not on the pneumonia panel, half of which were Stenotrophomonas maltophilia. (The others were Citrobacter freundii complex, Acinetobacter spp., Staphylococcus intermedius group, and Klebsiella variicola.) “However,” he said, “there were very few instances of standard-of-care positivity and BioFire negativity for the on-panel targets. If it was there by the standard-of-care method and it was included in the panel, the panel picked it up, suggesting a good negative predictive value.”

“What was interesting is that we had many instances”—92—“of unique pneumonia panel detections, the majority of which were Staphylococcus aureus and Haemophilus influenzae. One of our hypotheses,” he said, “was that perhaps these unique pneumonia panel detections were linked to either microorganism burden in the specimen and/or specimen type. So maybe there wasn’t enough there for the standard-of-care methods to detect it.”

For bacteria that were uniquely detected by the pneumonia panel, this was more common at low microorganism burden in the sample. “If there was not a lot of it there, it was more likely to be detected only by the BioFire.” For Staphylococcus aureus, the unique detections were more common in potentially polymicrobial specimens, such as sputum-type specimens. “It could potentially be that the S. aureus may be interpreted as part of normal microbiota in those specimen types and potentially overlooked,” Dr. Anderson said.

What is the significance of the unique pneumonia panel detections? Do they behave more like patients in which they were standard-of-care positive or patients in which they were standard-of-care negative?

They compared their sole detections to instances in which both methods were positive and instances in which both methods were negative, in terms of length of hospital stay and 30-day mortality, for S. aureus and H. influenzae. “The long and the short of it is that we didn’t have significant differences in length of stay or 30-day mortality for S. aureus or H. influenzae. However, our study wasn’t powered to answer this question, and it’s something that is in need of further research,” Dr. Anderson said.

When they looked at the methicillin-resistant S. aureus callout versus methicillin-susceptible S. aureus, the finding was similar. There was a lot of concordance, but the pneumonia panel detected more MRSA than culture. Eleven instances of MRSA detection were unique to the BioFire panel, and these were more common in polymicrobial samples. “The failure to recognize the resistant clone in mixed population in culture may explain these results,” Dr. Anderson said.

“We had a similar story when we looked at antibiotic resistance. When resistance was present, the pneumonia panel was very good at picking it up. We had great agreement between the pneumonia panel and standard of care in that regard. However, we did have 11 instances of unique detection of resistance in which a resistance organism was not identified by standard-of-care testing.”

In one instance, Klebsiella pneumoniae carbapenemase was detected in a tracheal aspirate. “By our standard-of-care method, this was called greater than 100,000 CFUs per mL of yeast and bacterial microbiota below the threshold for workup,” Dr. Anderson said. “The pneumonia panel gave results that also suggested a mixed population of microorganisms. We had Enterobacter cloacae and Pseudomonas aeruginosa, but we also had that KPC in there. I would argue that this is a very significant finding and a potential win for the pneumonia panel. For infection control implications alone, KPC is not something you want to miss.”

Dr. Anderson, Dr. Burnham, and colleagues compared the BioFire pneumonia panel to the BioFire upper respiratory panel when performed on the same specimen. “The reason we wanted to do this is because our lab, similar to many other labs, has validated the BioFire upper respiratory panel for off-label testing on lower respiratory specimens. There aren’t a lot of options for broad pathogen testing of lower respiratory specimens, so we have been using the upper respiratory panel for this purpose following appropriate validation.”

“Now that we have a large panel for lower respiratory testing, is it necessary to run both?” he asks. “Is there one that’s better than the other for specific targets, and, if so, we want to know that so we can adjust our workflow appropriately.” They tested the 200 specimens by the BioFire upper respiratory panel (RP2) and compared that to the pneumonia panel results. “We wanted to make sure we were comparing apples to apples, so both of these tests were performed on exactly the same specimen and then we just simply compared results.” There was strong agreement between both panels, with only eight discrepant results out of the 200 samples tested, for a 96 percent overall agreement for targets common to both panels.

Does the discrepant analysis favor one assay versus the other? “The short answer is no,” he said. “We had unique detections in favor of the pneumonia panel, and we had unique detections in favor of the upper respiratory panel. So this can be interpreted as there not being any difference in performance for the shared targets between the two panels. And that translates to no benefit in performing both assays in parallel on the same specimen. That’s an important piece of data we all need to keep in mind when we put together our workflows.”

The final data Dr. Anderson reported was the projected improvement in turnaround time with the pneumonia panel. Their study found it took about 44 hours to get an organism identification and another 14 hours to get phenotypic susceptibility testing with conventional methods. When they run a pneumonia panel, results are out in about 2.3 hours following specimen receipt. “In our study we performed the pneumonia panel retrospectively, so the estimated turnaround time was based on RP2.0 [upper respiratory pathogen panel] testing.”

“This is an estimated 42-hour savings in the amount of time it takes to turn around an organism ID, and a 56-hour savings in how long it takes to turn around a susceptibility.”

Dr. Burnham cited a study published this year that estimated the potential impact of the finding of good analytical performance characteristics for the pneumonia panel compared with standard-of-care methods on early modification of antibiotic therapy (Buchan BW, et al. J Clin Microbiol. 2020;58[7]:e00135–20). “And I say potential impact because this was theoretical based on retrospective review,” Dr. Burnham said.

“The findings were striking although not entirely surprising,” she said. “They found that in nearly half of patients that antimicrobials could be appropriately de-escalated or discontinued based on having the result more quickly. In a small proportion of patients, about four percent, the antibiotics should have been escalated or initiated, and although that’s a relatively small number of patients, I think that’s really important. We know that if there are delays in appropriate therapy, outcomes are compromised.”