Multiplex PCR for Microbial Detection in Spinal Fluid (BIOFIRE FILMARRAY Meningitis/Encephalitis Panel)
Expires: July 1, 2022
Robin Patel, M.D., is the Division Chair of Clinical Microbiology in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. She holds the academic rank of Professor of Medicine and Microbiology.
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Transcript and References
Hi, I’m Bobbi Pritt, Director of the Clinical Parasitology Laboratory and Vice Chair of Education in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s “Hot Topic,” my colleague, Dr. Robin Patel, will discuss the FilmArray meningitis/encephalitis (ME) panel (BioFire Diagnostics). Dr. Patel will describe the panel and her experience with it, including an algorithm for its use. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patient’s health care.
Thank you for that introduction.
Dr. Patel reports grants from CD Diagnostics, Merck, Contrafect, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Contrafect, TenNor Therapeutics Limited and Shionogi. Dr. Patel is or has been a consultant to Curetis, Specific Technologies, Next Gen Diagnostics, Selux Dx, GenMark Diagnostics, PathoQuest, Heraeus Medical, and Qvella; monies are paid to Mayo Clinic. In addition, Dr. Patel has a patent on Bordetella pertussis/ parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. Dr. Patel receives travel reimbursement from ASM and IDSA and an editor’s stipend from ASM and IDSA, and honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course.
FDA-Approved Multiplex PCR Assay—Spinal Fluid BioFire FilmArray ME Panel
The BioFire FilmArray ME panel is a U.S. Food and Drug Administration approved and cleared multiplex PCR test that detects DNA or RNA of 15 pathogens, including 7 viruses, 6 bacteria, and 1 yeast from spinal fluid. Specifically, the assay detects cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella zoster virus, Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, and Cryptococcus neoformans/gattii.
Herpes simplex virus 1 is one of the most common causes of viral encephalitis, and herpes simplex virus 2 is a significant cause of meningitis. Enteroviruses are also common causes of meningitis and encephalitis.
Cytomegalovirus is nearly ubiquitous in the population worldwide, and a rare cause of central nervous system infection. Likewise, human herpesvirus 6 is very common in the population worldwide, causing roseola in infants, but central nervous system infection is rare and most commonly reported in severely immunosuppressed individuals. Human parechoviruses can rarely cause encephalitis or meningitis, and varicella zoster virus is another occasional cause of encephalitis or meningitis.
E. coli K1 and S. agalactiae cause neonatal meningitis, and H. influenzae, N. meningitides, and S. pneumoniae are vaccine-preventable causes of acute bacterial meningitis. L. monocytogenes is a foodborne illness that can result in meningitis in immunosuppressed patients, pregnant women, neonates, fetuses, and the elderly.
C. neoformans and C. gattii are causes of central nervous system infection usually in immunocompromised hosts.
The BioFire FilmArray ME panel was approved and cleared by the FDA in 2015 and can be performed on one of several instruments. Here, the test is shown being performed on a FilmArray Torch instrument. Hydration buffer and the sample in sample buffer are loaded into a reagent pouch, and then the pouch is placed on the instrument. Testing takes about an hour.
Evaluation of BioFire FilmArray ME Panel
We evaluated the panel using 291 cerebrospinal fluid specimens originally submitted to our laboratory for routine microbiology testing that were positive for at least one target represented on the panel (e.g., by bacterial culture or individual real-time PCR assays). Following routine testing, samples were refrigerated or frozen until testing by the panel was performed. For the majority of specimens that were positive for viral or C. neoformans/gattii targets, the storage period was a week. However, the majority of specimens (76 out of 80) were positive for a bacterial target that had been stored frozen for three decades (with collection dates ranging from March 1975 to June 1997; 58 out of 76 [76%] of which were collected between 1980 and 1986).
The overall percent positive agreement was 90.1% for viruses, 97.5% for bacteria, and 52% for C. neoformans/gattii.
Comparison of BioFire FilmArray ME Panel to Routine Testing
161 samples were selected due to a virus being identified during routine testing. Of these, a virus was correctly identified in 145. The panel was uniquely positive for a viral target in 5 samples, including 1 each for herpes simplex virus 1 and 2 and varicella zoster virus and 2 for human herpesvirus 6. The panel was negative for a viral target in 16 specimens that were positive for a virus, including 2 positive for enterovirus and 7 positive each for herpes simplex virus 1 and 2 by routine testing.
After discordant analysis with additional testing, the adjusted, overall percent positive agreement for viral targets was 94.8% (147 out of 155).
Comparison of BioFire FilmArray ME Panel to Routine Testing, continued
80 specimens were positive for a bacterial pathogen by routine testing, and these pathogens were accurately detected by the panel in 78 (97.5%). Target-specific positive percent agreements ranged from 90% for N. meningitidis to 100% for S. agalactiae and E. coli K1. S. pneumoniae was uniquely detected by the panel in three specimens, while S. agalactiae, N. meningitidis, and L. monocytogenes were each detected in 1 specimen that was negative for these targets by routine methods. Interestingly, unique detections of S. pneumoniae, N. meningitidis, and L. monocytogenes were part of coinfections, as determined by the panel. After discordant analysis, the adjusted, overall percent positive agreement for bacterial targets was 100%.
The panel was positive for C. neoformans/gattii in 26 of 50 specimens (52%) that were positive for cryptococcal antigen by a lateral flow assay. The percent positive agreement was 92.3% (12 out of 13), however, when results were compared to those of routine fungal smear or fungal culture.
Apparent coinfections were detected in 8 of 291 (3%) samples tested. In 7 of the 8 cases, the expected, previously characterized target was detected. For the additional targets detected, discordant analysis and medical record review were performed, where possible. In case 1, which was positive for H. influenzae by counter immunoelectrophoresis, L. monocytogenes was also detected by the panel. The additional detection of L. monocytogenes was unable to be confirmed due to the lack of an available confirmatory test and no access to the patient’s medical record. Among the next six cases, four of the additional detections (including the two additional detections of S. pneumoniae in cases 2 and 3, the additional detection of human herpesvirus 6 in case 4, and the additional detection of herpes simplex virus 1 in case 5) were not confirmed by alternate molecular testing. In two cases, an additional target (human herpesvirus 6 in case 6 and varicella zoster virus in case 7) for which routine testing was not initially ordered was detected by an alternate molecular test. Finally, in case 8, testing using an alternate molecular method confirmed the findings of S. pneumoniae and N. meningitidis found by the panel.
Detection of multiple pathogens in cerebrospinal fluid should be considered a rare event, and coinfections reported from this source by multiplex molecular tests should be interpreted with caution and ideally confirmed using a second assay.
There are several limitations to the BioFire FilmArray ME panel. Human herpesvirus 6 and cytomegalovirus do not typically cause central nervous system infections in immunocompetent hosts, so their detection should be interpreted accordingly. Moreover, human herpesvirus 6 may be detected as a result of germline integration. Many organisms that cause central nervous system infection are not part of the panel, including, but not limited to, Staphylococcus epidermidis, Cutibacterium acnes, many Gram-negative bacilli, Mycobacterium tuberculosis, Borrelia burgdorferi, Histoplasma capsulatum, Coccidioides immitis/posadasii, Blastomyces species, West Nile virus, Powassan virus, Jamestown Canyon virus, California encephalitis virus, Eastern equine encephalitis virus, St. Louis encephalitis virus, Western equine encephalitis virus, Ehrlichia species, Naegleria fowleri, Balamuthia mandrillaris, and Toxoplasma and Acanthamoeba species, to name a few.
False-positive results for S. pneumoniae have been attributed to amplified DNA contamination or S. pneumoniae colonization of health care workers or laboratory staff handling samples. False-positive results can lead to the real diagnosis being missed or delayed. For example, a case of tuberculous meningitis was diagnosed late because of a false-positive herpes simplex virus 1 result from an ME panel.
ME Panel Algorithm
The algorithm shown is a general guideline for testing in patients with findings of acute symptoms (i.e., symptoms for fewer than eight days) of meningitis or encephalitis. It does not apply to those with a cerebrospinal fluid shunt or central nervous system surgical site infection. Blood cultures should be obtained. Lumbar puncture should include measurement of opening pressure. Cerebrospinal fluid should be submitted for cell count and differential, protein, glucose, Gram stain, bacterial culture (with susceptibility testing), and, if indicated by patient risk factors, cryptococcal antigen testing. In a previously healthy patient, if the following is available on-site, it is recommended to perform a pneumococcal antigen, and herpes simplex virus and enterovirus and possibly varicella zoster virus PCR. If such testing is not available, or if the patient is immunocompromised or has received recent or current antibiotics, the ME panel is recommended.
If results are negative, additional testing should be considered based on clinical presentation, seasonality, geographic location, exposure history, prevalence of disease, cerebrospinal fluid parameters, and patient risk factors. Ideally, extra cerebrospinal fluid should be collected during the lumbar puncture to enable such additional testing if results of the panel testing are not informative. Examples of additional tests are shown here and on the next slide.