ME multiplex panel: debating the tradeoffs

Anne Paxton

June 2019—Meningitis and encephalitis have been called by some the most terrifying diseases in medicine, in part because of the difficulty of diagnosing their underlying pathology. The clinical stakes of laboratory testing are high for diagnosing and treating the inflammation that meningitis/encephalitis (ME) causes within the central nervous system. While an FDA-approved panel exists for molecular testing of cerebrospinal fluid for viruses, bacteria, and one fungus known to be associated with ME, questions surround the panel’s use.

BioFire’s FilmArray ME panel, which detects 14 targets directly from CSF obtained by lumbar puncture, is one of the most recently approved syndromic panel-based tests. But as the causes of ME in different patient populations have shifted in recent years, studies are showing, the bacterial targets included in the panel increasingly fail to reveal the etiology of a patient’s disease. At the same time, nucleic acid amplification testing is known to fall short as a testing modality for the significant viral causes of ME.

In a point-counterpoint presentation at the Association for Molecular Pathology annual meeting last year, panelists debated this question: “Meningitis/Encephalitis Syndromic Testing in the Clinical Setting: Is It Ready for Prime Time?” Jennifer Dien Bard, PhD, D(ABMM), and Kevin Alby, PhD, D(ABMM), took on three topics: whether panel testing is appropriate when diagnosing ME, how serious the risks are of false-positives and false-negatives, and whether multiplexed panel testing for ME should be restricted.

The ME panel tests for six bacteria (E. coli K1, H. influenzae, S. pneumoniae, L. monocytogenes, S. agalactiae, and N. meningitidis), seven viruses (HSV-1 and -2, enterovirus, HHV-6, VZV, HPeV, and CMV), and the fungus C. neoformans/gattii. But the panel approach does not meet the current epidemiology of ME, so the chances of false-positives and false-negatives are significant, said Dr. Alby, currently associate director of clinical microbiology at the University of North Carolina.

Overall, bacterial meningitis rates have been dropping, and at least two of the panel’s targets are falling off the grid as important causes of ME. “There’s been a precipitous decline in both pneumococcal and meningococcal meningitis as the causative agent of bacterial meningitis in the U.S.,” he said, crediting vaccination programs as the primary reason. In addition, two organisms or groups of organisms missing from the ME panel are significantly related to ME. “Gram-negative meningitis and staphylococcal meningitis are now equal to or eclipsing meningococcal meningitis” as causes of ME, said Dr. Alby, citing a chart of the incidence of bacterial meningitis by organism in the U.S. from 1997 to 2010 (Castelblanco RL, et al. Lancet Infect Dis. 2014;14[9]:813–819). “Many other bacteria are more likely to be causes of ME in many patient populations.”

He noted that infections often stem from neuroinvasive procedures and are considered health care associated. Over an 11-year period at one hospital, there were about 80 bacterial infections and half were community onset, half health care associated (Srihawan C, et al. J Am Geriatr Soc. 2017;65[12]: 2646–2650). The pathogens linked to health-care–associated infections “are just as important but are not present, by and large, on the panel.”

Obtaining a negative FilmArray ME panel, then, does not indicate that the patient can be taken off antibiotics because of the other potential bacterial pathogens. “Just because you have a negative ME test, you have not excluded infectious meningitis or encephalitis,” he pointed out. In fact, in nearly 70 percent of cases of ME, whether acute or subacute, the causative agent of the diagnosis is unknown. Histoplasma, other fungi (native or iatrogenic), and other bacteria, especially with indwelling devices or zoonotic exposures, should also be considered.

Viral causes, in addition, are a significant cause of morbidity and mortality but cannot be diagnosed by nucleic acid methods, Dr. Alby said, citing arboviruses as a prominent example. In the Northeast, there was a particularly high rate of West Nile virus in 2016 (Burakoff A, et al. MMWR Morb Mortal Wkly Rep. 2018;67[1]:13–17). In Pennsylvania, “through 2018 we’re already fourfold above our moving mean over the last five years.” If those patients come in with encephalitis, they may get tested and be found to be negative for a viral pathogen, then go undiagnosed. “They may not get that additional testing they need to diagnose their disease.”

So just using the ME panel with targeted organisms on it is not enough and even carries dangers, in his view. “It is not an exhaustive panel by any stretch of the imagination,” Dr. Alby said. “We still don’t know what’s going on because we don’t know a lot about this disease process.”

But Dr. Dien Bard, director of clinical microbiology and the virology laboratory at Children’s Hospital Los Angeles (CHLA), takes a different view based on her laboratory’s experience using FilmArray ME on 745 patients over more than two and a half years, with an overall positivity of 12.3 percent. “Panel testing is appropriate when diagnosing meningitis/encephalitis because it can rule out the majority of the pathogens of concern,” she said. Enterovirus is the No. 1 cause of meningitis that is identified, followed by bacterial meningitis, she noted. She believes a lot of the specific pathogens involved are included within the Film-Array ME panel. Adding herpesvirus as a cause “accounts for about 73 percent of all cases that you would see” (Hasbun R, et al. Clin Infect Dis. 2017;65[3]:​359–363).

That there are other significant causes of meningitis not included in the panel is not an argument against using the panel, she said. “If anything, the panel allows the provider to comfortably rule out meningitis caused by such pathogens as enterovirus or Streptococcus pneumoniae. The clinician can then investigate other etiologies based on the patient’s clinical presentation.” Enterovirus and bacterial meningitis are the top two causes of ME in pediatric patients, so the pathogens included in the panel are also highly relevant for children (Hasbun R, et al. Ped Infect Dis J. 2019;38[1]:37–41).

Contrary to Dr. Alby’s argument, she said, common pathogens in community-acquired CNS infections are included in the FilmArray ME panel. A study of 2,500 patients in 20 countries looked at community-acquired CNS infections and found “there were two top pathogens recovered: S. pneumoniae, which is part of the panel, and M. tuberculosis.” In addition, “within the groups of the elderly and the immunocompromised, Listeria, also in the panel, was a significant finding, along with VZV and S. pneumoniae.”

Some targets are not included in the panel, she conceded. “But this is not a standalone test. It’s not going to provide you with all the answers. However, it would help for a great majority of the most common viruses and bacteria that you would want to rule out in the beginning of the patient workup.”

Before CHLA brought in the Film­Array, the majority of positives found were on target pathogens included in the panel, she said. “There are a variety of others that are identified, but this would of course require alternative samples, serological testing, etc.” Post implementation, with standard-of-care testing, all targets detected within the patient population of 135 were included in the panel. “In the patients that did not have a FilmArray ME panel, we had a total of three pathogens that were identified, two of which were targets included in the panel and an Enterococcus faecium, which is not included in the panel.”

Dr. Dien Bard believes that underuse of current diagnostic testing may be indicating misleadingly a decline in certain pathogens. “The decline may be due to the fact that the appropriate tests are just not being ordered,” she said. A study of test orders showed that enterovirus PCR was performed only for about 60 percent of all patients presenting with aseptic meningitis in adults, and for 74 percent of children. “For HSV PCR,” she said, “only about 39 percent of adults were tested, and only 16 percent of children.” A great number of patients still did not have any sort of diagnosis (Shukla B, et al. J Clin Virol. 2017;94:110–114). Only 18.5 percent of the infectious etiologies were identified, and Dr. Dien Bard believes this may be due to test ordering patterns: “If you’re not going to look for it, you’re not going to find it.”

A U.K. study published last year showed that prospective CSF testing on all of 638 patients showed a much higher positivity rate than is seen in other published reports; she believes that is “because they’re testing it.” With the targets that they performed on every single sample, “There are 29 cases of Neisseria meningitidis out of the 638, which is nothing to scoff at. The same with S. pneumoniae. The fact that they were able to identify these pathogens emphasizes the need for these types of broad-spectrum panels” (McGill F, et al. Lancet Infect Dis. 2018;18[9]:992–1003).

Standardizing ordering practices is necessary, Dr. Dien Bard said. “There’s definitely no rhyme or reason in how certain tests are ordered or not ordered, and I think that’s one of the main reasons why we’re not confirming more cases.” A study of the epidemiology of CNS infections in 358 children admitted to CHLA showed that etiology was identified in almost 60 percent of the patients. And, she noted, “all of the pathogens that were identified by standard-of-care methods, except for one Staphylococcus aureus, were included in the FilmArray ME panel” (Felsenstein, et al. Poster presentation at 25th ECCMID Congress, 2015).

For another patient at CHLA who had amyotrophic lymphocytic leukemia, the FilmArray ME identified CMV. The eventual diagnosis—meningoencephalitis as well as CMV retinitis—“was identified and treated appropriately purely because of this FilmArray result that we were able to provide them right away,” Dr. Dien Bard said, showing the value of the FilmArray ME in detecting other significant pathogens associated with ME. “I do believe that a variety of these pathogens we’ve identified in our patients would not have been identified had we not performed the FilmArray ME, because even though we offered laboratory-developed tests for all the viral targets, the tests were rarely part of the initial sets of orderables.”

“With these types of panels, you may actually increase the detection of bacteria that’s not being recovered by culture. Inclusion of viral targets in the panel may also increase the chances of detection,” she added.

In the decision to use or not use the FilmArray ME, should the risks of false-positives and false-negatives be a deal-breaker? That was the second question Dr. Alby and Dr. Dien Bard addressed.

The performance characteristics of the current ME panels, Dr. Alby said, increase the chance for patient harm. “Why do I say this? Well, there are high rates of false-positive bacterial detections that could lead to inappropriate antimicrobial therapy, false-positive viral detections can distract from other potential causes of infection, and then false-negative results, of course, can prolong diagnosis or inappropriately stop antimicrobial therapy.”

A large multicenter evaluation of the ME panel showed that despite careful attention to standard of care and the discrepant analysis, 43 samples had results that were not present in the standard-of-care testing, a few falsely negative and a larger majority falsely positive (Leber AL, et al. J Clin Microbiol. 2016;54[9]:2251–2261). Extra detections by the FilmArray ME may be picking up more pathogens, he said. “But they are as likely to be a false-positive as to be a true positive.”

Dr. Alby believes traditional testing works well. During his time at the Hospital of the University of Pennsylvania, he and colleagues evaluated previous positive CSF samples using the ME panel and confirmed five HSV-2, four enterovirus, and eight varicella-zoster virus. As expected, there were three additional targets detected—one HSV-2, one CMV, and one H. influenzae—that were negative upon repeat testing. The lab then tested 44 samples semi-prospectively. Most were negative for all targets, but two were positive for Cryptococcus, confirmed by culture; one was positive for enterovirus, confirmed by PCR. A sample that was positive for human herpesvirus 6 was confirmed by a secondary PCR test.

But false-positives posed problems. “The thing that held us back were the last two—one that was positive for Haemophilus influenzae and one that was positive for Streptococcus pneumoniae, neither confirmed by culture.” Repeat ME testing showed a negative result in both cases. “Then we looked at what was going on with the patients,” he said.

The patient with positive H. influenzae presented with altered mental status and left-sided weakness, and was discharged with a diagnosis of stroke, never given antimicrobial therapy, and recovered as expected for someone who had a stroke. The patient with false-positive S. pneumoniae had no pleocytosis and had a history of cutaneous T-cell lymphoma; that patient was discharged to hospice with no definitive diagnosis because other findings made clinicians think this was a transformation of the T-cell lymphoma.

False-negatives also carry dangers. A Mayo study of the ME panel on archived samples showed relatively good performance with strong positive agreement of the ME results with previously characterized samples for a number of targets. The ones raising concern were the HSV-1 target and the Cryptococcus target, Dr. Alby said. There was a particularly large number of false-negative Cryptococcus results. In one case, negative by the ME panel but positive by cryptococcal antigen and culture, “the patient was actually sent home, because they had a negative ME panel and when the bug started growing, they were called back to the hospital to be admitted and treated.”

In the second case, the clinician did not believe the result and requested a separate cryptococcal antigen test to be done concurrently with the ME panel; that test was positive. There was no change in therapy, however, because the physician didn’t believe the result initially (Liesman RM, et al. J Clin Microbiol. 2018;56[4]:e01927-17).