Molecular diagnosis of central nervous system infections

INTRODUCTION — Molecular diagnostic tests and nucleic acid amplification tests (NAATs) are used synonymously in reference to test methods that detect DNA or RNA specific to infectious organisms (eg, bacteria, viruses) as a means of diagnosis. Such tests have dramatically impacted both the diagnosis and management of infectious diseases [1]. This is particularly true for central nervous system (CNS) infections where rapid, accurate identification of a pathogen and prompt initiation of antimicrobial therapy are potentially lifesaving.

The increasing availability and use of molecular tests for the detection of microorganisms from cerebrospinal fluid has redefined our approach to common CNS infections, such as meningitis, encephalitis, and brain mass lesions (particularly in HIV-infected individuals or other immunocompromised hosts), and improved our ability to identify the etiologic agent responsible for other CNS syndromes, such as transverse myelitis.

The unique aspects of molecular testing as applied to CNS infections and guidance in the use and interpretation of molecular testing for pathogens commonly encountered in the management of patients with CNS infections will be reviewed here. Management of specific CNS infections is discussed in the appropriate topics. (See "Aseptic meningitis in adults" and "Viral meningitis in children: Clinical features and diagnosis" and "Viral encephalitis in adults" and "Acute viral encephalitis in children: Clinical manifestations and diagnosis" and "Herpes simplex virus type 1 encephalitis" and "PCR testing for the diagnosis of herpes simplex virus in patients with encephalitis or meningitis" and "Japanese encephalitis" and "Arthropod-borne encephalitides" and "Clinical features and diagnosis of acute bacterial meningitis in adults".)

GENERAL CHARACTERISTICS — Molecular methods are particularly well suited for the diagnosis of central nervous system (CNS) infections because pathogens are generally not present outside disease states, and infections, when present, are typically monomicrobial (with the notable exception of bacterial brain abscess). Furthermore, CSF typically lacks common inhibitors of nucleic acid amplification methods (eg, polymerase chain reaction [PCR]) such as heme, endonucleases, and exonucleases that can lead to false-negative results [2]. As a result, direct detection of nucleic acids from CNS samples may be less prone to common causes of false-positive (eg, contamination or presence of nonpathogenic colonization) or false-negative (eg, inhibition) results compared with other body sites.

Types of tests — There are several types of molecular tests:

●Targeted tests – Targeted nucleic acid detection methods are often more sensitive than conventional culture-based or antigen detection methods and may detect organisms that are nonviable or uncultivable. However, except for herpes simplex virus (HSV) and JC virus, the true clinical sensitivity of most molecular tests for CNS infections is not known because there are few studies utilizing a reference standard (eg, brain biopsy) for comparison. In addition, nucleic acid tests may be less sensitive for West Nile virus and Borrelia burgdorferi, and they are not specific for cytomegalovirus, Epstein Barr virus, human herpesvirus 6 (HHV-6), and SARS-CoV2.

●Multiplex tests – Multiplex or panel-based nucleic acid amplification tests (NAATs) combine multiple individual NAATs into a single test, thereby allowing clinicians to test for an array of potential pathogens that may cause a clinical syndrome at the same time. In 2015, the first commercial multiplex NAAT for infectious causes of community-acquired meningitis and encephalitis (BIOFIRE^® FILMARRAY^® Meningitis/Encephalitis [ME] Panel) was cleared by the US Food and Drug Administration (FDA) for use as an aid in the diagnosis of these conditions [3]. This multiplex NAAT detects 14 bacterial, viral, and fungal pathogens in just over one hour, including Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae (ie, group B Streptococcus), Escherichia coli (K1 serotype only), Listeria monocytogenes, enterovirus, HSV-1, HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), HHV-6, human parechovirus, and Cryptococcus neoformans/gattii. A second multiplex NAAT, the QIAGEN QIAstat-Dx Meningitis/Encephalitis Panel, was approved in the European Union in 2022.

However, multiplex NAATs do not detect all causes of CNS infection or provide antimicrobial susceptibility results. In addition, both false-negative and false-positive results can occur. As an example, a multiplex NAAT should not be used as the sole basis for management decisions when a CNS device or neurosurgery procedure or wound is the suspected source of infection, since the relevant etiologic agents are not targeted by the test. Similarly, the sensitivity of multiplex PCR testing is low for other pathogens (eg, cryptococcal antigen, HSV-1/HSV-2 PCR). (See 'Viruses' below and 'Fungi and parasites' below and 'Importance of clinical correlation' below.)

To address the limitations of multiplex NAAT testing, culture should be performed whenever multiplex NAATs are used. In addition, it is important that multiplex NAAT results are interpreted in the context of other clinical and laboratory findings (eg, CSF parameters) as well as the likelihood of infection. Bacterial false positives can be common, and even predominate, when the population incidence and clinical probability of bacterial meningitis is low [4-6].

The sensitivity of the FilmArray ME Panel was best evaluated in a 2022 meta-analysis of 19 diagnostic accuracy studies [6]. In this analysis, the FilmArray ME Panel was only moderately sensitive. The sensitivity for S. pneumoniae was 87.5 percent (95% CI 77-94 percent) but ranged from 64.9 to 74.5 percent for other bacterial targets (H. influenzae, L. monocytogenes, E. coli, S. agalactiae, N. meningitidis). For viral infections, it was 75.5 percent (95% CI 51-90 percent) sensitive for HSV-1, although the sensitivity for VZV, Enterovirus, and HSV-2 was higher (91.4 to 94.4 percent). In other studies, detection of Cryptococcus neoformans/gattii was suboptimal [7,8].

In another report, nearly one in six positive results of the FilmArray ME panel were classified as false positives after analysis of discrepant results, with 12 of 16 (75 percent) of S. pneumoniae detections being unconfirmed by culture and 7 of 16 (44 percent) unconfirmed by a second targeted nucleic acid test [4]. In a different study, clinical and laboratory adjudication of the FilmArray ME panel for H. influenzae detection at two Swiss hospitals classified 14 of 18 (78 percent) as likely false positives over three years [5]. Similarly, most positive HHV-6 detections in immunocompetent adults do not represent active clinical disease [9].

●Metagenomic next-generation sequencing – Metagenomic next generation sequencing (NGS) is another rapidly evolving molecular technology that has the potential to provide a direct, unbiased analysis of the microbial composition of clinical samples without reliance on traditional culture or targeted molecular tests. For example, in a prospective study of 204 pediatric and adult patients with idiopathic meningitis, encephalitis, or myelitis who did not have a diagnosis at time of enrollment, the etiologic agent was identified by conventional testing alone in 45 percent of patients, by metagenomic NGS alone in 22 percent of patients, and by both methods in 33 percent of patients [10]. Notably, receipt of antecedent antibacterials was not reported but may have compromised CSF culture results for those cases where NGS alone detected bacteria that are commonly associated with CNS infections. Application of metagenomic NGS likely has utility in select clinical contexts but its routine use is not indicated. Additional studies are needed to clearly define clinical and laboratory criteria that will optimize the value of metagenomic NGS in CSF infection. (See "Next-generation DNA sequencing (NGS): Principles and clinical applications".)

Commercial versus noncommercial assays — Many commercially available molecular tests for CSF or CNS specimens have not been approved or cleared by the FDA and are laboratory-developed tests that have been validated for CNS specimens in laboratories certified to perform high complexity testing. As such, these assays are not standardized between laboratories, and interlaboratory variability is common. Analytical sensitivity may vary significantly, and quantitative values (eg, viral load, copy number) are not comparable between laboratories. For both commercial and noncommercial assays, testing specificity for a given organism depends on the uniqueness of the target being amplified.

False-positive and -negative results — Despite advances in test design and understanding, false-negative and false-positive results may still occur and do not always indicate laboratory error or test failure. Potential causes for false-negative test results (ie, failure to detect nucleic acid despite active infection due to the targeted pathogen) include:

●Insufficient analytical sensitivity of a laboratory's assay for the specific microorganism (eg, concentration of the organism [nucleic acid] is below the functional limit of detection)

●"Inhibition" of amplification (most commonly due to a "bloody tap," CSF fluid with high number of erythrocytes)

●Failure of the assay to amplify or detect the intended microorganism in the absence of inhibition (rare, usually due to unrecognized species genetic variation or high rate of mutation, as with RNA viruses)

●Insufficient clinical sensitivity (eg, concentration of the microorganism is low in the submitted sample due to pathophysiology of the disease [eg, West Nile virus], prolonged treatment, and/or immune clearance)

●Sampling error (occurs at low nucleic acid concentration and is related to the inherently small test volumes used in molecular testing)

●Failure to test for the causative organism (eg, appropriate pathogen-specific test not performed, as most molecular tests detect only a single targeted pathogen)

Likewise, a positive NAT result for a specific pathogen may not always indicate CNS disease due to that pathogen and should be interpreted with caution. Due to the extreme sensitivity of these assays, there is an inherent risk of false-positive results due to:

●Amplification of a contaminating organism introduced at the time of specimen collection (eg, nonsterile technique, blood contamination of CSF) or post-collection (eg, in the laboratory)

●Amplification of a noncausative "bystander" (eg, microorganism present in circulating leukocytes that cross the blood-brain barrier during acute inflammation)

●Amplification of nucleic acid from a latent (not active) infection

●Lack of specificity of assay primer or probe sequences

Importance of clinical correlation — Test results should be analyzed in the context of the probability of infection. In the absence of an elevated leukocyte count or elevated protein level in the CSF, the likelihood of a true positive molecular result for an infectious pathogen is significantly reduced, while the number of false-positive and uninterpretable results increase. Limiting utilization of CSF molecular tests to those patients with a moderate to high pretest probability of having CNS disease based on clinical presentation, disease prevalence, and CSF parameters increases the predictive value of test results and reduces cost.

The efficacy of using screening criteria before molecular diagnostic testing was illustrated by a retrospective review of protein and leukocyte counts for 974 CSF specimens submitted for PCR analysis [11]. Among the samples tested for HSV, all 13 of the patients with a positive HSV PCR result had either an elevated CSF white blood cell count or protein level, while none of the 202 samples with normal CSF parameters were positive. However, screening based on the presence or absence of abnormal CSF parameters may not be appropriate for patients who are immunocompromised, neonates, or for those with intraparenchymal (but not meningeal) inflammation (ie, encephalitis) [12,13].

ROLE OF MOLECULAR TESTING BY CENTRAL NERVOUS SYSTEM SYNDROME

Meningitis — The evaluation of a patient with suspected meningitis routinely includes molecular testing, particularly when viral etiologies are considered. Traditional nonmolecular testing remains clinically useful, however, and in many cases should be used in conjunction with molecular testing (eg, culture or serologic evaluations). Similarly, cerebrospinal fluid (CSF) cell count, glucose, and protein analyses maintain traditional predictive value and have been examined for their potential in screening CSF samples prior to the performance of molecular testing (eg, enteroviruses, herpes simplex virus [HSV]). (See 'Importance of clinical correlation' above.)

A summary of molecular tests available for specific pathogens and the recommended test method(s) for diagnosis in patients with suspected meningitis is presented in the following Table (table 1).

Bacteria — Gram stain and culture remain the methods of choice for diagnosis of bacterial meningitis. The US Food and Drug Administration (FDA)-cleared multiplex nucleic acid amplification test (NAT), which detects most common causes of community-acquired (including neonatal) bacterial meningitis, may be useful as an adjunct to culture, especially in patients who have already received antibiotic treatment [3]. Nucleic acid amplification testing may also be useful in select cases when uncultivable or fastidious organisms are suspected (eg, Mycoplasma spp, Brucella spp, or Tropheryma whipplei), although these assays are not widely available.

Mycobacteria — Direct detection of M. tuberculosis from CSF (eg, tuberculous meningitis) is routinely available from reference laboratories and recommended as an adjunct to traditional mycobacterial culture when clinical suspicion for tuberculosis meningitis is high. Mycobacterial culture should also always be ordered when a diagnosis of tuberculous meningitis is suspected given the variable sensitivity of molecular testing from CSF. (See "Central nervous system tuberculosis: An overview".)

Rickettsia and spirochetes — Molecular testing for Rickettsia and spirochetes is investigational and not routinely available. Serum and/or CSF evaluations for serologic response and titers remain the primary methods of diagnosis. (See "Diagnosis of Lyme disease" and "Syphilis: Screening and diagnostic testing".)

Viruses — Use of molecular methods for the detection of enteroviruses and Herpesviridae (eg, HSV, varicella-zoster virus [VZV], cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) is standard of care when central nervous system (CNS) infection due to these viruses is suspected. Targeted single-pathogen NATs are typically used, although an FDA-cleared multiplex NAT, which detects enterovirus, HSV-1 and -2, VZV, CMV, human herpesvirus 6 (HHV-6), and parechovirus simultaneously, is also available [3]. However, this multiplex NAT is less sensitive for HSV-1 than highly sensitive singleplex assays, making it necessary to test specifically for HSV-1 when clinical suspicion for HSV infection is high [7]. Similarly, most enteroviral NATs do not detect parechoviruses, making it necessary to order specific NATs for these when they are suspected clinically. (See "PCR testing for the diagnosis of herpes simplex virus in patients with encephalitis or meningitis" and "Enterovirus and parechovirus infections: Clinical features, laboratory diagnosis, treatment, and prevention".)

Molecular testing for the arthropod-transmitted viruses is less well characterized, with the possible exception of West Nile virus (WNV), the only agent for which NAT is routinely available. However, evaluation of serum and/or CSF for the presence of an antibody response to WNV is preferred to molecular tests for this virus unless patients are immunocompromised. In cases when CNS disease due to respiratory virus is suspected, testing of alternative non-CNS sources (eg, respiratory specimens) alone or in conjunction with CSF optimizes diagnostic yield.

Molecular testing for SARS-CoV-2 from CSF is generally not available and not indicated clinically as most patients with neurologic and neuropsychiatric symptoms during or after COVID-19 infection do not have active viral replication in the CNS or virus detectable in the CSF or brain parenchyma [14].

Fungi and parasites — Fungal and parasite stains, culture, histology, cryptococcal antigen detection, and serology are preferred when infections with these agents are suspected clinically. Although the US FDA-cleared multiplex NAT includes Cryptococcus neoformans/gattii, it is less sensitive than high-performing cryptococcal antigen tests and can give false-negative results in patients with cryptococcal meningoencephalitis [3,7,15,16]. Information on cryptococcal antigen testing is presented elsewhere. (See "Clinical manifestations and diagnosis of Cryptococcus neoformans meningoencephalitis in patients without HIV", section on 'Cryptococcal antigen' and "Epidemiology, clinical manifestations, and diagnosis of Cryptococcus neoformans meningoencephalitis in patients with HIV", section on 'Cryptococcal antigen (CrAg)'.)

Other molecular assays for fungi and parasites are occasionally available but insufficiently characterized for formal recommendation.

Encephalitis — The differential diagnosis of encephalitis is extensive and includes both infectious and noninfectious causes [17]. Testing, in particular molecular testing, for specific infectious agents should be guided by epidemiology, risk factors, test availability, and clinical presentation. A combination of traditional diagnostic studies (eg, CSF analysis, imaging) potentially including microbiologic cultures of non-CNS sites should be employed prior to or concurrent with molecular testing. Molecular diagnostic methods should be included in the evaluation of suspected viral encephalitis whenever possible. However, with the exception of HSV encephalitis, the clinical sensitivity and predictive value of molecular testing from CSF for intraparenchymal disease remains largely undefined. Thus, while molecular testing of CSF or brain tissue is considered a primary method of diagnosis for many viral causes of encephalitis (eg, HSV), negative results may not exclude infection, and a combined diagnostic approach including serology, imaging, repeat molecular testing, and occasionally biopsy may be necessary to confirm the diagnosis. (See "PCR testing for the diagnosis of herpes simplex virus in patients with encephalitis or meningitis".)

Importantly, molecular testing should be negative in postinfectious encephalitis (ie, an immune-mediated disease). The clinical history, serology, and potentially other studies may be necessary to document recent primary infection outside the CNS (see "Viral encephalitis in adults"). A summary of molecular testing methods that may be useful in the evaluation of patients with encephalitis is provided in the Table (table 2).

Bacteria — Meningoencephalitis from bacteria is uncommon, but NATs may be useful in selected cases, such as when the exposure or patient history suggests a fastidious, slow-growing, or uncultivable bacteria such as Coxiella burnetii [18]. Mycoplasma pneumoniae may be a more common cause of nonviral encephalitis than was previously recognized, but NAT of CSF has a low diagnostic yield relative to serology. NAT of respiratory secretions may support the diagnosis [17,19,20]. (See "Mycoplasma pneumoniae infection in adults", section on 'Diagnosis'.)

Spirochetes, Rickettsia, and Ehrlichiae — NAT for spirochetes, Rickettsia, and Ehrlichiae spp are not widely available with the exception of state public health and reference laboratories. Serum and CSF testing for antibodies remain the primary means of diagnosis.

Viruses — Molecular testing (from CSF) is recommended for diagnosing encephalitis due to Herpesviridae, especially HSV (see "PCR testing for the diagnosis of herpes simplex virus in patients with encephalitis or meningitis"). For several herpesviruses, quantitative assays are increasingly used to aid in the distinction of latent infection of host leukocytes or chromosomal integration into host cells (eg, HHV-6) from active infection [21-23]. However, it is important to note that the sensitivity and negative predictive value of NATs from CSF for viruses other than HSV are not well defined. (See "Clinical manifestations, diagnosis, and treatment of human herpesvirus 6 infection in adults".)

Targeted single-pathogen NATs are typically used, although an FDA-cleared multiplex NAT, which detects enterovirus, HSV-1 and -2, VZV, CMV, HHV-6, and parechovirus simultaneously, is also available [3].

Polymerase chain reaction (PCR) testing of CSF for rabies virus is one of several tests to confirm a suspected case and is available from the United States Centers for Disease Control and Prevention (CDC) and some state public health laboratories. (See "Clinical manifestations and diagnosis of rabies".)

Although PCR tests are generally available for WNV, testing of serum and/or CSF for antibodies (especially IgM) is preferred for the diagnosis of WNV encephalitis in most patients since virus is usually cleared before clinical presentation. Patients with risk factors for poor antibody response and prolonged viremia (eg, hematopoietic cell or organ transplant recipients, patients receiving rituximab, patients with AIDS) should get antibody testing and PCR. (See "Clinical manifestations and diagnosis of West Nile virus infection".)

PCR tests for other arthropod-borne viral causes of encephalitis (eg, Colorado tick fever, California encephalitis virus,) are usually available from public health or national laboratories. Mumps and measles remain infrequent but potential causes of meningitis or encephalitis for which NAT is available from public health or national laboratories.

Molecular testing for SARS-CoV-2 from CSF is generally not available and not indicated clinically, as discussed above. (See 'Viruses' above.).

Fungi and parasites — As noted in the section on meningitis, NAT for most organisms remains investigational and not routinely available. If infection due to these organisms is a consideration, traditional methods of diagnosis should be used. A NAT for Cryptococcus neoformans/gattii is included in an FDA-cleared multiplex NAT, but this test has reduced sensitivity relative to cryptococcal antigen detection tests and fungal culture of CSF, as described above. (See 'Fungi and parasites' above.)

For certain pathogens, such as Toxoplasma gondii, molecular detection methods may be useful and rapid adjunctive tools to diagnose life-threatening encephalitis. (See "Epidemiology, clinical manifestations, and diagnosis of Cryptococcus neoformans meningoencephalitis in patients with HIV" and "Clinical manifestations and diagnosis of Cryptococcus neoformans meningoencephalitis in patients without HIV" and "Free-living amebas and Prototheca".)

Transverse myelitis — Although reports of success exist, there is relatively little published data from which to judge the sensitivity of molecular testing for the various infectious etiologies of transverse myelitis. Molecular tests should be negative when disease is postinfectious or due to noninfectious inflammatory causes. A Table summarizing the potentially useful molecular tests for the evaluation of transverse myelitis is provided (table 3).

Brain abscess — Nonmolecular methods (eg, stains, culture, histologic examination, and/or serology or antigen detection) remain the methods of choice to detect bacteria, mycobacteria, fungi (eg, Cryptococcus), and parasites (eg, amebic, Toxoplasma) from intraparenchymal lesions (table 4). However, molecular detection methods are available for some pathogens and may be helpful in certain clinical contexts (eg, suspected amebic brain abscess). (See "Pathogenesis, clinical manifestations, and diagnosis of brain abscess".)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Infectious encephalitis".)

SUMMARY

●Benefit of molecular testing for CNS infections – Molecular diagnostic tests have dramatically impacted both the diagnosis and management of central nervous system (CNS) infections where rapid, accurate identification of a pathogen and prompt initiation of antimicrobial therapy is potentially lifesaving. (See 'Introduction' above.)

Molecular methods are particularly well suited for the diagnosis of CNS infections because CNS samples are less prone to common causes of false-positive (eg, contamination or presence of nonpathogenic colonization) or false-negative (eg, inhibition) results compared with other body sites. (See 'General characteristics' above.)

Nucleic acid detection methods are often more sensitive than conventional culture-based or antigen detection methods and may detect organisms that are nonviable or uncultivable. (See 'General characteristics' above.)

●Limitations of molecular testing for CNS infections – Despite advances in test design and understanding, false-negative and false-positive results may still occur. Test results should always be analyzed in the context of the pretest probability of infection. (See 'Importance of clinical correlation' above.)

In addition, there remain few US Food and Drug Administration–approved assays for CNS samples, and the majority of assays in common use are not standardized between laboratories, and interlaboratory variability makes quantitative values (eg, viral load, copy number) not comparable between laboratories. (See 'Commercial versus noncommercial assays' above.)

●Specific tests – Molecular test availability for specific pathogens and the recommended test method(s) for diagnosis in patients with suspected meningitis, encephalitis, transverse myelitis, and brain abscess is presented in the Tables (table 1 and table 2 and table 3 and table 4). (See 'Role of molecular testing by central nervous system syndrome' above.)