|Year : 2020 | Volume
| Issue : 1 | Page : 3-7
Laboratory diagnosis of encephalitis: New insights into areas of uncertainty
Deema A Alokaili1, Mustafa A Salih2, Ali Mohammed Somily3
1 Saudi Center for Disease Control, Deputy Ministry of Public Health, Ministry of Health, College of Medicine, King Saud University, King Saud University Medical City, Riyadh, Saudi Arabia
2 Department of Pediatrics, Neurology Unit, College of Medicine, King Saud University, King Saud University Medical City, Riyadh, Saudi Arabia
3 Department of Pathology and Laboratory Medicine, Microbiology Unit, College of Medicine, King Saud University, King Saud University Medical City, Riyadh, Saudi Arabia
|Date of Submission||02-Feb-2019|
|Date of Decision||26-Mar-2019|
|Date of Acceptance||12-May-2019|
|Date of Web Publication||06-Jan-2020|
Ali Mohammed Somily
Department of Pathology and Laboratory Medicine, Microbiology Unit, College of Medicine, King Saud University, King Saud University Medical City, Microbiology (32), PO Box 2925, Riyadh 11461
Source of Support: None, Conflict of Interest: None
Encephalitis is a serious clinical syndrome, which presents with a wide range of severity. Some cases might be missed due to non specific symptoms, especially in old debilitated patients and immunocompromised host. In this review, we cover the various clinical presentations of this syndrome and provide an overview of laboratory testing that can be performed in routine clinical microbiology laboratories and reference laboratories. Recently, the advance in medical care and advancing age of our patient population, as well as increased number of immunocompromised conditions either due to post chemotherapy, human immunodeficiency virus, transplant and autoimmune diseases complicated both clinical and laboratory diagnosis of encephalitis. Other noninfectious differential diagnoses of encephalitis should be ruled out with appropriate tests. There has been tremendous development in advanced laboratory testing including multiplex polymerase chain reaction, and next-generation sequencing requiring tedious validation and cost-effectiveness study to justify wise clinical utilization for these tests in the management of patients with encephalitis. Understanding of the interpretation of these new tests by treating physicians though better communication with medical microbiologists is required. Whole-genome sequencing is a new molecular test that enables us to detect a rare pathogen or even a new pathogen with a high degree of sensitivity and specificity if the standard recommendations are followed. The use of these tests should be available for specific clinical indications in an accredited reference laboratory for better utilization. Preanalytical parameters such as type of sample collected, tests requested, transportation, and storing of sample, could all affect the result of the test performed.
Keywords: Cerebrospinal fluid, molecular test, viral encephalitis
|How to cite this article:|
Alokaili DA, Salih MA, Somily AM. Laboratory diagnosis of encephalitis: New insights into areas of uncertainty. J Nat Sci Med 2020;3:3-7
| Introduction|| |
Encephalitis is a severe neurologic syndrome associated with significant morbidity and mortality but can often be treated if diagnosed promptly. Encephalitis is a pathological process referring to inflammation of the brain parenchyma. The clinical presentation depends mainly on the location of the inflammatory process, and this makes the diagnosis of this syndrome complicated and hard to recognize in many cases., The patient with encephalitis usually presents with fever, headache, and altered level of consciousness and other clinical manifestations which vary from one patient to another such as seizures, focal paresis or paralysis, and behavioral changes. In some patients, encephalitis can be associated with meningeal irritation which is called meningoencephalitis.
Lack of reporting of all pathogens causing encephalitis makes accurate estimation or a precise calculation of the incidence of encephalitis very difficult. Several reported data of encephalitis incidence are hospital-based studies associated with bias and might not reflect the true incidence like population-based reports. A large study showed that the incidence of encephalitis to be 7.4/100,000 person-years. Another study using national discharge data for encephalitis identified an incidence rate of 7.3/100,000 population, leading to 230,000 hospital days and 1400 deaths annually.
| Pathogenesis And Causative Agents|| |
It is estimated that around 40%–60% of encephalitis cases are unexplained.,,, Encephalitis can be caused by direct infection of the brain parenchyma by microbial agents (bacteria, fungi, viruses, and parasites) or it can be due to noninfectious causes in about 10% of patients and it is usually found to be immune-mediated.,,, Immune-mediated encephalitis usually follows an infectious process such as acute disseminated encephalomyelitis (ADEM), typically following infection with measles, mumps, or rubella,, or immunization leading to a demyelinating disease of the central nervous system (CNS) which is called post- or parainfectious encephalitis diagnosed usually by exclusion when neuropathogenic agents are not detected.,,, Immune-mediated encephalitis can also be antibody-mediated such as those caused by antibodies to voltage-gated potassium channels or to the N-methyl-D-aspartate receptor (NMDAR)., ADEM is seen mainly in children and adolescents and is characterized by poorly defined white matter lesions on magnetic resonance imaging (MRI) that are enhanced following gadolinium administration.
The list of pathogens causing encephalitis is extensive and is commonly divided into viral and nonviral causes [Table 1]. Viruses are important yet often poorly understood cause of encephalitis. A range of viral agents has been implicated. Herpes simplex virus (HSV)-1, which can kill rapidly and needs urgent antiviral treatment with acyclovir, is most commonly implicated. However, other viruses can also cause encephalitis including herpesviruses (HSV-2, varicella-zoster virus, cytomegalovirus, Epstein–Barr virus, and human herpesvirus [HHV]-6 and HHV-7); paramyxoviruses (measles and rubella); orthomyxoviruses (influenza A virus); enteroviruses (EV-70 and EV-71 and polio-, echo-, and coxsackieviruses); flaviviruses (West Nile, Japanese encephalitis, dengue, and Zika viruses); retroviruses (human immunodeficiency virus [HIV]); alphaviruses (Venezuelan, eastern, and western equine encephalitis virus); bunyaviruses (La Crosse virus); rhabdoviruses (rabies virus); parvovirus (B19); and astroviruses.,, Several recent reviews explore more on new emerging viruses in different areas of the world.,,
| Clinical Diagnosis And Laboratory Investigation|| |
Most patients presenting to the acute medical unit with encephalitis manifest confusion which has a wide list of differential diagnosis. The clinical challenge is to differentiate causes of encephalopathy, which may mimic the presentation of encephalitis but results from metabolic disturbance caused by liver or renal failure, intoxications, systemic sepsis with encephalopathy, or anoxia, rather than from inflammation.,, A detailed history is critical in the evaluation of all patients with a presumptive diagnosis of encephalitis. Initial history should identify clues as to possible causes, including a full collateral history if available, to determine accurately the duration of the problem. Evidence should be explored regarding periods of change in level of consciousness or seizures (which may be subtle), and changes in personality or behavior. The physician should obtain travel history, including any contact with animals, fresh water, mosquito or tick bites, or exposure to illnesses in the community. Immune status of the patient must be determined, and risk factors for HIV should be established. Physical examination should establish the level of consciousness, seizure, or focal neurological findings attributable to the encephalitis or movement disorder. The patient's geographic location and time of year should be noted as they are associated with certain microbiological agents.
The most essential investigation for the diagnosis of CNS infection in all suspected patients is cerebrospinal fluid (CSF) analysis or brain tissue obtained at either brain biopsy or autopsy. Brain biopsy analysis can be performed using formalin fixation similar to routine histopathological examination, with the addition of special stains for infectious agents and immunohistochemistry stains. Nowadays, the role of brain biopsy in the diagnosis of encephalitis has declined since the advent of polymerase chain reaction (PCR) testing in CSF, and it does not form a part of the initial assessment. However, it can be still considered in patients without an established diagnosis following extensive investigation, particularly if there are focal abnormalities on imaging.
Lumbar puncture (LP) is often excessively delayed, primarily due to performing brain imaging to exclude increased intracranial pressure. Imaging is not always needed before LP, as there are only specific indications outlined by consensus guidelines.,, If it is indicated, then either a computed tomography scan or, preferably, MRI should be urgently performed, and if the radiological findings did not suggest any contraindications, LP should be performed as soon as possible. Brain imaging serves three purposes: to look for changes of encephalitis, to exclude alternative diagnosis, and to assess the patency of basal cisterns and an absence of mass effect so that LP can be performed.
Spinal fluid should be obtained and immediately submitted for cell count, glucose, and protein analysis, as well as the specific microbiologic studies detailed below. If possible, 2 ml of CSF should be saved and frozen at − 70°C for future testing. CSF cell counts associated with encephalitis are variable, but typically, the pleocytosis is relatively mild (50–1000 cells/mm3) compared to bacterial meningitis. Mononuclear cells usually predominate; however, early on, a transient neutrophilic pleocytosis may be seen. Protein levels may be elevated as well but rarely exceed 200 mg/dl.
All CSF samples should be cultured for bacterial pathogens, even when cell counts and clinical presentation are strongly suggestive of a viral process, to rule out other potentially treatable bacterial causes of meningoencephalitis. Mycobacteria, yeasts, molds, and occasionally parasites can variably be identified using specific stains performed on CSF spun (e.g., Gram stain and acid-fast stain, India ink). In cases of suspected amoebic encephalitis, a wet mount of the CSF may reveal motile protozoa, allowing rapid initiation of therapy.
Currently, viral culture and direct microscopic examination of the CSF have a very limited role in the diagnosis of encephalitis, and this is mainly because of the low sensitivity and poor yield of detection by this method as viral culture requires days to weeks and may fail in the event that the virus cannot be propagated in the cell line selected. In addition to that, the procedure is considerably labor intensive and the cost is high. Nevertheless, viral culture has an important role in the case of enteroviral infections where performing serotyping for epidemiological investigation is of great value.
The role of CSF PCR in the diagnosis of viral infections of the nervous system has been extensively reviewed. Fresh frozen tissue should be sent for viral culture and specific PCR studies. Several molecular techniques, including reverse transcriptase PCR, multiplex PCR, nested PCR, and real-time PCR, and more recently, high-throughput sequencing have revolutionized the diagnosis of encephalitis., Nowadays, we can detect as few as ten copies of HSV per reaction in CSF., The specificity and sensitivity of some PCR assays such as that for HSV-1 DNA are exceptionally high at 96% and 99%, respectively; other assays are not as robust. For example, while it was originally thought that PCR for CNS enteroviral infections was very sensitive at 95%, more recent data suggest that this figure may be considerably lower. A positive Epstein–Barr virus CSF PCR result may indicate infection or represent reactivation of previously latent disease sparked by a concomitant infection. Concordant results for HSV by PCR were seen in only 28%–30% of samples during an external quality assessment of nine European reference laboratories. Because of the various potential problems with PCR in neurovirological diagnosis, caution must be exercised when basing the identification of novel CNS viruses with this technique alone.
Extraneural sites (e.g., oropharynx, stool, and cutaneous vesicles) might be used to perform some diagnostic testing of specimens. Vesicular skin lesions can be sampled for direct fluorescent antibody or PCR to diagnose cutaneous varicella-zoster virus or HSV infections and enterovirus can be isolated from the respiratory and gastrointestinal tracts during acute infection. Clinicians should be aware that positive results in extra-CNS sites need to be interpreted cautiously because some might be incidental findings and clinical correlation of all laboratory results is mandatory.
Serology can succeed when both culture and PCR failed. However, the interpretation is one of the most difficult challenges for the clinician. The best approach for optimal titers interpretation is the collection of paired acute and convalescent samples which are tested in parallel when IgM is negative, and cross-reactive antibodies can confound specificity, as during the early phase of investigation of the 1999 West Nile virus outbreak in North America when St. Louis encephalitis virus was inaccurately implicated., Moreover, elevated antiviral antibodies may simply represent nonspecific polyclonal activation of memory B-cells from previous infection. If the serology is negative in the acute phase, this might indicate either absence of infection or insufficient time has elapsed for the patient to mount enough serological response. In such case, another serum sample should be collected for 2–4 weeks after the acute presentation to resolve this issue. Testing paired serum may allow retrospective confirmation of infection but has a limited role in diagnosis at the time of acute presentation. Acute infection as well as prior infection or even immunization can lead to elevated titer on the initial serum sample. Another significant application of serology is in the diagnosis of viral hemorrhagic encephalitis (flaviviruses) by the measurement of IgM antibodies in the CSF. Virus-specific IgG detection is less sensitive and specific, due to the delay in appearance and inability to diffuse across the blood–brain barrier, respectively. Various mathematical models can help in the discrimination between CNS- and blood-derived antibodies., The most simplified of these calculations is a ratio of serum antibody levels to those in CSF, with a value of ≤20 indicative of intrathecal synthesis.
In certain circumstances where CNS sampling is not possible or fails to provide clues to a causative agent, some clinicians and investigators survey blood, the oropharynx, feces, or urine. The identification of an agent in any sample type may lead to targeted assays of CSF or antibody tests, if seroconversion is consistent with acute infection.
If tests for an infective cause are negative, or if patients present with a recognizable phenotype of autoimmune encephalitis at the outset, then autoantibody testing should be considered. There are several antibodies associated with encephalitis, but the best-recognized phenotypes are those of NMDAR antibody encephalitis and LGI1-antibody encephalitis. The decision of which test to perform is best made in consultation with neurologists.
| Future Direction|| |
Despite thorough investigation for infective and autoimmune causes, between one-third and two-thirds of encephalitis cases do not have an etiological agent detected., Modern laboratory technologies, including proteomics, transcriptomics, and metabolomics, may enrich our insight and understanding of the immunological response in encephalitis, and this may help to stratify unknown patients into etiological groups.
New molecular technologies such as next-generation sequencing (NGS) for viral discovery provide a great opportunity to detect unexpected, previously unknown, or novel microbial pathogens in complicated cases of encephalitis as these techniques sequence-based methods can detect any microbial nucleic acid present in a biological specimen without any previous knowledge of the target sequence of the infectious agent. It has been utilized previously to successfully detect infectious agents in some rare cases of encephalitis of unknown etiology, such as novel lymphocytic choriomeningitis virus-related arenavirus and astrovirus in immunocompromised patients.,,, A new report confirmed the diagnosis of encephalitis by detection of parvovirus B4 in human blood and tissue as well as a novel emerging virus called cyclovirus in CSF specimens.,
| Conclusion|| |
The implementation of advanced genomic techniques such as multiplex PCR and NGS requires extensive laboratory validation and standardization to avoid any misleading result due to contamination or nonspecific reaction. For the best utilization of these new technologies for patient's management and cost-effectiveness, it should be limited to certain clinical setting like encephalitis in immunocompromised or complicated cases of encephalitis of unknown etiology after consultation with a neurologist and infectious disease specialist.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Roos KL. Encephalitis. Neurol Clin 1999;17:813-33.
Solomon T, Hart IJ, Beeching NJ. Viral encephalitis: A clinician's guide. Pract Neurol 2007;7:288-305.
Whitley RJ, Gnann JW. Viral encephalitis: Familiar infections and emerging pathogens. Lancet 2002;359:507-13.
Romero JR, Newland JG. Viral meningitis and encephalitis: Traditional and emerging viral agents. Semin Pediatr Infect Dis 2003;14:72-82.
Beghi E, Nicolosi A, Kurland LT, Mulder DW, Hauser WA, Shuster L. Encephalitis and aseptic meningitis, Olmsted county, Minnesota, 1950-1981: I. Epidemiology. Ann Neurol 1984;16:283-94.
Khetsuriani N, Holman RC, Anderson LJ. Burden of encephalitis-associated hospitalizations in the United States, 1988-1997. Clin Infect Dis 2002;35:175-82.
Granerod J, Ambrose HE, Davies NW, Clewley JP, Walsh AL, Morgan D, et al.
Causes of encephalitis and differences in their clinical presentations in England: A multicentre, population-based prospective study. Lancet Infect Dis 2010;10:835-44.
Kennedy PG. Viral encephalitis: Causes, differential diagnosis, and management. J Neurol Neurosurg Psychiatry 2004;75 Suppl 1:i10-5.
Davison KL, Crowcroft NS, Ramsay ME, Brown DW, Andrews NJ. Viral encephalitis in England, 1989-1998: What did we miss? Emerg Infect Dis 2003;9:234-40.
Granerod J, Tam CC, Crowcroft NS, Davies NW, Borchert M, Thomas SL. Challenge of the unknown. A systematic review of acute encephalitis in non-outbreak situations. Neurology 2010;75:924-32.
Glaser CA, Honarmand S, Anderson LJ, Schnurr DP, Forghani B, Cossen CK, et al.
Beyond viruses: Clinical profiles and etiologies associated with encephalitis. Clin Infect Dis 2006;43:1565-77.
Solomon T, Michael BD, Smith PE, Sanderson F, Davies NW, Hart IJ, et al.
Management of suspected viral encephalitis in adults – Association of British neurologists and British infection association national guidelines. J Infect 2012;64:347-73.
Chaudhuri A, Kennedy PG. Diagnosis and treatment of viral encephalitis. Postgrad Med J 2002;78:575-83.
Steiner I, Kennedy PG. Acute disseminated encephalomyelitis: Current knowledge and open questions. J Neurovirol 2015;21:473-9.
Garg RK. Acute disseminated encephalomyelitis. Postgrad Med J 2003;79:11-7.
Lancaster E, Martinez-Hernandez E, Dalmau J. Encephalitis and antibodies to synaptic and neuronal cell surface proteins. Neurology 2011;77:179-89.
Vincent A, Buckley C, Schott JM, Baker I, Dewar BK, Detert N, et al.
Potassium channel antibody-associated encephalopathy: A potentially immunotherapy-responsive form of limbic encephalitis. Brain 2004;127:701-12.
Young NP, Weinshenker BG, Lucchinetti CF. Acute disseminated encephalomyelitis: Current understanding and controversies. Semin Neurol 2008;28:84-94.
Debiasi RL, Tyler KL. Molecular methods for diagnosis of viral encephalitis. Clin Microbiol Rev 2004;17:903-25.
Ellul M, Solomon T. Acute encephalitis – Diagnosis and management. Clin Med (Lond) 2018;18:155-9.
Quan PL, Wagner TA, Briese T, Torgerson TR, Hornig M, Tashmukhamedova A, et al.
Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerg Infect Dis 2010;16:918-25.
Barzon L. Ongoing and emerging arbovirus threats in Europe. J Clin Virol 2018;107:38-47.
Muñoz LS, Garcia MA, Gordon-Lipkin E, Parra B, Pardo CA. Emerging viral infections and their impact on the global burden of neurological disease. Semin Neurol 2018;38:163-75.
Paixão ES, Teixeira MG, Rodrigues LC. Zika, chikungunya and dengue: The causes and threats of new and re-emerging arboviral diseases. BMJ Glob Health 2018;3:e000530.
Kennedy PG, Quan PL, Lipkin WI. Viral encephalitis of unknown cause: Current perspective and recent advances. Viruses 2017;9. pii: E138.
Wong SH, Jenkinson MD, Faragher B, Thomas S, Crooks D, Solomon T. Brain biopsy in the management of neurology patients. Eur Neurol 2010;64:42-5.
Michael B, Menezes BF, Cunniffe J, Miller A, Kneen R, Francis G, et al.
Effect of delayed lumbar punctures on the diagnosis of acute bacterial meningitis in adults. Emerg Med J 2010;27:433-8.
Tunkel AR, Glaser CA, Bloch KC, Sejvar JJ, Marra CM, Roos KL, et al.
The management of encephalitis: Clinical practice guidelines by the infectious diseases society of America. Clin Infect Dis 2008;47:303-27.
McGill F, Heyderman RS, Michael BD, Defres S, Beeching NJ, Borrow R, et al.
The UK joint specialist societies guideline on the diagnosis and management of acute meningitis and meningococcal sepsis in immunocompetent adults. J Infect 2016;72:405-38.
Barnett ND, Kaplan AM, Hopkin RJ, Saubolle MA, Rudinsky MF. Primary amoebic meningoencephalitis with Naegleria fowleri
: Clinical review. Pediatr Neurol 1996;15:230-4.
Polage CR, Petti CA. Assessment of the utility of viral culture of cerebrospinal fluid. Clin Infect Dis 2006;43:1578-9.
Steiner I, Schmutzhard E, Sellner J, Chaudhuri A, Kennedy PG; European Federation of Neurological Sciences. EFNS-ENS guidelines for the use of PCR technology for the diagnosis of infections of the nervous system. Eur J Neurol 2012;19:1278-91.
Salzberg SL, Breitwieser FP, Kumar A, Hao H, Burger P, Rodriguez FJ, et al
. Next-generation sequencing in neuropathologic diagnosis of infections of the nervous system. Neurol Neuroimmunol Neuroinflamm 2016;3(4):e251.
Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al.
Real-time PCR in clinical microbiology: Applications for routine laboratory testing. Clin Microbiol Rev 2006;19:165-256.
Allawi HT, Li H, Sander T, Aslanukov A, Lyamichev VI, Blackman A, et al.
Invader plus method detects herpes simplex virus in cerebrospinal fluid and simultaneously differentiates types 1 and 2. J Clin Microbiol 2006;44:3443-7.
Tang YW, Rys PN, Rutledge BJ, Mitchell PS, Smith TF, Persing DH. Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid. J Clin Microbiol 1998;36:2714-7.
Weinberg A, Bloch KC, Li S, Tang YW, Palmer M, Tyler KL. Dual infections of the central nervous system with epstein-barr virus. J Infect Dis 2005;191:234-7.
Schloss L, van Loon AM, Cinque P, Cleator G, Echevarria JM, Falk KI, et al.
An international external quality assessment of nucleic acid amplification of herpes simplex virus. J Clin Virol 2003;28:175-85.
Centers for Disease Control and Prevention (CDC). Outbreak of West Nile-like viral encephalitis – New York, 1999. MMWR Morb Mortal Wkly Rep 1999;48:845-9.
Nash D, Mostashari F, Fine A, Miller J, O'Leary D, Murray K, et al.
The outbreak of West Nile virus infection in the New York city area in 1999. N Engl J Med 2001;344:1807-14.
Kennedy PG. Neurovirological methods and their applications. J Neurol Neurosurg Psychiatry 2003;74:1016-22.
Reiber H, Lange P. Quantification of virus-specific antibodies in cerebrospinal fluid and serum: Sensitive and specific detection of antibody synthesis in brain. Clin Chem 1991;37:1153-60.
Monteyne P, Albert F, Weissbrich B, Zardini E, Ciardi M, Cleator GM, et al.
The detection of intrathecal synthesis of anti-herpes simplex IgG antibodies: Comparison between an antigen-mediated immunoblotting technique and antibody index calculations. European Union concerted action on virus meningitis and encephalitis. J Med Virol 1997;53:324-31.
Steiner I, Budka H, Chaudhuri A, Koskiniemi M, Sainio K, Salonen O, et al.
Viral encephalitis: A review of diagnostic methods and guidelines for management. Eur J Neurol 2005;12:331-43.
Graus F, Titulaer MJ, Balu R, Benseler S, Bien CG, Cellucci T, et al.
A clinical approach to diagnosis of autoimmune encephalitis. Lancet Neurol 2016;15:391-404.
Glaser CA, Gilliam S, Schnurr D, Forghani B, Honarmand S, Khetsuriani N, et al.
In search of encephalitis etiologies: Diagnostic challenges in the California encephalitis project, 1998-2000. Clin Infect Dis 2003;36:731-42.
Palacios G, Druce J, Du L, Tran T, Birch C, Briese T, et al.
A new arenavirus in a cluster of fatal transplant-associated diseases. N Engl J Med 2008;358:991-8.
Sato M, Kuroda M, Kasai M, Matsui H, Fukuyama T, Katano H, et al.
Acute encephalopathy in an immunocompromised boy with astrovirus-MLB1 infection detected by next generation sequencing. J Clin Virol 2016;78:66-70.
Cordey S, Vu DL, Schibler M, L'Huillier AG, Brito F, Docquier M, et al.
Astrovirus MLB2, a new gastroenteric virus associated with meningitis and disseminated infection. Emerg Infect Dis 2016;22:846-53.
Benjamin LA, Lewthwaite P, Vasanthapuram R, Zhao G, Sharp C, Simmonds P, et al.
Human parvovirus 4 as potential cause of encephalitis in children, India. Emerg Infect Dis 2011;17:1484-7.
Tan le V, van Doorn HR, Nghia HD, Chau TT, Tu le TP, de Vries M, et al.
Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections. MBio 2013;4:e00231-13.
Mbisa J, Tedder R. The Use of Genomics in the Clinical Diagnosis and Management of Viral Infections. London, UK: The Royal College of Pathologists; 2016. p. 158-62.