Clinical aspects

Description

	• Several technologies are used for the molecular diagnosis of infections of the nervous system.
	• Most of the tests are based on PCR, and next generation sequencing has also been found to be useful.

PCR-based tests. Construction of a DNA probe requires knowledge of the sequence of the suspected infective organism to be detected. This limitation is being rapidly overcome as the sequence of most infective organisms is known. Multiplex probes can be constructed to detect more than 1 organism. PCR kits are available commercially from several companies for the diagnosis of various infectious diseases. Clinical laboratories are now offering PCR tests to physicians for the diagnosis and monitoring of a variety of infectious diseases. Automated systems have been developed for this purpose as well as for sequencing.

RT-PCR is used for quantification of viruses and is particularly useful as a guide to therapy of AIDS. Other tests that are useful for quantification of HIV are branched DNA and nucleic acid sequence-based amplification.

FilmArray Panel, a commercially available test, is a rapid and fully automated multiplex PCR for the microbiological diagnostic workup of infectious meningitis/encephalitis, and the results are 90.9% concordant with conventional microbiological procedures but faster (29). Combination of FilmArray with microbiological diagnostic workup will improve the management of patients with suspected CNS infections. It gives reliable results even in cases where patients have received antibiotic treatment before lumbar puncture. One limitation is that it cannot determine antibiotic sensitivity of the pathogenic microorganisms.

Nucleic acid sequence-based amplification. Nucleic acid sequence-based amplification offers a simple and rapid alternative method for nucleic acid amplification. It can yield an RNA amplification of 109-fold in about 90 minutes. It was originally envisaged as an improved diagnostic method for the detection of RNA viruses but has now been developed into a technology with much wider applications. Unlike RT-PCR, nucleic acid sequence-based amplification can selectively amplify RNA sequences in a DNA background because DNA strands are not melted out. There are no false-positive signals due to dead bacteria.

Next generation sequencing. It is now possible to perform genomic sequencing on DNA from single microbial cells, which provides an alternative to culturing organisms for diagnosis. Next generation high-throughput sequencing methods generate data that can enable the assembly of microbial genome sequences in days. Sequencing of microbes provides valuable information for tracking epidemics, understanding pathomechanism of diseases caused by infections, study of drug resistance, and discovery of new antimicrobial agents.

Diagnosis of infectious agents and parasites causing meningitis as well as encephalitis can be challenging in many cases in which the cause is never identified, but these pathogens all have nucleic acids, and a sequencing approach is the fastest and most comprehensive way to detect that.

Branched DNA test. Branched DNA test is based on a highly branched form of synthetic DNA. This test can detect 500 viral equivalents per milliliter and promises to revolutionize the process of testing new antiretroviral agents.

Use of nanovesicles in molecular diagnosis. All cells continuously release nanoscale lipid membrane-enclosed packets, nanovesicles, which carry the signature of their cells of origin. Most of these vesicles originate from normal cells, but disease cells also release them. Nanovesicles can be detected in all body fluids, including blood, urine, and cerebrospinal fluid and are the basis of diagnostics for a wide spectrum of central nervous system diseases including infections (15).

The most common bacterial pathogens responsible for meningitis in Southeast Asia are Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis, and Mycobacterium tuberculosis. However, frequently patients with CNS infections do not have a causal diagnosis despite CSF culture and DNA molecular assays, partly because of low CSF pathogen density and previous antibiotic use (07). These drawbacks can be remedied by use of nanomolecular diagnostics for direct detection of microorganisms as such tests are becoming available at affordable cost.

Indications

PCR has helped to identify infectious disease etiologies for previously idiopathic cases and improved our understanding of pathogenesis. From the review of the application of molecular diagnostics, the most important uses are in the diagnosis of viral infections of the central nervous system. Standard laboratory techniques such as viral culture and serology provide only circumstantial or retrospective evidence of viral infections of the central nervous system. PCR is now considered to be the first-line diagnostic test for viral CNS infections such as herpes encephalitis, enterovirus meningitis, and other viral infections occurring in HIV-infected persons. A patient with positive PCR results is extremely likely to have a definite diagnosis of viral infection of the central nervous system. A negative PCR result can be used with moderate confidence to rule out a diagnosis of viral infection of the central nervous system. This also has economic benefits because patients hospitalized for suspicion of viral encephalitis can be discharged if PCR of the CSF is negative. Quantitative molecular diagnostic methods have provided a valuable additional tool for clinical management of diseases detected by these methods.

Advantages of the use of molecular methods in the diagnosis of infections of the nervous systems are as follows:

(1)	The sensitivity and specificity of these methods enable the detection of occult or latent disease where other methods of diagnosis have failed.
(2)	These methods can be applied to organisms that are difficult to culture or cannot be cultured at all. Organisms need not be viable at the time of examination.
(3)	Molecular methods enable detection of infection in the absence of immune reaction when antibody-based tests are negative.
(4)	Results are available within a few days, whereas a culture may take weeks.
(5)	They obviate the need for invasive procedures such as brain biopsy.
(6)	Examination can be done on extremely small amounts of tissue and body fluids.
(7)	Qualitative as well quantitative assessments are possible.
(8)	Testing is possible with archival tissues.
(9)	These methods can be applied in large scale, epidemiological studies.

Molecular methods have been found to be useful for various infections involving the central nervous system; these are shown in Table 1.

Table 1. Use of Molecular Methods for Diagnosis of Infections of the Central Nervous System

Viruses
Virus name: HIV
Neurologic manifestation Specimen Test	- AIDS dementia - serum - quantitative-PCR, branched DNA test, NASBA
Virus name: HSV-1
Neurologic manifestation Specimen Test	- herpes simplex encephalitis - CSF - PCR
Virus name: HSV-2
Neurologic manifestation Specimen Test	- neonatal herpes encephalitis - brain biopsy - PCR
Virus name: HSV-2
Neurologic manifestation Specimen Test	- Mollaret meningitis - CSF - PCR
Virus name: Cytomegalovirus
Neurologic manifestation	- encephalitis and polyradiculitis; opportunistic infection involving the CNS in AIDS and immunosuppressed transplant patients
Specimen Test	- CSF, brain - competitive quantitative PCR
Virus name: human T lymphocyte virus-1
Neurologic manifestation Specimen Test	- myelopathy - serum, CSF - PCR
Virus name: Enteroviruses
Neurologic manifestation Specimen Test	- aseptic meningitis; encephalitis; poliomyelitis - CSF - PCR
Virus name: Epstein-Barr virus
Neurologic manifestation	- focal encephalitis; aseptic; meningitis; transverse myelitis; peripheral neuropathy
Specimen Test	- brain, blood - PCR, in situ hybridization
Virus name: Papova viruses (JC and BK)
Neurologic manifestation Specimen Test	- progressive multifocal leukoencephalopathy - CSF, brain - PCR
Virus name: Varicella-Zoster
Neurologic manifestation Specimen Test	- Ramsay Hunt syndrome - tissue obtained during surgical decompression - PCR
Virus name: Measles
Neurologic manifestation Specimen Test	- subacute sclerosing panencephalitis - brain tissue - PCR
Virus name: West Nile virus
Neurologic manifestation Specimen Test	- encephalitis - CSF, blood - PCR, NASBA, immunoassay to detect antibodies in serum
Virus name: Dengue virus
Neurologic manifestation Specimen Test	- hemorrhagic fever, encephalitis in some cases - blood - PCR, immunoassay to detect antibodies in serum
Virus name: SARS corona 2
Neurologic manifestation	- encephalitis, cerebral infarction, NeuroCovid
Specimen	- blood, CSF
Test	-real-time PCR
Bacteria
Bacteria name: Borrelia burgdorferi
Neurologic manifestation Specimen Test	- Lyme neuroborreliosis; encephalitis; polyneuropathy; myopathy - CSF, urine, muscle tissue - PCR
Bacteria name: Tropheryma whippelii
Neurologic manifestation Specimen Test	- Whipple disease - jejunal biopsy - PCR
Bacteria name: Listeria monocytogenes
Neurologic manifestation Specimen Test	- meningitis - CSF - PCR
Bacteria name: Mycobacterium tuberculosis, Neisseria meningitidis
Neurologic manifestation Specimen Test	- tubercular meningitis - CSF - PCR
Bacteria name: Treponema pallidum
Neurologic manifestation Specimen Test	- syphilitic meningitis - CSF - PCR
Bacteria name: Haemophilus influenzae
Neurologic manifestation Specimen Test	- meningitis - CSF - PCR
Fungi
Fungi name: Cryptococcus neoformans
Neurologic manifestation Specimen Test	- cryptococcal meningitis in AIDS - CSF - PCR
Fungi name: Coccidioides immitis
Neurologic manifestation Specimen Test	- coccidioidal meningitis - CSF - PCR
Protozoa
Protozoa name: Toxoplasma gondii
Neurologic manifestation Specimen Test	- toxoplasmosis of CNS in AIDS - brain - PCR
Protozoa name: Toxoplasma gondii
Neurologic manifestation Specimen Test	- congenital toxoplasmosis; cerebral calcification; hydrocephalus - amniotic fluid - PCR
Prion diseases
Neurologic manifestation Specimen Test	- variant Creutzfeldt-Jacob disease - brain - enzyme immunoassay
Screening for variant Creutzfeldt-Jacob disease in symptomatic individuals
Specimen Test	- blood - direct immunodetection of prion proteins

Examples of special situations where molecular diagnostics have been useful are:

Bacterial meningitis. The introduction of molecular diagnostics has changed the approach to laboratory diagnosis of CNS infections. Bacterial antigen testing for the diagnosis of acute bacterial meningitis rarely impacts patient management and is not routinely needed. CSF shunt infections differ from usual meningeal infections and require rapid diagnosis, whereas tubercular meningitis remains a difficult disease to diagnosis but may be confirmed first by PCR testing of CSF. A prompt diagnosis is important so that the treatment can be started as soon as possible. A low-cost and rapid closed PCR system, Xpert MTB/RIF, has good accuracy in smear-negative pulmonary tuberculosis. A study has indicated that Xpert MTB/RIF is a test for the diagnosis of tubercular meningitis in HIV-infected individuals (27).

Methods such as microscopic examination, culture, and antigen detection of CSF samples are commonly used but may have low sensitivity, particularly when antibiotics have been given already. Several PCR methods are available for individual infectious organisms and are more sensitive than usual methods of diagnosis. There were no false negatives in culture positive specimens in a study of the use of oligoprobes on amplified DNA in the diagnosis of bacterial meningitis due to Neisseria meningitidis, Haemophilus influenzae, Streptococcus spp, and Mycobacterium tuberculosis. A multi-target real-time PCR assay has been developed that can rapidly identify 6 different microorganisms in a single CSF specimen: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes, and Cryptococcus neoformans (11).

A novel real-time PCR-hybridization assay has been developed for the rapid (ie, less than 1 hour) detection of penicillin susceptibility in Streptococcus pneumoniae. It is more sensitive than culture, microscopy, or antigen detection and provides susceptibility data, even in culture-negative cases. PCR for staphylococcal cassette chromosome mec, accessory gene regulator typing, and Panton-Valentine leukocidin loci are done on cerebrospinal fluid specimens in meningitis due to methicillin-resistant Staphylococcus aureus (01).

Peripheral blood RNA gene expression profiling in patients with bacterial meningitis using quantitative RT-PCR has shown strong activation of immune response at the transcriptional level that is influenced by the type of pathogen (21).

Diagnosis of brain abscesses. Culture-based methods for diagnosis of infective agents have limitations and do not provide a thorough documentation of the frequently polymicrobial nature of brain abscesses. PCR technology is a better alternative to culture-based methods for diagnosing brain abscesses because of its speed, sensitivity, and specificity. Molecular typing based on sequencing is available for several microorganisms and has increased the number of bacteria and fungi detected in brain abscesses (24). In immunocompromised patients with opportunistic infection, a PCR assay for Toxoplasma gondii should be performed in addition to smears and cultures of CSF or material aspirated from abscess for mycobacteria, nocardia species, and fungi (05).

Diagnosis of brain lesions in AIDS. Central nervous system opportunistic diseases are a major cause of morbidity in AIDS patients. A presumptive diagnosis for toxoplasmic encephalitis can usually be made only when it responds to specific treatment, but the lack of improvement after therapy does not rule out the disease. For the diagnosis of other opportunistic lesions, histological examination of the brain biopsy specimen is usually required. PCR performed on cerebrospinal fluid has been shown to be a sensitive and specific method for detecting genomic sequence of many opportunistic infections. Analysis of PCR products by restriction fragment length polymorphism has been reported to lead to rapid identification of type I allele at B1 gene of Toxoplasma gondii (02). The combination of PCR and neuroimaging techniques may obviate the need for brain biopsy in selected cases of focal neurologic disease in patients with acquired immunodeficiency syndrome.

Borrelia burgdorferi. The wide spectrum of clinical manifestations includes meningitis, encephalitis, and polyneuropathy in 20% of cases. Direct detection of spirochetes by microscopy rarely gives a positive result because few organisms are present. Borrelia burgdorferi is cultured from the CSF of patients with meningitis. The mainstay of diagnosis has been serology using immunofluorescence or enzyme immunoassay methods to detect antibodies. This has limitations, because antibody responses are slow to develop and there is cross reactivity with other spirochetes. Hydrogel microparticles can sequester and concentrate bacterial antigens to increase the sensitivity of urinary tests for Lyme disease (08).

The sequence of chromosomal DNA specific to Borrelia burgdorferi has been identified, and a highly specific and sensitive assay for Borrelia burgdorferi has been developed using PCR. This has helped in understanding the pathogenesis of Lyme disease by providing evidence that spirochetes are active in the chronic form of the disease and can be detected by PCR. Even adequate antibiotic treatment may not eradicate the disease, and a positive PCR has been shown to correlate with relapse and a positive culture. A method based on sequencing of the Borrelia burgdorferis 16S ribosomal RNA gene can detect and confirm diagnosis from blood samples of patients who have off-season spirochetemia with low bacterial counts to enable early start of therapy and prevent tissue damage (18).

Mycobacterium tuberculosis. In cases of tubercular meningitis, where mycobacterial cultures and stains are often negative, PCR of the cerebrospinal fluid seems to be highly sensitive. This is particularly useful in patients that have responded to antimicrobial therapy, and a negative PCR in the cerebrospinal fluid indicates cure. PCR, along with the suggested clinical criteria, offers a rapid and fairly accurate diagnosis of tubercular meningitis. Nested PCR can be used to detect mycobacterium tuberculosis DNA in CSF for assessing the clinical course of tuberculous meningitis during antituberculosis treatments, when results convert from positive to negative, correlating with the improvement of the patient's clinical condition. A single-tube method is available for the isolation of PCR-compatible DNA from Mycobacterium tuberculosis using Chelex-100 chelating resin, which does not require organic solvents, facilitates early and reliable diagnosis of tubercular meningitis (25).

Tuberculomas may be found without meningitis. PCR of the cerebrospinal fluid specimen may provide a noninvasive diagnosis even when cultures of the cerebrospinal fluid are negative. Immunohistochemistry methods have some operational advantages over PCR and are more suited to laboratories in developing countries for establishing a tuberculous etiology of these lesions.

Antibodies against Mycobacterium tuberculosis antigens can be detected by ELISA in CSF of patients with tuberculous meningitis. The detection rate of antibodies in the CSF of patients with positive culture is lower than in patients with negative culture because detection of antibodies in CSF tends to decrease as bacillary load increases.

Neurobrucellosis. LightCycler real time PCR assay in CSF samples is more rapid and sensitive than conventional microbiological tests and could be useful for the rapid diagnosis of neurobrucellosis.

Neuroleptospirosis. In a patient with severe combined immunodeficiency suspected of having a severe CNS infection, brain biopsy was not helpful in diagnosis. Next-generation sequencing of the CSF identified sequence reads corresponding to leptospira infection (36). Recovery followed targeted antimicrobial therapy, and PCR confirmed evidence of Leptospira santarosai infection although clinical assays for leptospirosis were negative.

Neurosyphilis. Diagnosis of syphilis involving the central nervous system is particularly difficult. With the use of PCR, it is possible to detect Treponema pallidum directly within cerebrospinal fluid. Rarely, cerebral syphilitic gumma can occur in the brain as space-occupying lesions. The diagnosis of this can be facilitated by PCR because Toxoplasma gondii, Mycobacterium tuberculosis, and lymphoma are more likely to cause a space-occupying lesion in the brain of a patient with HIV infection. No diagnosis can be made in many cases of diffuse encephalitis because of sampling problems. Most infants with T pallidum infection of the central nervous system can be identified by physical examination, conventional laboratory tests, and radiographic studies; but confirmation of diagnosis of such infants requires the use of additional tests, including IgM immunoblotting and PCR assay.

Whipple disease involving the brain. Central nervous system Whipple disease is a treatable bacterial infection that may be associated with normal jejunal histology due to minimal or patchy gastrointestinal involvement. The diagnosis is difficult and is frequently made postmortem. A PCR-based technique enables the detection of the causative organism Tropheryma whippelii; therefore, the diagnosis of this disease can be made in the living patient when the clinical suspicion is high and tissue biopsy is nondiagnostic. PCR assay of cerebrospinal fluid may be useful in a patient with suspected Whipple disease. Synovial fluid PCR can be used to aid the diagnosis of central nervous system Whipple disease in patients who present with arthritis as 1 of their symptoms.

Detection of viral infections of the nervous system. PCR is for detection of DNA viruses and RT-PCR for detection of RNA viruses. Techniques based on amplification of viral genome-specific fragments by multiplex RT-PCR and their subsequent detection via hybridization with microorganism-specific binding probes on microarrays enable simultaneous detection of multiple viruses in a single clinical sample (19). A multiplex PCR assay has been developed that detects the 4 most common causes of viral meningitis and encephalitis: (1) herpes simplex virus type 1, (2) herpes simplex virus type 2, (3) varicella-zoster virus, and (4) enteroviruses. A reverse transcription PCR DNA microarray test (Clart Entherpex kit) enables rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses in CSF specimens: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (20). It is possible to perform a comprehensive analysis of a large panel of antiviral antibodies against all known human viruses, known as systemic viral epitope scanning, although this procedure is not yet commercially available. Next-generation sequencing-based tests are commercially available to identify pathogens in CSF or brain tissue (33). Individual infections are described in the following sections.

Epstein-Barr virus. The diagnosis of Epstein-Barr virus of the nervous system is often entertained but difficult to prove. Laboratory confirmation usually relies on serological detection of antibodies; this method is inadequate. Epstein-Barr virus antibodies have been detected in the cerebrospinal fluid, but they could have been transported from the blood. Epstein-Barr virus DNA is usually identified in brain samples by Southern blotting or by in situ hybridization. PCR has been used to amplify and detect Epstein-Barr virus in blood and biopsy specimens from various tissues. Real-time quantitative PCR on the LightCycler (LC-PCR) instrument was developed to measure the Epstein-Barr virus load in clinical samples. Epstein-Barr virus load measurement with LC-PCR is helpful in monitoring the management of Epstein-Barr virus-associated primary central nervous system B-cell lymphoma. Quantitative PCR of CSF can establish the presence of lytic cycle Epstein-Barr virus mRNA (a marker of viral replication) in CSF of patients with Epstein-Barr virus-associated neurologic disease.

Herpes simplex encephalitis infections of the brain. Herpes simplex encephalitis is 1 of the most common viral brain disorders of immunocompetent individuals. Unlike most viral encephalitides, herpes simplex encephalitis responds to antiviral therapy (acyclovir). If untreated, the disease is fatal; a rapid and reliable diagnosis of the disease is, therefore, essential. Fortunately, nested PCR amplification of the herpes simplex virus sequences from CSF offers a rapid, sensitive, inexpensive, and less invasive method for establishing the initial diagnosis of herpes simplex encephalitis and for monitoring the response to therapy. If antiviral therapy has been started before drawing the CSF, PCR is still of diagnostic value, as herpes simplex virus sequences are typically detectable for up to 5 days after.

PCR of the CSF is the gold standard for diagnosis of herpes simplex virus encephalitis. PCR can also differentiate between herpes simplex virus-1 and herpes simplex virus-2 by use of primers specific for each during PCR, or by Southern hybridization analysis of the amplification products with probes containing sequences specific for herpes simplex virus-1 or herpes simplex virus-2. Quantitative real-time PCR analysis of CSF is important for the diagnosis of various human herpes viruses as quantitative data can be correlated with varying clinical manifestations to obtain more information on the role of the virus in pathogenesis (12). The use of PCR in combination with the detection of a specific intrathecal antibody response to herpes simplex virus currently represents the most reliable strategy for the diagnosis and monitoring of the treatment of adult patients with herpes simplex encephalitis. Even though herpes simplex virus-1 burden in the CSF does not distinctly correlate with the severity of clinical signs or the degree of cranial imaging finding, quantitation of herpes simplex virus-1 copies by PCR continues to be a rapid and reliable method of monitoring antiviral therapy. The European Concerted Action on Virus Meningitis and Encephalitis recommends that the antiviral treatment for herpes simplex virus encephalitis should be monitored by PCR detection of herpes simplex virus in CSF. Herpes simplex virus can be detected in CSF for up to 20 days in 50% of patients with herpes simplex virus encephalitis despite standard treatment with acyclovir.

Mollaret meningitis is a benign recurrent form of meningitis with spontaneous recovery and is most frequently associated with herpes simplex virus-2 infection. It can be diagnosed with PCR examination of the CSF.

Herpes simplex virus DNA has been demonstrated in surgically excised tissue from human epileptic foci by use of PCR. The frequency of this finding is significantly different from that of nonepilepsy control specimens, suggesting an association of the virus with seizure activity. Establishment of whether herpes simplex virus is in an active or latent phase, and in which cell the virus is located, are the next steps in determining viral etiology of epilepsy. If meaningful association is found, it may open the possibility of treatment of epilepsy with acyclovir.

Real-time PCR enables rapid detection of herpes simplex virus-2 DNA in CSF.

A sensitive multiplex PCR method has been developed for the simultaneous detection of 6 human herpesviruses: cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, Epstein-Barr virus, varicella-zoster virus, and herpesvirus 6. The method simplifies detection and reduces time as well as costs.

Varicella-zoster virus infections of the nervous system. Latent infections with virus are associated with a large variety of neurologic disorders. Detection of the virus in neurons, oligodendrocytes, meningeal cells, ependymal cells, or the blood vessel wall often requires a combination of morphologic, immunohistochemical, in situ hybridization, and PCR methods. The PCR analysis of CSF remains the mainstay for diagnosing the neurologic complications of varicella-zoster virus during life. PCR examination of CSF in varicella-zoster virus infections of the nervous system has shown that viral loads are higher in patients with encephalitis and acute aseptic meningitis than in other neurologic syndromes caused by this virus (28).

Human cytomegalovirus infections. These are the leading cause of infectious complications in immunocompromised patients, particularly in organ-transplant patients and those suffering from AIDS. Neurologic manifestations are peripheral neuropathy, radiculomyelopathy, and encephalitis. Active infection is observed in 60% of solid organ recipients. Episodes of acute and chronic rejection increase the probability of opportunistic infections in these patients. Classical diagnosis is based on viral detection in polymorphonuclear leukocytes. The most specific diagnostic tool is the detection of cytomegalovirus DNA by PCR in the CSF. For quantification of cytomegalovirus in the affected tissues, a quantitative competitive PCR with capillary gel electrophoresis is used.

PCR is more useful than clinical and neuroradiologic findings for documenting cytomegalovirus infection of the central nervous system in patients with AIDS.

Use of PCR for measurement of CSF viral sequences for the evaluation of central nervous system white-matter lesions in AIDS patients, along with clinical findings and MRI, can enable a definite diagnosis to be made of HIV-1 and cytomegalovirus-related manifestations, and to evaluate treatment with zidovudine plus foscarnet.

Nucleic acid sequence-based, amplification-based quantitative cytomegalovirus-RNA assays are available and can be useful for monitoring the effect of antiviral drugs on cytomegalovirus disease.

Hepatitis C virus in the central nervous system. In patients with chronic hepatitis C, reverse transcriptase PCR has revealed hepatitis C virus RNA negative strands in the brain tissue, suggesting that this virus can replicate in the central nervous system, probably in cells of the macrophage or monocyte lineage. Hepatitis C virus has also been detected in the CSF of HIV or hepatitis C virus positive patients and raises the possibility that the central nervous system may act as a reservoir site for hepatitis C virus.

Polyomavirus viruses. Only 2 of these viruses are pathogenic in humans and are named after the initials of the patients in whom these were first isolated (JC and BK). Distribution and localization of JC and BK viruses in the central nervous system and CSF of AIDS patients have been studied. Only HIV-positive patients with clinically evident progressive multifocal leukoencephalopathy and JC-DNA in the brain have PCR detectable JC in their CSF. BK has been reported to produce encephalitis in an immunocompetent patient; the diagnosis is established by PCR. This virus should also be included in the screening program for viruses that produce encephalitis. LightCycler real-time PCR assay provides rapid and specific quantification of polyomavirus load.

JC virus DNA in the brain of progressive multifocal leukoencephalopathy patients contains various progressive multifocal leukoencephalopathy-type regulatory regions that are generated from the archetypal regulatory region during persistence. PCR can efficiently amplify the regulatory region from most JC virus subtypes prevalent in the world. Because the structures of progressive multifocal leukoencephalopathy-type JC virus regulatory regions are unique to individual patients, the current PCR can eliminate false positives that may arise from contamination if the amplified fragments are sequenced. Evaluation of the diagnostic techniques revealed that stereotactic biopsy-based PCR diagnosis combines speed and sensitivity with the highest specificity available. Although the noninvasive technique of JC virus detection in CSF by PCR is even more sensitive, leading to detection of about 20 genome equivalents per 1 µL of CSF, the specificity of the method is limited by subclinical presence of JC virus DNA in CSF of neurologically asymptomatic HIV-infected patients. Very low copy numbers of JC virus nucleic acid can be detected in paraffin sections by the specific and highly sensitive in situ PCR.

Enteroviruses. The gold standard for the diagnosis of enterovirus infections is viral cell culture. Drawbacks of the conventional laboratory diagnostic procedures are: (1) tissue culture has only 65% to 75% sensitivity, and some enteroviruses do not grow well (if at all); (2) average time required for culture of CSF is 6 days; and, (3) most of the conventional techniques are cumbersome, labor-intensive, and require specialized personnel.

A PCR-based test for enterovirus is now commercially available. The assay time is about 5 hours. The sensitivity and specificity are 96.3% and 99.0% respectively. These tests can detect enteroviral meningitis at a consistent sensitivity of 1000 copies of enterovirus RNA/mL of CSF. The more simple-to-handle one step nucleic acid sequence-based assay is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples.

Due to genetic differences, human parechovirus type 1 genome is not detected with an enterovirus-specific reverse transcriptase (RT)-PCR procedure. Hence, a rapid, specific, and sensitive RT-PCR assay has been developed that can be used for the detection of human parechovirus type 1 (formerly called ECHO virus 2) in clinical samples. This virus can cause central nervous system infections as well as mild gastroenteritis and respiratory infections. The sensitivity and reproducibility of real-time quantitative RT-PCR assay used in combination with internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections (35).

Enteroviruses are the causative agents in over 85% of cases of aseptic meningitis. The signs and symptoms are those of meningitis, but no bacterial organisms can be cultured. Enteroviruses can be isolated from the CSF in the first few days after the onset of meningitis, but rarely after the first week. The course of the disease is usually benign, and no specific treatment is required. If diagnosis can be made early, the patient need not be hospitalized any longer. The economic impact of saving hospitalization costs by making an early diagnosis is obvious and may be significant.

A proof-of-principle study has shown that enteroviral meningitis is associated with a distinctive protein profile that may be directly detectable by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) in CSF specimens (32).

West Nile encephalitis virus. This flavivirus is 1 of the most important emerging viruses known to man. Infection with the virus can cause mild disease with flu-like symptoms or more severe disease characterized by encephalitis or meningitis. Reverse transcriptase-PCR and TaqMan assays are used for the detection of West Nile virus in the CSF as well as the blood. Nucleic acid sequence-based amplification assay has also been used for the detection of this virus. Compared to reverse transcriptase-PCR and TaqMan, the nucleic acid sequence-based amplification assay demonstrates exceptional sensitivity and specificity, yielding results in less than 1 hour. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methods and as a tool in conducting flavivirus surveillance in the United States. The FDA-cleared West Nile immunoassay test kit provides results faster than existing methods. It detects West Nile virus antibodies in the serum from people with suspected symptoms of the disease. The genome of the West Nile virus has been sequenced.

Dengue fever. The Centers for Disease Control has developed a test to detect the presence of dengue virus in people with symptoms of dengue hemorrhagic fever and its complications, including encephalitis. The test, called the CDC DENV-1-4 Real Time RT PCR Assay, has been authorized by the FDA and can be performed using equipment and supplies many public health laboratories already use to diagnose influenza. The new test will help diagnose dengue within 1 week after symptoms of the illness appear when most people are likely to see a health care professional and the dengue virus is likely to be present in their blood. The test can identify all 4 dengue virus types. This is the first FDA-approved molecular test for dengue that detects evidence of the virus itself. A clinical study has shown that it is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses (06). The other available FDA-approved test detects IgM antibodies to dengue virus. Most patients begin to develop these antibodies 4 days after they become ill. However, because not everyone develops these antibodies until a week after they get sick, the antibody test might not recognize dengue early in a patients illness.

Neurologic complications of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the 2019 coronavirus disease (COVID-19) outbreak, enters host cells by means of the envelope spike protein, which binds to angiotensin-converting enzyme 2 receptors, which are highly expressed in the heart, lungs, and brain. Neurologic complications of COVID-19 include meningoencephalitis and acute necrotizing encephalopathy (26). Diagnosis is well established by RT-PCR-based tests for SARS-CoV-2 systemic infection on nasopharyngeal and throat swabs. Patients' CSF may be devoid of viral particles even when they test positive for COVID-19 on a nasal swab as the presence of virus in the CSF may depend on systemic disease severity and the degree of the virus neurotropism (03). Molecular diagnostic tests are not useful in identifying persons who have already recovered from COVID-19, as they will no longer have detectable levels of viral RNA in their body. These recovered patients will, however, have antibodies that fight off the virus circulating in their blood. An ELISA has been developed using recombinant antigens derived from the spike protein of SARS-CoV-2, which is sensitive and specific, enabling screening and identification of COVID-19 seroconverters using human plasma/serum as early as 3 days after onset of symptoms (04).

The spectrum of neurologic complications in COVID-19 is not yet sufficiently understood and remains to be discovered and explained through more extensive studies. One case proved to be challenging in terms of differential diagnosis, and the authors hope that future research will shed light on the pathophysiological mechanisms through which SARS-CoV-2 interacts with and affects the nervous system (34). Patients with neurologic manifestations of COVID-19 were included in some studies, and the most common neurologic diseases were COVID-19-associated encephalopathy (30.2%), acute ischemic cerebrovascular syndrome (25.7%), encephalitis (9.5%), and Guillain-Barré syndrome. Neurologic manifestations appeared after the first COVID-19 symptoms, with a median delay of 6 (3 to 8) days (22). Brain magnetic resonance imaging of encephalitis patients showed heterogeneous acute nonvascular lesions in 14 (66.7%) of 21 patients. Cerebrospinal fluid was analyzed, with pleocytosis found in 18.6% and a positive SARS-CoV-2 PCR result in 2 patients with encephalitis.

Variant Creutzfeldt-Jacob disease. Current diagnosis is by application of enzyme immunoassay to brain tissue obtained by biopsy or at autopsy. A blood-based test has been developed by concentrating disease-associated prion proteins and coupling this to direct immunodetection of surface-bound material (09). It will be useful for diagnosis of variant Creutzfeldt-Jacob disease in symptomatic individuals, as well as for large-scale screening of individuals with asymptomatic variant Creutzfeldt-Jacob disease prion infection.

Neurocysticercosis. Human neurocysticercosis, caused by Taenia solium larvae, can lodge in the central nervous system. The usual diagnosis is by radiology, but if the findings are uncertain, immunological assays are often also used. The sensitivity of serologic testing for T solium is almost 100% in patients who have multiple cysts, but it is less useful in patients with solitary CNS lesions in whom diagnosis can be confirmed after identification of T solium DNA in brain biopsy tissue with use of a global DNA screening platform (13). A PCR test, based on the noncoding HDP2 sequence of T saginata, has been developed for detecting DNA from T solium cysticerci and confirming the diagnosis of neurocysticercosis (14).

Results

Molecular diagnosis has led to improvements in clinical outcome and patient care in addition to providing a better understanding of the natural history and clinical spectrum of the viral infections of the nervous system. PCR is important for the diagnosis of acute viral infections. Chronic infections induce an intrathecal humoral immune response and the appearance of antibodies directed against the causal infectious agent. The use of PCR in combination with the detection of a specific intrathecal immune response would be the most reliable strategy for the diagnosis of chronic viral infections of the central nervous system. Molecular diagnostics of CNS infections help in determining the prognosis of the illness.

All patients with positive PCR results have a definite diagnosis of CNS viral infection, but a negative result did not rule out the possibility of viral infection of the CNS. Limitations of amplification methods for detection of infections are as follows:

(1)	DNA/RNA sequences must be known and unique.
(2)	False positives and false negatives can occur.
(3)	Quantification is difficult with target amplification alone. Alternative techniques such as RT-PCR and branched DNA test need to be used for this purpose.
(4)	It is difficult to distinguish between colonization and invasion.
(5)	Cultures are not available for further typing, eg, characterization
(6)	Results are usually not available within an hour and may take a few hours. This is not suitable for diagnosis of infections in an emergency or outpatient setting.

Various pitfalls of molecular diagnostics are as follows:

(1)	Cross contamination is a potential problem due to the high sensitivity of this method, but this can be prevented.
(2)	False positives and false negatives can occur, though the percentages are low.
(3)	Presence of latent viruses, such as herpes simplex virus-1, does not necessarily correlate with the clinical presentation, which may be due to another cause.
(4)	Current PCR-based diagnostics cannot detect single bacteria or virus particles in specimens.
(5)	No microorganisms can be identified in the CSF in postinfectious encephalitis, which requires brain imaging for diagnosis (30).

According to the recommendations of the European Federation of Neurological Sciences, PCR technology is currently a reliable method for the diagnosis of viral as well as bacterial (except tuberculosis) infections, but only for some protozoal infections and helminthic infestations (31). There are not enough data to recommend the routine use of PCR in fungal infections.

The limitations are being addressed in nanotechnology-based molecular diagnostics that enable detection of single bacteria or a few virus particles in a specimen, and the procedures can be performed in half an hour or less (16).

Adverse effects

There are no adverse effects of the in vitro diagnostic, but an invasive procedure to obtain specimen for examination may have adverse effects.

Special considerations

Pregnancy. PCR-based tests on amniotic fluid enable the confirmation of some infections involving the nervous system of the fetus, such as cytomegalovirus, herpes simplex virus, syphilis, and toxoplasmosis.

In case of congenital toxoplasmosis, the affected fetus can develop serious complications, such as chorioretinitis, cerebral calcifications, hydrocephalus, and neurologic damage. Prenatal diagnosis of congenital toxoplasmosis is based on ultrasonography, amniocentesis, and fetal blood sampling. The accuracy of serum toxoplasma-specific IgM test is questionable. Transmission can occur from the mother to the fetus, but the finding of a high IgM titer in maternal serum does not necessarily indicate infection in the fetus. Confirmation of the diagnosis requires culture of the amniotic fluid or the fetal blood, which requires about a week, and only half of the cases show a positive result. Several DNA oligonucleotide primers directed at toxoplasma-specific DNA sequences have been designed. PCR test targets the B1 gene of Toxoplasma gondii, uses an internal control, and can be completed in a day. PCR test of the amniotic fluid gives better results than the conventional parasitology methods (sensitivity, 97.4% vs. 89.5%; negative predictive value, 99.7% vs. 98.7%). This has practical importance if treatment of the affected fetus is being considered or for decision-making regarding termination of the pregnancy.

Since the Zika virus infection outbreaks were first reported in South America in 2013, this mosquito-borne flavivirus has spread throughout the Americas. A causal link has been established between Zika virus infection of pregnant women and major fetal malformations such as microcephaly. Testing for Zika virus infection during pregnancy is important as only 20% to 25% of infected women have clinical symptoms. Nucleic acid detection by RT-PCR targeting the nonstructural protein 5 genomic region has been the primary means of diagnosis. Serological tests, including immunofluorescence assays and ELISA may indicate the presence of anti-ZIKV IgM and IgG antibodies. A Zika virus immunoassay was developed by the CDC, and several commercial real-time, reverse-transcriptase PCR assays are available. Limitation of molecular diagnostics include cross-reaction of immunoglobulin serologies with other endemic flaviruses, such as dengue, and persistent viremia in pregnancy weeks to months after primary exposure (10). Fetal brain malformations precede the sonographic detection of microcephaly.

Pediatrics. In children with suspected central nervous system infections, the FilmArray Meningitis Encephalitis Panel, a multiplex PCR for testing of cerebrospinal fluid, has a diagnostic yield comparable to conventional methods but reduces time-to-diagnosis with potential for more judicious use of antimicrobials (23).

Scientific basis

PCR is a method for producing large amounts of a specific DNA fragment of a defined sequence and length from a small amount of a complex template. It can selectively amplify a single molecule of DNA or RNA several million times in a few hours. Analyses can be performed even on a few cells present in the body fluids, so the need for preparation of large amounts of DNA from tissue samples is eliminated.

Several methods of detection are used for the amplified DNA: Southern blotting, detection by enzyme DNA conjugates, fluorescence, and chemiluminescence.

RNA can also be studied by making a DNA copy of the RNA and using the enzyme reverse transcriptase. This approach enables the study of messenger RNA in cells that are using the molecule to synthesize specific proteins or for detecting the genome of RNA viruses.