CONTINUING EDUCATION
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LEARNING OBJECTIVES
1. Describe the current symptoms and laboratory tests which aid in the diagnosis of CNS disease.
2. List new testing methods which serve for quick and accurate identification of the microorganisms that cause CNS disease.
3. Discuss the factors that will be addressed with the use of molecular testing in the diagnosis of CNS disease.
4. Discuss the conclusions of the research studies that are included in the article.
Ideas to improve turnaround time and increase diagnostic yield
Infections of the central nervous system (CNS) such as meningitis or encephalitis can be caused by myriad microorganisms and may be life-threatening. Patients with acute CNS infections generally present with similar findings of fever, headache, and neurological changes. Given the similarity in symptomology, it is often difficult to distinguish bacterial and viral infection based on clinical presentation alone. As a result, obtaining a rapid and accurate diagnosis is important for proper patient management. Indeed, rapid identification of CNS pathogens is critical for antimicrobial treatment in cases of bacterial or herpes simplex virus (HSV) infection.1,2 Any delays in appropriate therapy can lead to poor patient outcomes, including death.3-5 The aim of this Continuing Education article is to review the current landscape for diagnostic testing of cerebrospinal fluid (CSF) in acute CNS infections, present the potential impact of rapid identification, and discuss methods to increase the diagnostic yield in uncertain cases. It is anticipated that new technologies will aid in providing rapid and accurate pathogen identification, potentially leading to better patient outcomes, improved antimicrobial stewardship, and decreased hospital costs.
Current diagnostics for meningitis/encephalitis
Analysis of patient CSF is crucial for diagnosis and management of CNS infection. For cases of acute meningitis/encephalitis, CSF is obtained via lumbar puncture (LP). Clinical findings such as cell count, glucose, and protein concentrations can provide clues regarding infection.6 Ninety percent of patients with bacterial meningitis will exhibit elevated white blood cell (WBC) counts > 100 WBC/mm3, and approximately 60 percent will have cell counts greater than 1,000 WBC/mm.3,7 WBC counts should be normal to elevated (0-500 cells/mm3) with viral, fungal, or tuberculosis meningitis. Glucose concentrations may be decreased in bacterial, fungal, or tuberculosis meningitis, but should be normal in cases of viral infection.8 Protein concentrations are generally elevated in all cases. These differential indicators are useful to aid in diagnosis, but because of overlap between etiologies, they are not sufficient to guide targeted therapy.
The standard for the diagnosis of bacterial meningitis/encephalitis is Gram stain and CSF culture.9 Gram stain has a sensitivity between 60 percent and 90 percent, while CSF culture can detect a microorganism in a majority of bacterial cases, provided that the patient has not received antibiotics prior to lumbar puncture.9 The sensitivities of CSF culture and Gram stain decrease after antibiotic therapy.9 Other methods for detection and identification of bacterial pathogens such as direct antigen testing or polymerase chain reaction (PCR) are not yet commonly used as first-line diagnostics, but have recognized value in patients with clinical findings consistent with a bacterial CNS infection who have negative Gram stain and culture results.9 The U.S. Food and Drug Administration (FDA) has cleared tests for both methods that target the most commonly CNS-infecting bacteria such as Group B Streptococcus (GBS), Haemophilus influenzae, S. pneumoniae, Neisseria meningitis, Listeria monocytogenes, or Escherichia coli K1.10,11
In contrast to bacterial detection, PCR has become the standard for detecting viruses associated with aseptic meningitis or encephalitis.12 Depending on the age and immune status of the patient, many different viruses can cause infection, including HSV, varicella zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), enterovirus (EV), and parechovirus (HPeV).13 Additional viruses that may be associated with CNS disease but are less frequently observed in immunocompetent adults include HIV and arboviruses, such as West Nile, St. Louis encephalitis, Eastern equine encephalitis, and Japanese encephalitis viruses. In some cases, serological testing may be more appropriate for suspected arbovirus infections, since immunocompetent patients may not have these viruses in their CSF at the time of presentation.14
Given the importance of these pathogens in CNS disease, it is perhaps not surprising that many highly sensitive and specific laboratory-developed and commercial, FDA-cleared tests are available for viral CSF testing.12,15,16 In addition to these single-target or multiplex viral detection assays, a rapid commercial multiplex PCR test for the detection of 14 pathogens (six bacteria [E. coli K1, GBS, H. influenzae, L. monocytogenes, N. meningitis, and S. pneumoniae], seven viruses [CMV, EV, HHV-6, HPeV, HSV-1/2, and VZV] and two fungi [Cryptococcus neoformans and C. gattii]) received FDA clearance for use as an aid to diagnosis of meningitis/encephalitis in October 2015.17,18 This closed multiplex PCR system demonstrated analytical limits of detection (LOD) around 103 gene target copies/mL and prospective clinical sensitivities ≥ 95.7 percent for most FDA-cleared targets. Clinical specificities were ≥ 99.2 percent for all assays.19 The addition of this type of panel-based syndromic testing to CNS disease allows detection of a wide range of pathogens in a clinically actionable timeframe (about one hour) and has demonstrated high diagnostic value with other infectious disease syndromes.19-21
Benefits of a rapid turnaround time
Due to the potential severity of CNS disease, early and effective treatment is critical to reducing morbidity and mortality; however, many current diagnostic methodologies are technically cumbersome and/or time-consuming to perform. CSF culture has a turnaround time (TAT) of 24 to 48 hours, and TATs for standard PCR tests can range from a few hours to several days, depending on whether the test is performed in-house or has to be shipped to a reference lab. Given the approximate one hour sample-to-answer run time with the commercial multiplex test mentioned above, a STAT result could be reported within 1.5 to two hours of specimen collection. A faster TAT has been previously reported with implementation of rapid respiratory panel testing, resulting in a five- to six-hour savings over the standard of care testing.22 Beyond providing a STAT result, improving TAT can likely improve antimicrobial stewardship, infection control practices, and healthcare costs associated with CNS infections.
In a study on identification of blood culture pathogens, Pardo et al reported a decrease in time to appropriate targeted therapy for patients infected with vancomycin-resistant enterococci when using a rapid, multiplex PCR system.23 For CNS infections, a similar narrowing of antimicrobial therapy could potentially be made if an appropriate diagnosis were made within a few hours. A rapid diagnosis also has the potential to benefit infection control practices. For example, droplet precautions, including isolation or cohorting, are required to prevent the spread of N. meningitidis.24 Faster identification of N. meningitidis would allow timelier implementation of appropriate infection control measures as well as guide community outbreak notification.
Finally, reductions in the scope of diagnostic testing required to make a definitive diagnosis have the potential to reduce length of hospitalization and overall healthcare costs. Indeed, rapid identification of blood culture pathogens using a commercially available multiplex panel reduced length of hospitalization and cost of care for patients whose cultures were positive with contaminating bacteria.23 It is likely that having rapid identification of CNS pathogens could have a similar impact. Based on the multiple studies that have already demonstrated cost savings, reduced length of hospitalization, and reduced exposure to unnecessary antibiotics, associated with molecular testing for early identification of pathogens in CSF (in particular Enterovirus), it is hypothesized that faster results through multiplex PCR testing will also have an impact for CNS disease.25
Increasing diagnostic yield
Even with an improved potential TAT for CNS infection diagnosis, many cases of acute meningitis/encephalitis are Gram stain and CSF culture negative. Negative Gram stains are observed in 90 percent of patients presenting with acute meningitis reporting to the emergency department.26 Because no underlying cause can be readily identified, patients are often unnecessarily hospitalized and treated with empiric antimicrobial therapy, even those later determined to have a viral infection or other condition not requiring intervention.26,27 As a result, viral meningitis-associated hospitalization is a significant healthcare burden.28 Currently, there are no epidemiological data or diagnostic algorithms available that incorporate modern molecular diagnostic methods to guide physicians for patients presenting with acute meningitis/encephalitis with a negative CSF Gram stain.29 Investigations beyond CSF culture and Gram stain are often biased by physician experience.29
Two recent studies have demonstrated that performing additional CSF PCR tests will increase the diagnostic yield in culture/Gram stain negative, acute meningitis cases. In the first study, performing PCR tests for HHV-6 and HPeV along with EV and HSV on CSF in infants less than six months of age increased the diagnostic yield by 14 percent compared to performing PCR tests for EV and HSV alone.30 Confirming a microbial etiology in febrile infants may result in an earlier discharge, decreased exposure to antimicrobials, and decreased cost of hospitalization, as has been shown when EV is identified by clinical testing.30,31
In the second study, performing additional PCR-based CSF testing for EV, HSV, and VZV, combined with arbovirus serology, increased the diagnostic yield by 20 percent in the culture/Gram stain negative adult patients.29 Some of the identified etiologies, such as VZV, HSV, and acute HIV are treatable, while others such as West Nile virus and EV still do not have treatment options. However, detection of these non-treatable pathogens may still influence patient care decisions.29 A subset of these samples were further evaluated using the rapid multiplex PCR test described above, and additional treatable microorganisms not previously detected, including VZV, HSV, and S. pneumoniae, were identified.32 These pathogens were likely not discovered during the initial testing because the routine evaluation of those samples did not include specific assays for those targets. The S. pneumoniae positive patient was treated with intravenous antibiotics prior to the CSF specimen collection, which could have confounded the culture results.32
While expanded PCR testing on CSF specimens may provide better diagnostic yield, the presence of viral nucleic acid in CSF does not necessarily exclude an alternative primary etiology that could have triggered a secondary latent viral reactivation.33,34 For example, it is well-established that herpesviruses exhibit lifelong persistence in CNS cells and can be reactivated.35 In an effort to understand frequency and contribution of HSV, VZV, EBV, CMV, HHV-6, and EV in various CNS disorders, an 11-year retrospective analysis of CSF PCR testing was performed (2001-2012; n=16,496 PCR tests performed) with pediatric and adult patients, comparing CSF PCR detection with CNS disorder.35 Four major CNS disease groups were identified: patients with symptoms typical for CNS infections, patients with further inflammatory CNS disease, patients with impaired immune systems, and patients with further neurological diseases.
Viral nucleic acids were detected in 125 of the 3,085 patients across all four CNS disease groups. Of these 125 positive detections, 70 (57 percent) were observed in patients exhibiting symptoms typical for CNS infection. Detection of EV, HSV, and VZV were highest in this group and were associated with cases of encephalitis as well as aseptic meningitis. Detection of EBV and HHV-6 were more commonly observed in the three remaining CNS disease groups, perhaps suggesting that detection of these viruses may be independent of an active clinical infection, and more likely associated with latent viral reactivation.36 It is important to consider, however, that even if these viruses were not the causative agents, they may still contribute to the course of the host response during CNS disease.37,38 For example, co-detection of high EBV titers in CSF with bacterial meningitis is associated with an increased risk of death.39 Viral quantification may be useful in determining relevance of latent herpesvirus detection in CSF, and results should be reviewed in context with other medical information.12
Conclusion
Recent advances in molecular technology may be ushering in a new era of diagnostics for acute meningitis and encephalitis. In addition to the new multiplex PCR system, other techniques such as next generation sequencing have great promise to increase diagnostic yield.38 Faster and more comprehensive results could lead to improved patient outcomes through decreased TAT for diagnosis and better antimicrobial stewardship. Improved diagnosis may also lead to lower hospital costs through decreased patient admission and/or shorter length of stay. Additional studies are needed to demonstrate these outcomes as well as determine the relationships between new virus results and CNS disease progression. Further, the healthcare community should work together to determine when these new tests are most appropriate to balance diagnostic yield with laboratory costs.39
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Andrew C. Hemmert, PhD, serves as Associate Director of Biochemistry at BioFire Diagnostics, LLC. He has been involved in the development of a number of FilmArray Multiplex PCR panels including the Meningitis/Encephalitis (ME) and Blood Culture Identification (BCID) Panels.
Jeremy J. Gilbreath, PhD, MLS(ASCP)CM, serves as a Clinical Research Scientist at BioFire. He is involved in the design and execution of clinical research studies and clinical evaluations of FilmArray diagnostic panels.