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Clinical Microbiology Reviews, October 2004, p. 903-925, Vol. 17, No. 4
0893-8512/04/$08.00+0 DOI: 10.1128/CMR.17.4.903-925.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Pediatrics, Division of Infectious Diseases,1 Department of Neurology,2 Department of Medicine, Microbiology and Immunology, University of Colorado Health Sciences Center,4 Denver Veterans Administration Medical Center, Denver, Colorado3
SUMMARY INTRODUCTION DISTINGUISHING ENCEPHALITIS FROM ENCEPHALOPATHY AND POSTINFECTIOUS ENCEPHALITIS Encephalitis versus Encephalopathy Encephalitis versus Postinfectious Encephalomyelitis GENERAL PRINCIPLES OF MOLECULAR DIAGNOSTIC METHODS AS APPLIED TO THE DIAGNOSIS OF CNS INFECTION CSF PCR General comments. CSF PCR for diagnosis of viral CNS infection. CSF specimen handling and preparation. Detection of amplified products. (i) Qualitative PCR. (ii) Quantitative PCR techniques. Sensitivity. Specificity. Positive and negative predictive values. Multiplex and Consensus Primer PCRs Pan-herpesvirus assays. Herpesviruses with enteroviruses. Others. CNS Tissue PCR SPECIFIC ENTITIES FOR WHICH MOLECULAR DIAGNOSTIC METHODS MAY BE CONSIDERED Viruses for Which PCR Is of Proven Efficacy in Diagnosis and Is Widely Available Herpes simplex virus (HSV). (i) General comments. (ii) Sensitivity, specificity, and predictive value. (iii) Kinetics of PCR positivity. (iv) Effect of antiviral therapy on PCR positivity. (v) Quantitative HSV PCR. (vi) Role of HSV PCR in special clinical situations. Enteroviruses (EV). (i) General comments. (ii) Sensitivity and specificity of EV RT-PCR compared to culture. (iii) Impact on hospitalization and patient management. JC virus (JCV). Epstein-Barr virus (EBV). (i) Immunocompetent hosts. (ii) Immunocompromised hosts. Cytomegalovirus (CMV). Viruses for Which PCR Has Potential Efficacy in Diagnosis but Is Less Widely Available Other human herpesvirus infections. (i) Varicella-zoster virus (ii) Human herpes virus 6. Other viruses. (i) Human immunodeficiency virus. (ii) Rabies virus. (iii) Human T-cell lymphotrophic virus. Viral Entities for Which Molecular Diagnostic Methods Are Not Readily Available and/or Are of Unproven Sensitivity or Specificity Arboviruses. (i) West Nile virus. Measles virus. Adenovirus. BK virus. Use of PCR in the Diagnosis of Infections Which Can Mimic Viral CNS Disease M. pneumoniae. B. burgdorferi (Lyme disease). Toxoplasma. Mycobacterium tuberculosis meningitis. REFERENCES
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From a practical point of view, a clinician confronted with a patient with fever, headache, and altered mental status must initially distinguish encephalitis from noninfectious causes of brain dysfunction (encephalopathy). Having made this distinction, it is next necessary to distinguish cases in which brain injury is a direct consequence of viral infection from cases in which it occurs as a consequence of a postinfectious immune-mediated process (e.g., acute disseminated encephalomyelitis). Finally, the goal in cases of encephalitis is to identify a specific etiologic agent, with particular emphasis on diseases that require acute treatment, such as herpes simplex encephalitis (HSE) (17, 64, 190, 250, 252).
Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies, such as the PCR to amplify viral nucleic acid from CSF (65, 230, 244, 261). Despite the use of newer techniques, including CSF PCR, up to 70% of cases of encephalitis remain of unknown etiology in modern surveys (100).
| DISTINGUISHING ENCEPHALITIS FROM ENCEPHALOPATHY AND POSTINFECTIOUS ENCEPHALITIS |
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| GENERAL PRINCIPLES OF MOLECULAR DIAGNOSTIC METHODS AS APPLIED TO THE DIAGNOSIS OF CNS INFECTION |
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CSF specimen handling and preparation. Minimal amounts of specimen are required for CSF PCR testing; however, the sensitivity of PCR assays is related to the volume of specimen tested. Most assays can be run on a minimum of 30 µl of sample, but 100 to 200 µl may yield better results. The PCR is optimally run on freshly obtained CSF specimens; however, positive results have been obtained from archival specimens frozen for years. In general, the stability of RNA viruses in CSF samples is less that of DNA viruses. The decline in sensitivity over long-term storage has not been rigorously studied, but there is no appreciable loss in yield with brief storage at 4 or –20°C. If long-term archival storage is anticipated, specimens should be stored at –70°C and multiple freeze-thaw cycles should be avoided.
Prior to entry into the PCR, nucleic acids present in CSF must be made accessible. Although simple methods such as exposure to high temperature or repetitive freeze-thawing have been utilized to release nucleic acids, nucleic extraction and purification techniques, such as phenol-chloroform or spin column-based (Qiagen) techniques, have the advantage of providing pure nucleic acid for entry into the amplification reaction, along with removal of potential inhibitors of the PCR amplification reaction as well as substances that may degrade nucleic acid and reduce yield.
Detection of amplified products. (i) Qualitative PCR. Traditionally, the amplified PCR product is electrophoresed on an agarose gel and is transferred to a nitrocellulose membrane. The membrane is hybridized with a specific probe complementary to the sequence of the desired PCR product and visualized with either radioactivity or chemiluminescence. More recently, colorimetric detection methods have been developed, entailing the use of biotinylated primers used within the amplification reaction and resulting in labeled amplicon PCR products. Unlabeled "capture" oligonucleotide probes (complementary to the internal sequence of the amplicon) are preapplied to 96-well plates, and the labeled PCR product is incubated with the plate. Avidin-horseradish peroxidase conjugate is added to detect bound amplicon. A colorimetric readout is utilized, which can be quantified by measuring absorbance at 450 nm in a microwell plate reader.
(ii) Quantitative PCR techniques. An additional concern which may have an impact on the positive predictive value of a CSF PCR is that viruses which associate with peripheral blood mononuclear cells (PBMCs) (such as Epstein-Barr virus [EBV]) might be carried into CSF in a setting of inflammation, be detected by PCR ("bystander"), and lead to a spurious viral diagnosis. In actuality, this may be only a theoretical concern. In one study, only 11 of 2,222 specimens were positive for EBV by PCR, and in all cases the patients had illness consistent with EBV disease (CNS lymphoma, primary EBV infection, or bone marrow transplantation with fever) (180a). Quantitative PCR and reverse transcription-PCR (RT-PCR) have increasingly been applied to clinical samples. Several studies have suggested that nucleic acid copy number (viral load) may be a marker for the severity of disease or may help predict outcome, particularly in immunocompromised patients, although it remains to be determined whether this is true for all infections (2).
Qualitative PCR assays can be modified for quantification if an internal control is added at a known concentration or a standard curve is run in parallel with clinical specimens. However, real-time quantitative PCR techniques are more frequently utilized in clinical practice. In these assays, reagents that are capable of generating a fluorescent signal within the PCR amplification reaction can be monitored and quantified to reflect the starting amounts of nucleic acid template at each cycle of the PCR. Probe-based quantitative PCR methods rely on the use of a fluorescent reporter molecule that increases as product accumulates with each cycle of amplification (137) (Fig. 2). The reporter molecule probe is composed of a sequence-specific oligonucleotide with an integrated fluorophore and quencher, as in molecular beacon probes (e.g., Scorpion probes) or TaqMan probes. Molecular beacon probes are designed as a hairpin loop configuration with the fluorophore and quencher adjacent (nonfluorescent complex) in the unbound state (3, 80). Upon extension of the amplicon and probe hybridization, physical separation of the fluorophore and quencher occur and fluorescence is emitted. In the case of TaqMan probes, as primers anneal to the nucleic acid target and the PCR product is generated, the 5'-3' nuclease activity of Taq polymerase cleaves the fluorescent probe, releasing the quencher from the fluorophore and leading to emission of fluorescence (3, 80, 137, 154).
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Regardless of the probe-based method used, real-time analysis at each step of amplicon production is monitored by assessing fluorescence intensity, which is directly proportional to the quantity of nucleic acid that was amplified from the original sample.
As an alternative to probe-based techniques, SYBR Green-based quantitative PCR utilizes a sensitive DNA binding dye that it binds to any double-stranded product generated during the PCR (Fig. 3). Thus, the same dye (and master mix) can be used in different quantitative PCR applications, in contrast to the case for probe methods that require synthesis of specific probes for each pathogen. Although intercalating dyes allow detection of nonspecific PCR products, the inclusion of a hot-start mechanism in the PCR reduces or eliminates mispriming events, which increases reaction specificity and helps ensure that only the desired product is measured (122, 144, 255). Postamplification melting curve analysis of PCR products, which detects sequence differences in target amplicons, is an additional safeguard to ensure the specificity of the PCR.
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A variant of classical PCR termed nested PCR is utilized for analysis of specimens in which very few viral particles are presumed to be present (such as CSF), with the goal of substantially increasing the sensitivity and specificity of the PCR (224). In nested PCR, the first PCR is followed by an additional amplification with a second set of primers which are complementary to sequences internal to the sequence targeted by the first set of primers.
Replicating virus and viral nucleic acid do not persist indefinitely in infected patients, particularly in immunocompetent patients who mount an effective neutralizing antibody response. However, most CSF testing is performed in the clinical setting of suspected meningitis or meningoencephalomyelitis within 1 to 2 days following onset of neurologic symptoms, a time at which the yield from PCR testing is likely to be at its peak. Positive CSF PCR test results have been noted up to 4 weeks after onset of clinical symptoms, depending on the pathogen (244).
Specificity. The specificity of the PCR must also be considered. The exquisite sensitivity of PCR is both its greatest virtue and greatest potential limitation. A positive CSF PCR result indicates the presence of viral nucleic acid. In general, a positive CSF PCR result indicates recent or ongoing active viral infection of the CNS by that particular pathogen, especially in immunocompetent individuals. Possible exceptions, in which a positive CSF PCR result does not indicate true CNS viral infection (false-positive result), include cases in which a breakdown of the blood-brain barrier (e.g., in severe bacterial meningitis) or contamination of the CSF with blood has occurred. In these cases, infectious nucleic acid may be carried into the CNS and detected as a contaminant, rather than reflecting local infection of the CNS itself. Some experts have also suggested that this scenario might also theoretically occur in the case of viral pathogens which preferentially infect leukocytes: viral genome present in circulating leukocytes may be carried into the CSF as bystanders during the host inflammatory response and detected by PCR (128, 244). Several other factors may contribute to the low specificity seen in some PCRs, such as nonspecific binding of primers to nontarget DNA, cross-reaction of primers with related viruses, and contamination of specimens. Methods to increase specificity (decrease false positives) of the PCR include stringent laboratory standards to avoid and detect contamination, such as inclusion of duplicate samples and positive and negative controls in each PCR run. A dedicated space for the PCR should be used, and positive pressure tips should be used for pipetting samples and reagents to limit the chance of carryover contamination. In addition, the specificity of all amplified PCR products should be confirmed by hybridizing the gel on which the product was electrophoresed and visualized with pathogen-specific probe.
Positive and negative predictive values. The predictive value of any test depends not only on the specificity and sensitivity of the test but also on the prevalence of the pathogen or disease in the population in which it is being tested. Positive CSF PCR results may be more difficult to interpret for neurotropic pathogens which also cause non-CNS disease with a high baseline prevalence in the general population, since the frequency with which viral nucleic acid is detectable in CSF in the absence of clinical CNS disease is not well delineated. A good example of this is with regard to CSF PCR testing for human herpesvirus 6 (HHV-6) in immunocompetent hosts. CSF PCR testing for HHV-6 inherently has a low positive predictive value, since the viral genome has been detected in the CSF of healthy children in the absence of CNS disease (see "HHV-6" section below).
Pan-herpesvirus assays. Several groups has developed assays that contain primers for detection of HSV-1, HSV-2, varicella-zoster virus (VZV), CMV and EBV, and HHV-6 in various combinations (28, 35, 39, 86, 147, 158, 218). These assays may prove to be clinically useful in the diagnosis of HIV patients presenting with a CNS disease, since multiple herpesviruses are capable of causing neurologic symptoms in this subset of patients (177).
Herpesviruses with enteroviruses. Since HSV and enterovirus (EV) are the two most commonly identifiable etiologies of sporadic encephalitis in immunocompetent individuals and since PCR testing for both diseases has become the gold standard for diagnosis (see "HSV" and "EV" sections below), there has been interest in the development of assays that can simultaneously detect these pathogens. Several groups have developed multiplex PCR assays that are capable of detecting HSV-1 and -2, EV, and VZV in a single CSF specimen (36, 37, 180).
Others. Multiplex and/or consensus PCR has also been developed for detecting multiple polyomaviruses (JC virus [JCV], BK virus, and simian virus 40) (82) in immunocompromised patients, for codetection of EBV and Toxoplasma in AIDS patients (183), and for detection of various agents of arboviral infection for purposes of both clinical diagnosis and mosquito surveillance (107, 207).
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The widespread use of PCR amplification of HSV DNA from CSF has led to modification of our understanding of the signs and symptoms of HSE and has dramatically broadened the recognized spectrum of HSV CNS infections (70, 71, 83). The clinical features of PCR-defined HSE are generally similar to those previously defined for biopsy-proven disease, although the overall severity of illness identified in PCR-proven cases appears to be considerably milder than that in biopsy-proven series. This difference is also reflected in the fact that in PCR-based series approximately 15 to 20% of patients are identified as having mild or atypical forms of HSE that would almost certainly have previously gone unrecognized.
(ii) Sensitivity, specificity, and predictive value. In cases of HSE, virus is cultured from CSF in fewer than 5% of cases. However, in the overwhelming majority of cases, HSV-specific DNA is present and can be detected by PCR (139, 174, 175). Although previously considered the optimal diagnostic approach for patients suspected of having HSE (214), brain biopsy is now only rarely employed for diagnosis of HSE, since amplification of HSV DNA from CSF by PCR has provided a noninvasive test with speed, specificity, and sensitivity equivalent to that of brain biopsy but without the associated risk of complications. In the most comprehensive study to date, CSF PCR was positive in 53 of 54 patients (98%) with biopsy-proven HSE and was negative in 94% of biopsy-negative cases. This results in a sensitivity of 98%, a specificity of 94%, a positive predictive value of 95%, and a negative predictive value of 98% (139). A recent review of the use of PCR for diagnosis of HSE that combined results achieved in several studies estimated that the sensitivity of CSF PCR for the diagnosis of HSE was 96% and that the specificity was 99% (229). These values can be used in Bayesian decision analysis to determine the effects of both positive and negative CSF PCR results on the likelihood of HSE (Table 3) (229).
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(a) False negatives. The potential of generating false-negative PCR results is obviously of concern, as this may result in failure to treat or to complete treatment in patients who would benefit from therapy. In a study by Lakeman and colleagues (139), 164 CSF specimens were tested for PCR-inhibitory activity by spiking them with 200 copies of HSV DNA and then testing the capacity of PCR to detect this added DNA. Two of the 164 specimens (1%) contained inhibitory activity. In another similar study, only 1 of 64 CSF specimens (1%) was found to have PCR-inhibitory activity (159). In the study by Lakeman and colleagues, one of the PCR-inhibitory CSF specimens contained hemolyzed erythrocytes, and the other was quite xanthochromic. It has been suggested that porphyrin compounds derived from the degradation of heme in erythrocytes may account for some false-negative PCR results (16). This suggests that caution should be used in interpreting negative PCR results in bloody CSF specimens. Rare CSF specimens contain inhibitory activity for the Taq polymerase, even in the absence of hemolyzed erythrocytes or their breakdown products (16, 68). Determination of intrathecal synthesis of HSV-specific antibody (118) may complement PCR, especially in patients who have already been ill for a week or more at the time that the first CSF specimen becomes available for diagnostic testing (52). Antibody tests are insensitive early after onset of illness but become positive in the majority of patients 2 weeks after onset of illness and later (14). For this reason, especially for patients with symptoms that have lasted a week or more, both CSF PCR and CSF or serum antibody studies should be performed.
(b) False positives. The incidence of false-positive CSF PCR results for HSV meningitis and encephalitis seems to be exceedingly low when the studies are performed in experienced laboratories using accepted standards and methods to avoid inadvertent contamination. Isolated examples of laboratories with high numbers of false-positive tests have been traced to improper procedures resulting in sample-to-sample contamination (84), but fortunately this problem can be eliminated by appropriate attention to approved laboratory practices. As noted, in the series by Lakeman and colleagues 3 of 47 brain biopsy-negative patients were found to be CSF PCR positive for HSV DNA. If all of these patients are considered false positives, this represents an incidence of about 6%. This is likely to be a substantial overestimate, as it is likely that most, if not all, of these cases were actually examples of false-negative brain biopsy results (139). In another study, none of 83 patients with "other neurologic disorders" and none of 30 patients with non-HSV-related meningitis or encephalitis had positive HSV CSF PCR results (159). It is important to recognize that these studies assume that PCR testing was done in an appropriate clinical setting (e.g., for a patient with suspected acute meningitis or encephalitis).
(iii) Kinetics of PCR positivity.
It is vital in evaluating the diagnostic utility of PCR for HSE to understand when viral DNA initially appears in the CSF, how long it remains detectable, and what effects antiviral therapy have on CSF HSV DNA levels. Initial studies of CSF PCR in HSE suggested that results became positive early after infection. However, recent studies have found that CSF PCR may be negative when performed during the first 72 h. In one recent study, 3 of 11 patients were found to have an initially negative CSF PCR performed at 1 to 3 days after onset of symptoms, which subsequently became positive when repeated 4 to 7 days later (247). This result is significantly higher than the rate of 
5% seen in earlier studies (102). Until further data are available, it would seem prudent to interpret negative CSF PCRs with caution when they are obtained within 72 h of symptom onset. Patients for whom no alternate diagnosis is identified should have their HSV CSF PCR repeated between days 4 and 7. Acyclovir therapy should not be discontinued on the basis of a negative CSF PCR obtained early (<72 h) after onset of symptoms for patients in whom the clinical suspicion of HSE remains high. Despite these concerns, retrospective studies of patients with negative HSV PCR testing suggest that fewer than 0.4% of these cases may have been false negatives (examples of HSE missed by PCR) (176).
(iv) Effect of antiviral therapy on PCR positivity. Lakeman and colleagues (139) found no effect of antiviral therapy lasting 1 week or less on detection of HSV DNA in CSF. However, only 47% of specimens from patients who received 8 to 14 days of antiviral therapy and only 21% of specimens from those receiving more than 2 weeks of therapy remained positive for HSV DNA. For the majority of patients included in this study, antiviral therapy consisted of vidarabine rather than acyclovir, so caution should be used in extrapolating these results to patients treated with acyclovir. However, in another study in which approximately two-thirds of the patients were treated with acyclovir (the remaining one-third having received vidarabine), HSV was shown to persist for at least 5 days after the start of antiviral therapy (212). Interestingly, it has been suggested that the amount of HSV DNA in the CSF actually increases during the first 5 to 6 days of acyclovir therapy and then subsequently declines (102, 174). Abortive replication of HSV in the presence of acyclovir may generate large quantities of small viral DNA fragments that facilitate PCR amplification (102, 174). In the majority of treated cases of HSE there is a sharp decline in the number of PCR-positive cases after 2 weeks, and HSV DNA is virtually never detected after 30 days (16, 212). As a result of these findings, it was recently suggested that CSF PCR should be performed upon completion of 10 to 14 days of acyclovir therapy and that patients who still have detectable HSV DNA in their CSF should receive an additional course of therapy (52).
(v) Quantitative HSV PCR. Few studies evaluating the potential utility of quantitative HSV PCR in determining prognosis of HSV encephalitis currently exist (69, 182, 253). In one study (69), patients with >100 copies of HSV DNA per µl of CSF (high copy number) had poorer outcomes than those with lower levels. In general, larger amounts of HSV DNA were found in CSF of patients with a longer duration of symptoms. For example, patients with high copy numbers had a mean of 6.6 ± 3 (standard deviation) days of symptoms, compared to 3.9 ± 3 days for those with low copy numbers. Patients with higher DNA copy numbers also tended to be older (mean age of 45 ± 19 years, compared to 25 ± 19 years). A higher copy number also reflected disease severity, with all patients having high copy numbers having a reduced level of consciousness, compared to only 43% of those with low copy numbers. All patients with high copy numbers also had CT scan abnormalities, compared to only 14% of patients with low copy numbers. As might be expected from these data, patients with high copy numbers also had a significantly worse prognosis than those with low copy numbers. Mortality was 11% in those with high copy numbers, and 63% of the survivors had moderate or severe impairment at a 3-month follow-up. By contrast, all patients with low copy numbers survived, and all had normal function at a 3-month follow-up. However, in a separate study (182), initial HSV DNA levels were not related to severity of clinical symptoms or predictive of outcome.
Quantitation of HSV viral load in the setting of neonatal HSV infection has been studied and may be useful for assessing prognosis, as well as provide additional information for clinical management (127, 182). Patients with disseminated infection had higher viral loads in their sera, whereas patients with CNS infection exhibited higher CSF viral loads. Serum viral loads were significantly higher in patients who subsequently died, and HSV-2-infected patients had higher CSF viral loads than HSV-1-infected patients, along with more CNS involvement and neurologic impairment.
(vi) Role of HSV PCR in special clinical situations. (a) Management of recurrent disease. A small number of adult patients with confirmed HSE will have a recurrence of symptoms following antiviral therapy (126, 238). The incidence of recurrent disease has been <10% in most reports of adult cases, but it may be significantly higher in children (126) and particularly in neonates (125) (see below for further discussion of neonatal HSV). It may be difficult to distinguish between postinfectious demyelination and ongoing infection as causes of recurrent symptoms. The absence of HSV DNA detectable by CSF PCR and the presence of white matter lesions on MRI suggest an immune-mediated postinfectious encephalopathy. A persisting positive CSF PCR or reisolation of virus on brain biopsy should prompt retreatment. Acyclovir-resistant isolates of HSV may arise as a result of mutations in the genes encoding either the viral thymidine kinase or DNA polymerase.
(b) HSV brain stem encephalitis. HSV is probably the most important cause of sporadic viral brain stem encephalitis (BSE) (186, 208). The clinical presentation of HSV BSE is indicative of multifocal involvement of the brain stem from midbrain to medulla. Several cases have been reported in patients with AIDS (104, 161). HSV CSF cultures are positive in a minority of cases (191), but evidence of increased intrathecal synthesis of HSV-specific antibody is supportive of the diagnosis. CSF PCR has been positive in isolated cases of both acute and recurrent brain stem encephalitis (43, 156, 163). CSF PCR has been used to amplify HSV-1-specific DNA from the CSF of a patient with recurrent episodes of BSE (235). These reports suggest that the diagnosis, as well as the clinical and epidemiological spectra of HSV BSE, may become more clearly delineated with the more widespread use of CSF PCR for antemortem diagnosis.
(c) Neonatal disease. The application of PCR to neonatal HSV disease has expanded our understanding of the extent to which CNS involvement occurs in each of the three presentations of neonatal HSV disease. In a retrospective study of 77 neonates with HSV (125), HSV DNA was detected in the CSF of 93% of infants with disseminated infection and 76% of infants with CNS disease, but also in 24% of infants with disease presumed to be limited to skin, eye, and mouth (SEM disease). This finding has led to the appreciation that infants diagnosed with SEM disease may also be at increased risk for long-term neurologic sequelae, particularly with three or more SEM recurrences in the first year following diagnosis. CSF PCR was also analyzed as a prognostic marker in that study: 95% of infants with positive PCR following completion of 10 days of antiviral therapy experienced significant morbidity and mortality. Quantitative PCR techniques are currently being studied to provide more detailed information about the kinetics of neonatal HSV disease and impact of viral load on outcome. In a subsequent prospective study of 26 neonates with SEM HSV disease, the efficacy of suppressive acyclovir against recurrences of SEM disease was evaluated for 6 months following resolution of the initial neonatal HSV diagnosis. Eighty-one percent of the infants remained recurrence free, in comparison to 54% of historical controls who did not receive suppressive therapy, and one infant with SEM recurrence had detectable HSV DNA in CSF by PCR (124).
Enteroviruses (EV). (i) General comments. Enteroviruses are RNA viruses (plus sense) of the picornavirus family. Sixty-four unique serotypes have been identified, which have been subgrouped as poliovirus, coxsackievirus, echovirus, or enteroviruses 68 to 71. Enteroviral infection occurs predominantly as a summer and fall illness (July to October), with a higher incidence in children than in adults (198, 203-205). Although EV infection may present as a nonspecific febrile illness with or without rash, the virus is neurotropic and CNS infection may occur. The spectrum of CNS disease is broad, including benign aseptic meningitis, which leads to 75,000 cases and 50,000 hospitalizations per year in the United States and accounts for 80 to 92% of cases where an etiologic agent is identified (189). Fifty percent of infants and children with febrile illness due to EV have concomitant meningeal involvement. Enteroviral encephalitis occurs as either a focal or a diffuse process and is the third most commonly identified cause of all encephalitis, after herpes simplex virus and arboviruses (194). EV may also cause a severe sepsis syndrome in newborns, consisting of meningoencephalitis, myocarditis, and hepatitis, leading to severe morbidity and mortality in that population (5). Chronic meningoencephalitis may occur in agammaglobulinemic patients (246).
(ii) Sensitivity and specificity of EV RT-PCR compared to culture. The 5' nontranslated of the EV genome is useful for detection by RT-PCR, since it is a highly conserved sequence among all enterovirus serotypes. This region contains elements essential for replication of the viral genome as well as translation of its protein-coding region. Enteroviral RT-PCR allows rapid detection (<24 h) of EV in clinical samples, in contrast to the 4 to 8 days required for viral culture (192, 193). Rapid distinction of polioviruses from other enteroviruses is possible by utilizing restriction analysis of PCR products. Colorimetric, nonquantitative methods are highly accurate and provide results within 4 h (200). Single-tube, quantitative detection of the EV genome by using real-time EV RT-PCR has been demonstrated in a linear range spanning 5 log units, with a sensitivity of 96 to 100% and a specificity of 96% compared to viral culture and with results available in 4 h (160, 178, 239, 240).
Although EV RT-PCR is readily available at most academic institutions, NASBA is an additional molecular method that may be considered as a suitable alternative for sensitive amplification and detection of enterovirus sequences in a range of clinical specimens, including CSF (85). NASBA is an isothermal, in vitro transcription-based amplification method which does not require specialized equipment (such as a thermal cycler) and has been developed into convenient kits for use in clinical laboratories in which RT-PCR methodology is not available.
There are multiple reasons why nonmolecular methods of enteroviral detection are suboptimal (160, 178, 194, 239, 240). Viral culture is insensitive (65 to 75%), likely due to the presence of neutralizing antibody, the relatively low viral load at the time of diagnosis, and the fact that several EV serotypes are simply not cultivable. Prolonged time is required for isolation of EV from CSF by tissue culture, often up to 8 days. Culture is labor-intensive, requiring cultivation on multiple cell lines. Culture of throat swabs or stool specimens provides only circumstantial evidence of etiology, not causality, in the setting of CNS disease, since prolonged excretion from these sites (4 weeks from the oropharynx and 16 weeks from the gastrointestinal tract) may occur following EV infection. Serologic approaches are hampered by the diversity of EV serotypes. Because of these problems, there is often difficulty in distinguishing EV disease from bacterial meningitis or sepsis. This leads to excessive use of empirical intravenous antimicrobial interventional procedures and tests and prolonged hospitali-zation.
The sensitivity of EV RT-PCR greatly exceeds that of culture techniques, and it has replaced culture as the gold standard for diagnosis of EV infection of the CNS. Enterovirus PCR has high sensitivity and specificity, which are both estimated at >95%, and has been validated in multiple independent studies (160, 178, 187, 195, 200, 239, 240). The sensitivity of CSF PCR exceeds that of serum PCR (approximately 81 to 92% sensitivity) and urine PCR (62 to 77% sensitivity). Several strains of EV which are not cultivable are detectable by EV RT-PCR. This is one explanation for the observations in one study that several clinical samples with negative results by culture showed detectable EV genome by RT-PCR (206). Despite the excellent sensitivity of the CSF EV RT-PCR, results may be negative in the setting of EV71 CNS infection (unpublished observation).
EV PCR is a sensitive diagnostic modality for EV CNS disease in the pediatric population. Up to 50% of children less than 1 month of age with clinical illness compatible with EV infection but without CSF pleocytosis have been noted to have detectable EV genome in their CSF by RT-PCR. EV PCR of serum and urine is also highly useful in the diagnosis of neonatal enteroviral disease. In one study, a colorimetric (PCR) assay was demonstrated to be more sensitive than viral culture in identifying viral infection in initial serum and urine specimens from 16 enterovirus-infected newborn infants, and it remained more sensitive throughout their illnesses, with a combined sensitivity of serum and urine PCR of 88% and a specificity of 100% (6).
(iii) Impact on hospitalization and patient management. Enterovirus PCR has had a profound impact on the management of patients with aseptic meningitis (217). In a large study in 1998, a retrospective chart review was performed for 276 inpatients for whom CSF PCR for enterovirus had been performed (179). Diagnoses in these patients included aseptic meningitis, convulsions, fever, and unspecified illness. The impact of EV PCR on clinical parameters, including length of stay, time to discharge following the PCR result, use of medications, and ancillary tests, was evaluated. In this study, 187 of 276 patients (70%) had the EV PCR result available prior to hospital discharge; 95 of these patients were positive, and 92 were negative. When the 95 patients with positive test results were compared to the 92 patients with negative test results, significant reductions (P < 0.001 to 0.005) were found in length of hospital stay (42.4 versus 71.5 h), time from PCR test to hospital discharge (5.2 versus 27.4 h), number of ancillary tests performed (head CT or MRI, 10 versus 71.2%; EEG, 1 versus 18%), and days of intravenous antimicrobial exposure (2 versus 3.5 days). Several groups have attempted to estimate potential cost reductions related to management of patients with enteroviral meningitis with use of RT-PCR (148, 165, 168, 185). Marshall's group (148) found a 59% reduction in overall hospital costs in one study of infants <6 months of age with CNS pleocytosis. Robinson's group (185) found a mean cost savings of >$2,000 per patient in a study of pediatric aseptic meningitis. An important proviso is that these cost reductions are only possible with rapid turnaround of results to the clinician, which is feasible only when in-laboratory-developed testing is available.
While the availability of EV RT-PCR in cases of aseptic meningitis has had an impact on hospital costs and reduced unnecessary use of antimicrobials and imaging studies, EV PCR has also allowed the rapid identification of cases of more severe EV CNS infection and neonatal sepsis, allowing for initiation of specific antiviral therapy. Pleconaril, a drug which integrates into a hydrophobic pocket on the surface of the EV capsid and inhibits viral receptor binding and uncoating, has undergone extensive clinical testing and appears to be both safe and effective (4, 188, 196, 199). In a recent study of life-threatening enteroviral infections, 12 of 16 patients with chronic enteroviral meningoencephalitis, 2 of 3 patients with paralytic poliomyelitis (two vaccine associated and one wild type) and 2 of 3 patients with encephalitis and/or transverse myelitis showed clinical improvement following treatment. In most cases in which data were available, clinical improvement was associated with virological responses, including sterilization of positive cultures or conversion of positive PCRs to negative (197, 201). Although currently limited predominantly to research studies, Pleconaril is available on an open-label compassionate-use basis and should be considered for patients with potentially life threatening EV infections, including severe encephalitis, flaccid paralysis, neonatal sepsis, and chronic meningoencephalitis (197, 216).
JC virus (JCV). JC virus is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating CNS infection primarily affecting patients with AIDS. The utilization of CSF PCR to detect JCV DNA has become an important minimally invasive diagnostic test for this disease, which previously required brain biopsy for definitive diagnosis (63, 202). CSF PCR for JCV has a sensitivity of 50 to 75% and a specificity of 100% for the diagnosis of PML as demonstrated in prospective analyses of HIV-infected patients presenting with focal neurologic signs and symptoms (106, 245). Performing PCR on pellets of centrifuged CSF may be even more sensitive than analysis of CSF supernatants (133). JCV viral loads in urine and blood are not predictive of clinical CNS infection due to JCV, as viruria is found as frequently in HIV-positive individuals as in uninfected control subjects, and JCV viremia correlates only with the level of immunosuppression and not with the presence of PML (133).
A potential correlation between the JCV viral load in CSF and the PML prognosis has also been investigated by using quantitative PCR techniques (76, 81, 88, 89, 133, 225, 226, 258). In a recent study, CSF samples from 12 patients with PML were noted to have wide variations in JCV load (>6 log units), with values of >4.7 log units associated with shorter patient survival time. No correlation was found between CSF viral load values and the global volume of damaged brain tissue, however (89). Another group studying patients with PML found differences between CSF and tissue JCV DNA concentrations of approximately 4 orders of magnitude and highly variable JCV DNA viral loads in CSF samples taken at comparable states of disease (72). CSF JCV loads were not related to absolute CD4 cell counts or plasma HIV viral loads in at least one study (226).
Other groups have used quantitative PCR to investigate the effect of highly active antiretroviral therapy (HAART) (72, 88), as well as cidofovir treatment (226), on JC virus load in CSF and to determine the clinical outcomes of patients with AIDS-associated PML. A recent multicenter analysis of 57 such patients revealed that among HAART-treated patients with baseline JCV levels of <4.7 log units, reaching undetectable levels after therapy predicted longer survival (67). Similar findings have been reported by other groups (67, 157).
Epstein-Barr virus (EBV). (i) Immunocompetent hosts. EBV has been implicated as an etiologic agent of benign aseptic meningitis, encephalitis, cerebellar ataxia, myelitis, and ADEM in immunocompetent hosts. Meningoencephalitis can complicate infectious mononucleosis or can occur as the sole manifestation of EBV infection. There are no distinctive clinical features of EBV meningoencephalitis. Patients are often children or young adults. CSF PCR has been extremely useful in diagnosis of EBV CNS infections (87, 110, 119, 141, 219-221, 233). One study detected EBV DNA in the CSF of 7.4% of immunocompetent individuals with CNS disease of unknown etiology, using nested PCR (171). Surprisingly, given that seropositivity approaches 90% in older adults, CSF PCR is not positive in patients with latent EBV infection, and false positives rarely occur in EBV-seropositive individuals who develop non-EBV meningoencephalitis. Quantitative PCR studies are performed at some research laboratories, and determination of the EBV DNA copy number may help further distinguish true-positive active infections from rare cases with false-positive results from latent virus or rare patients with dual-positive CSF PCRs (220).
(ii) Immunocompromised hosts. EBV infection is associated with AIDS-related CNS lymphomas in HIV-infected patients, including primary CNS lymphoma (PCNSL) and AIDS-related non-Hodgkin lymphoma with CNS involvement (CNS-NHL). Patients with PCNSL and CNS-NHL often have positive EBV CSF PCR results, and the test appears to be a sensitive indicator of the presence of a tumor, with nearly 100% sensitivity and 98.5% specificity (44, 50, 56, 63, 170, 223). Using quantitative techniques, high CSF EBV DNA levels have been found in these settings, and viral load may be clinically useful, with high plasma EBV DNA levels also potentially predictive of CNS involvement (26, 106, 155). The availability of EBV PCR, in conjunction with JCV PCR (see "JCV" section above) in AIDS patients presenting with an intracranial mass has been a major advance in the management of these patients. PCR, in combination with clinical and neuroradiologic characteristics, provides another noninvasive tool to help differentiate CNS lymphomas from brain masses due to Toxoplasma infection or PML (JCV infection) without requiring brain biopsy in many cases (10).
In a recent study, real-time PCR was performed to quantify EBV DNA in CSF and plasma from 42 patients with AIDS-related NHL, with and without CNS involvement, as well as 20 patients with PCNSL and 16 HIV-infected controls with other CNS disorders (11, 26). EBV DNA was detected in the CSF from 8 of 12 (67%) patients with CNS-NHL and 16 of 20 (80%) patients with PCNSL, in contrast to only 7 of 22 (32%) patients with systemic NHL and 2 of 16 (13%) of the controls. EBV DNA levels were significantly higher in the CSF from patients with PCNSL or CNS-NHL compared to patients with systemic NHL or controls. No difference in plasma viral load was found between patient groups.
Quantitative EBV PCR has also recently been utilized to stratify the risk of EBV-associated CNS disease, including CNS lymphoma, encephalitis, and postinfectious neurologic complications. CNS lymphoma patients had high EBV loads (4.8 ± 0.2 log10 DNA copies/ml) and low CSF leukocyte counts, whereas encephalitis patients had high EBV loads (4.2 ± 0.3 log10 DNA copies/ml) and high leukocyte counts and postinfectious patients showed low EBV loads (3.0 ± 0.3 log10 DNA copies/ml) and high leukocyte counts. Additionally, lytic-cycle EBV mRNA, a marker of viral replication, was shown to present only in patients with CNS lymphoma and encephalitis (248).
PCR has also been utilized to monitor the response to therapy in HIV-infected patients with AIDS-related primary CNS lymphoma. Reductions in CSF EBV DNA burden following chemotherapy, as monitored by semiquantitative nested PCR, has been significantly associated with prolonged survival in these patients., suggesting that EBV DNA monitoring might be helpful in predicting response to chemotherapy (11, 12).
In contrast to the association of EBV with CNS lymphomas in HIV patients, other authors have indicated uncertainty about the role of EBV in other neurologic illnesses in these patients. One study of HIV-infected patients with CNS disorders distinct from and unassociated with primary CNS lymphoma revealed detectable EBV DNA in the CSF of 10 of 79 patients. However, only 1 sample in 10 had EBV as the sole pathogen detected, whereas 6 samples in 10 were also positive for other microbial agents of recognized neurologic pathogenicity. None of six tested samples had a detectable intrathecal EBV antibody response, despite the presence of the EBV genome. The authors hypothesized that the presence of EBV DNA in the CSF of a large fraction of these patients could be an incidental event associated with EBV reactivation in host PBMCs, rather than indicating EBV-associated CNS disease (170).
Cytomegalovirus (CMV). Although 80% of immunocompetent individuals are seropositive for CMV by adulthood, CMV meningoencephalitis is largely a disease of congenitally infected newborns and immunocompromised individuals, including organ transplant recipients and those with advanced HIV infection. In immunocompromised individuals, infection results predominantly from reactivation of latent virus. Nervous system complications include diffuse ventriculoencephalitis, focal periventricular disease, polyradiculomyelitis peripheral neuropathies, and retinitis (51). CSF PCR has proven to be an extremely useful technique for detecting CMV CNS infections in immunodeficient hosts, with a excellent sensitivity (82 to 100%) and specificity (86 to 100%) in HIV-infected patients (21, 30, 51, 55, 256, 257). CSF PCR appears to be a specific indicator of CNS infection, including retinitis (31, 237). The use of DNA PCR to detect CMV in CSF has greatly enhanced the ability to diagnose and differentiate CMV from other AIDS-related CNS diseases (98, 220, 221).
Quantitative CMV PCR is available in a number of laboratories, and CMV DNA copy number may provide a better index of disease severity (13, 211, 254). The CMV viral load in peripheral blood leukocytes has also been monitored as a method to predict which immunosuppressed patients might develop end-organ disease. The clinical utility of the test in the setting of congenital CMV infection is also being investigated, particularly with respect to correlating neurologic outcome with CSF viral load and efficacy of antiviral therapy (103, 121).
CMV PCR of CSF has also been utilized to identify the presence of antiviral-resistant strains from patient isolates (215, 256). Mutations within the UL97 region, which encodes a human CMV protein kinase, have been found to confer ganciclovir resistance and can be detected in plasma and CSF by direct sequencing of PCR products.
Other molecular techniques have been applied to the diagnosis of CNS CMV infection, including detection of CMV transcripts (human CMV mRNA). Transcription of late human CMV genes occurs only after viral replication, and therefore analysis for late gene transcripts should distinguish between active and latent infection. The pp67 late-gene transcript assay has been studied and compared to PCR and culture techniques for diagnosis of CNS CMV infection (21, 98, 260), and superior sensitivity has been suggested in some studies (98).
As with EBV PCR, CMV-seropositive individuals with latent infection do not have detectable DNA based on PCR testing. In one small study, 9% of immunocompetent CMV-seropositive individuals (1 out of 11) developed a positive CMV CSF PCR during bacterial meningitis, suggesting that other CNS infections can cause false-positive CMV PCRs, although the frequency with which this occurs in the population as a whole is unclear (221).
VZV is rarely cultured from CSF, but a positive CSF PCR for VZV DNA has been found in 60% of meningoencephalitis cases and, if present, distinguishes this from ADEM. Lower rates of CSF PCR positivity (25%) have been reported in later analyses which included all CNS symptoms associated with VZV, not limited to meningoencephalitis, regardless of whether symptoms occurred in the setting of primary or reactivated infection (75, 134, 135). Intrathecal synthesis of VZV-specific antibody or the presence of anti-VZV immunoglobulin M (IgM) antibody in the CSF is also specific for encephalitis and may be positive when PCR studies are negative. With the availability of PCR, it is increasingly being recognized that both varicella meningitis and encephalitis can occur in the absence of a detectable rash: recent studies have reported that only about one-half of patients with acute CNS symptoms related to VZV had an associated typical rash (75, 97, 112, 134). In patients without a rash, VZV CSF PCR may play a pivotal role in making early diagnosis, which is necessary for successful treatment of CNS VZV.
(b) Zoster. In immunocompromised patients, and much more rarely in apparently immunocompetent individuals, meningoencephalitis can occur as a complication of zoster reactivation. Pathologically, a variety of discrete processes can be identified. A vasculopathy of large arteries (granulomatous arteritis) occurs in older patients (>60 years of age) following an outbreak of trigeminal zoster. Pathological studies have shown viral antigen and nucleic acid in the affected arteries. Encephalitis also results from a small-vessel vasculopathy that produces multiple infarcts in both cortical and subcortical gray and white matter combined with deep white matter ischemic or demyelinating lesions (8, 93, 95, 96, 129). Zoster infection in immunocompromised patients also may result in a necrotizing infection of ependymal cells resulting in ventriculitis and periventriculitis, usually presenting with hydrocephalus, altered mental status, and gait difficulty. CSF PCR is positive for VZV DNA in the majority of these cases, and intrathecal synthesis of VZV-specific antibody occurs. Other groups have documented lower sensitivity of CSF PCR in the setting of subacute to chronic CNS VZV infection (myelitis and encephalitis) and have stressed that intrathecal synthesis of VZV-specific antibody may be a more reliable diagnostic measure in such cases, particularly with intervals of weeks to months between zoster and onset of neurologic disease (94).
In a recent study of HIV-infected patients, a favorable influence of HAART on the prevalence of neurologic complications caused by VZV among AIDS patients was noted, with a large reduction in the prevalence of VZV-related disease noted since 1996, the point at which HAART became widely available. The sensitivity of PCR in detecting VZV DNA in CSF from these patients with known VZV neurologic disease was 80%, with a specificity of 98%, in the small number of patients that were available for evaluation (59). A larger multicenter retrospective study of the neurologic complications of VZV infection in 34 HIV-infected adults (including encephalitis, myelitis, radiculitis, and meningitis) reported CSF PCR positivity in 100% of cases, compared to culture positivity in only 33% (66).
(ii) Human herpes virus 6. HHV-6 is a member of the herpesvirus family that, like CMV, is classified in the betaherpesvirus subfamily. Epidemiological studies suggest that infection occurs in early childhood (6 to 12 months), with two-thirds of children being seropositive by 1 year and up to 95% being seropositive by adulthood. Viral invasion of the CNS appears to be a common event during primary infection (38) and may account for the fact that one-third of infants with primary HHV-6 infection develop seizures. CSF PCR was positive for HHV-6 in 9 of 10 children with roseola in one study (132). A more recent study found that 10% of febrile infants under 3 months of age undergoing evaluation for sepsis had evidence of HHV-6 infection in plasma, CSF, or both, but HHV-6 DNA was found in infants with and without alternative explanations for their fevers (34).
HHV-6 has also been implicated as a cause of febrile convulsions. In one study, 80% of children with >3 febrile convulsions had HHV-6 DNA detectable in CSF, suggesting the possibility that HHV-6 can cause a syndrome of recurrent febrile convulsions (132). However, the incidence of primary HHV-6 infection is similar in patients with febrile seizures and age-matched controls (109), and HHV-6 DNA was not detectable in CSF from any of 23 prospectively analyzed febrile seizure patients in a separate study (228).
These results are consistent with results of PCR on brain tissue in which HHV-6 DNA is detectable in >75% of normal brains (42, 145). In a PCR-based study to investigate the role of herpesviruses in neurologic syndromes, HHV-6 was detected in 1% of adult immunocompetent patients presenting with a variety of neurologic symptoms and, in some of the cases, concurrent with dia
