ME multiplex panel: debating the tradeoffs

A 2017 case report showed that other patients may not be so lucky. The patient had a positive HSV-1 on an ME panel but showed no improvement with antimicrobial therapy. After being transferred to a different hospital where additional testing was performed, including PCR for tuberculosis, that testing was positive and the culture was positive for M. tuberculosis two weeks later (Gomez CA, et al. Open Forum Infectious Diseases, 2016).

The lesson: “Everyone was fixated on the positive HSV-1 as the cause of this patient’s illness and they stopped looking for anything else,” Dr. Alby said. “It wasn’t until the patient did not improve over the course of several days and was transferred to another hospital that the investigation continued. So this diagnosis was delayed because of the positive result.”

Dr. Dien Bard admits the ME panel has downsides. However, she argued that the benefits of the ME panel outweigh those limitations, and the cases Dr. Alby cited raise other issues worth noting. “I think the ME panel was a bit of a scapegoat in both cases,” she said. In the case of the false-negative HSV-1 patient, she contended that some fault may lay in the fact that the patient was presenting at a community hospital, where usually no infectious disease physicians are on staff and the care may not be at as high a level as at a tertiary care hospital—though, she noted, even at the tertiary care hospital, it took five days before ID was consulted and additional testing was ordered. “The patient was immunocompromised, moved from Vietnam 40 years back, and was at high risk for TB and Crytoptoccus infection. And these pathogens should have been on the differential diagnosis from the beginning.”

When she analyzed as many studies as she could find comparing the FilmArray HSV results against an alternative PCR assay, she found that for the most part “the results were pretty comparable.” Of almost 3,000 samples, there were nine false-negatives, one true positive, and three false-positives. “Most of the studies were just one or two off and often involved weak positives or preanalytical errors such as contamination,” she noted. And, she wondered, referring to the PCR test’s cycle threshold, “Is a Ct value of 45 positive really that significant clinically?”

The Mayo study finding of seven false-negatives was more significant. But Dr. Dien Bard wanted to highlight that discrepant results are assay- and site-specific. “It’s very dependent on what you’re using as a comparator method,” she said. “There are issues with the panel, but I think there is more to the story than just the fact that there was one test that made the biggest mistake—letting everything else off the hook.”

Her laboratory tested 32 samples with the exact same assay used in the Mayo study and picked up an additional HSV-positive on the ME panel, which was, after repeat testing on the direct alternative assay, found to be positive at a Ct of 39.8, a very, very weak positive, she said. “So at least in our institution we have not identified an issue with HSV sensitivity.”

Benefits like a significant decline in turnaround time—now at 2.4 hours at CHLA—outweigh the limitations of the ME panel, “which is why we’re still using the tests,” she said. One patient, a six-day-old, presented at an outside hospital where the HSV PCR was sent out on a CSF sample because it was not performed in-house. After the patient was transferred to CHLA two days later, the blood test was positive and the patient was treated with acyclovir, but died a week later. “The CSF result came back seven days later. I just don’t think that’s appropriate—seven days to wait for an HSV PCR result,” Dr. Dien Bard said.

A multicenter retrospective study on neonates using the Film­Array ME looked at 16 patients who were positive for enterovirus by the ME panel as well as conventional methods to determine whether providing the result early on would have an impact on length of stay. Nineteen percent of the patients were discharged in 24 hours, likely due to the 17-hour TAT for conventional method enterovirus, and an additional 69 percent would discharge in less than 48 hours. But shortening length of stay to zero is even possible. “We speculate that if we’re able to have a test that offers a rapid result like this and get a positive for enterovirus, you may not even have to admit the patient.”

If laboratories are finding a lot of false-positives with the ME panel, “you should look at your process and see how you can fix it,” Dr. Dien Bard advises. “It’s important to remember that the ME panel is a PCR test and you’re at risk for contamination like any other PCR test. The downfall of contamination is even more important because you’re testing CSF. So it’s extremely important to be aware of the risks.”

“Generally, to deal with potential contaminants, we review the Gram stain, the CSF parameters, the clinical presentation, and patient history. Always communicate with the provider, and if possible get an ID consult. If we need to, we’ll customize the reports to include comments.” Repeat testing is a thing of the past in her laboratory. “We don’t repeat-test any longer. If anything, we find it just adds to the confusion, where the result may be just a really weak positive or, due to some sampling variabilities, it’s just not picked up the second time. So we’ve eliminated that.”

She described the case of a child who was transferred from an outside hospital, together with a CSF sample, as an example of how the lab deals with false-positives and the importance of correlating with clinical findings and potentially other lab findings to avoid misdiagnosis. “It was a bloody CSF tap, so it wasn’t great. Streptococcus pneumoniae was detected and the ID attending at the time and I agreed that this is likely not consistent with bacterial meningitis. We decided to report it anyway because they couldn’t necessarily rule out ME, so we reported it with a cautionary comment that the sample could have been potentially contaminated.” This case illustrates that contamination is a true concern, Dr. Dien Bard said, but can be prevented with proper measures.

Based on the wide range of pros and cons, Dr. Alby and Dr. Dien Bard last considered this: Should multiplex panel testing for meningitis/encephalitis be restricted?

“The ME panel opens the door for many laboratories that did not have the skill set or the infrastructure to perform these types of higher-level molecular testing,” Dr. Alby said. “But the panel is not a test for all comers; it’s not for all institutions.” He believes CSF testing should be used only in limited circumstances with appropriate pre-test probability—that is, the probability of a patient having the target disorder—and ancillary testing services available. “And there needs to be some type of control over this test if it’s going to be used.”

A positive viral detection can be misleading, in part because some viruses are present in normal tissue, he noted. A patient who has a number of different primary CNS tumors, for example, may have HHV-6 in those tumors. The underlying concern are the tumors, not the potential presence of HHV-6 DNA in the CSF, he pointed out. “This is a good example of where detection of HHV-6 DNA in the CSF can lead someone down the wrong path.”

The same thing could happen with patients in whom the panel detects HSV-1, HSV-2, VZV, or HSV-6. If these patients had some other cause or some other viral infection, given the biology of herpes viruses, that infection or inflammation can produce low-level reactivation of those viruses, Dr. Alby said. “The virus is there, the DNA is there, and if our assays are detecting it and we don’t have appropriate controls and interpretations in place, clinicians will act on those results, even though they don’t implicate the causative agent of the ME.”

On the whole, meningitis/encephalitis is a rare event, Dr. Alby said. “Positivity in adults ranges from about one to two percent in terms of being able to diagnose the common meningitis viruses.” Seasonality is also a factor since time of year predicts positivity, as one study has shown with a chart of enterovirus results by date (Wilen CB, et al. J Clin Microbiol. 2015;53[3]:887–895).

So he believes in a more selective approach to ME testing. In the example reported in the same 2015 study, the institution set two criteria. “The patient had to have at least 10 white blood cells in their spinal fluid if they’re immunocompetent and then they could only do enterovirus testing if it was in the enterovirus season, which they classified as April through October.”

The effect of implementing this policy was that about a third of the tests for HSV-1/2, VZV, CMV, and enterovirus that were requested were not performed because they did not meet criteria. “This led to an annualized savings of more than $60,000 at that institution,” Dr. Alby said. An additional benefit, he noted, is that when fewer tests are performed where there is a low chance of positivity, “you won’t have to handle false-positive tests if you don’t perform the tests in the first place.”

Dr. Dien Bard, however, pointed out that meningitis can occur even in the absence of pleocytosis. She does not believe that multiplexed testing for ME should be restricted. “The approach at our institution is no restriction. Quite honestly, because the test was so new, we had no idea how to handle it, so we figured let’s just try it out and see what happens,” she said. “That being said, though, this is a test that we watch like a hawk, and it’s probably one of the few tests that we really communicate with physicians on.”

She cited two cases to illustrate why a lack of restriction was important. One was a six-week-old child with fever, irritability, and vomiting, with extremities poorly perfused, with tachycardia and tachypnea. “The CNS was completely benign and the white blood cell count was one. But this patient had enterovirus meningitis.”

A 34-day-old child with fever and fussiness also had completely normal CSF parameters, other than there being a lot of red blood cells in the CSF. This patient had parechovirus meningitis. As Dr. Dien Bard noted, a study of children under age 18 years demonstrated that in cases of bacterial meningitis, about 16 percent of the patients had a normal WBC count (Lin WL, et al. J Microbiol Immunol Infect. 2016;49[5]:723–728). In all of these cases, “if you were basing the decision to test solely on the presence of pleocytosis, the patient would not have been tested,” she said.

In fact, patients without CSF pleocytosis have a high rate of unfavorable outcome, another recent study showed. “In the adult population, particularly for HSV and meningoencephalitis, eight percent of the patients had an absence of pleocytosis, and the sequelae for them were high at 39 percent, with death for 21 percent” (Erdem H, et al. Int J Infect Dis. 2017;65:107–109). A CHLA study found, using the ME panel, that “Although pleocytosis was predictive of our ability to confirm bacterial meningitis in most cases, only about half of the patients who were positive for a viral target had any sort of pleocytosis.”

Restricting ME panel testing based on age, comorbidities, or other factors may make sense, Dr. Dien Bard noted. “I think that can be very institution-specific.” But she pointed to another reason why turning to a multiplexed panel for ME could appeal to institutions: cost-savings. A recent study developed an economic model to compare cost-effectiveness of three approaches—testing FilmArray ME on all CSF patients, performing standard-of-care testing, or Film­Array ME in the presence of pleocytosis (Duff S, et al. Future Microbiol. 2018;13[6]:617–629). “They found the cost per patient was lower when FilmArray ME was performed on all pediatric patients presenting with ME.”

Dr. Alby pointed out, on the other hand, that estimates of cost savings where more heavily multiplexed, more expensive tests are involved must take into account the cost of false-positives and treatment decisions that ensue from laboratory results. He cited a study that modeled the cost value of PCR screening for HSV, comparing the choice to screen all immunocompetent patients or only those with abnormal CSF findings. It found that increasing the significance of a positive test reduces the benefit of screening and the point at which pretest probability dictates screening varies depends on the cost of the PCR test (Hauser RG, et al. J Clin Microbiol. 2017;55[5]:​1566–1575).

As the point-counterpoint debate confirmed, critics and advocates of multiplexed panel testing for ME can agree that with ME patients, time to diagnosis is critical. But the best way to use panel testing to get to that diagnosis continues to involve tradeoffs that can be highly complex.

Anne Paxton is a writer and attorney in Seattle.