key: cord-0008180-di23dkn5
authors: Cinque, Paola; Bossolasco, Simona; Lundkvist, Åke
title: Molecular analysis of cerebrospinal fluid in viral diseases of the central nervous system
date: 2002-12-21
journal: J Clin Virol
DOI: 10.1016/s1386-6532(02)00173-7
sha: b4f0eaf115485c10a73d51d2e3b3734cad437cda
doc_id: 8180
cord_uid: di23dkn5

The use of nucleic acid (NA) amplification techniques has transformed the diagnosis of viral infections of the central nervous system (CNS). Because of their enhanced sensitivity, these methods enable detection of even low amounts of viral genomes in cerebrospinal fluid. Following more than 10 years of experience, the polymerase chain reaction or other NA-based amplification techniques are nowadays performed in most diagnostic laboratories and have become the test of choice for the diagnosis of several viral CNS infections, such as herpes encephalitis, enterovirus meningitis and other viral infections occurring in human immunodeficiency virus-infected persons. Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin and to recognise unusual or poorly characterised CNS diseases. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise genome characterisation. Current efforts are aiming at further improvement of the diagnostic efficiency of molecular techniques, their speed and standardisation, and to reduce the costs. The most relevant NA amplification strategies and clinical applications of to date will be the object of this review.

Cerebrospinal fluid (CSF) examination is an essential part of the diagnostic work-up of patients with suspected central nervous system (CNS) viral infections. It is often the means to achieve an etiological diagnosis, either by the direct identification of the responsible virus, or by the demonstration of an intrathecal specific immune response. Traditional direct virological techniques, including virus isolation, antigen detection and microscopy examination, usually have low sensitivity. Only virus isolation in cell culture may be of some value for diagnosis of aseptic meningitis, with enteroviruses found in almost half of the cases. Virus isolation, however, is insensitive for other viruses, such as herpes simplex virus type 1 (HSV-1) and most arboviruses. Virus antigen detection techniques and light or electron microscopy are of limited value, as they require a high number of CSF-infected cells for virus identification (Rubin, 1983) . On the other hand, indirect diagnosis by detection of intrathecally produced antibodies generally has poor sensitivity during the early stages of the disease.

Over the last decade, nucleic acid (NA) amplification-based techniques, primarily the polymerase chain reaction (PCR), have revolutionised the diagnosis of CNS infections, especially those caused by viruses (Fredricks and Relman, 1999) . Their advantages, accounting for their success in diagnostic neurovirology, have been the extraordinary sensitivity and rapidity. These techniques were first applied to CSF in the early 90s, for the diagnosis of herpes simplex encephalitis (HSE), and enteroviral meningitis (Puchhammer-Stöckl et al., 1990; Rotbart, 1990) . Since then, the number of scientific reports in this field has rapidly increased and NA amplification-based assays are now routinely performed in most diagnostic laboratories.

NA amplification-based techniques have become the test of choice for some viral CNS infections, such as HSE (Darnell, 1993; Tyler, 1994; Weber et al., 1996; Cinque et al., 1996a) . Furthermore, they have been useful to establish a viral etiology in neurological syndromes of dubious origin, e.g., Mollaret's meningitis (Tedder et al., 1994) , or to help recognise unusual or poorly characterised CNS diseases, such as mild forms of herpes encephalitis (Schlesinger et al., 1995a; DeVincenzo and Thorne, 1994; Domingues et al., 1997; Fodor et al., 1998) or cytomegalovirus (CMV) ventriculoencephalitis in human immunodeficiency virus (HIV)-infected patients (Arribas et al., 1996) . Molecular methods have made it possible to identify viruses in CSF normally causing extracerebral infections, such as rotavirus, parvovirus B19, CMV or human herpesvirus 6 (HHV-6), thus supporting their etiological role in inducing CNS disease (Kondo et al., 1993; Suga et al., 1993; Cassinotti et al., 1993; Ushijima et al., 1994; Studahl et al., 1995; McCullers et al., 1995; Barah et al., 2001) . Finally, molecular techniques have been of unique value for identification of novel viruses responsible of CNS disease (Chua et al., 2000; Cardosa et al., 1999) .

2.1. Methods

CSF treatment is usually required prior to NA amplification, in order to release NA from cells and to remove substances that may degrade NA or inhibit amplification. However, the relatively simple CSF composition may obviate, at least for DNA viruses, the need for NA purification. The simplest approaches include heating to high temperatures or repeated freeze-thawing of the specimens, procedures that facilitate cell membrane disruption and release of DNA (Table 1) . These procedures have the advantage of being rapid, requiring small CSF volumes, and reducing the risk of sample contamination during NA purification steps. However, the lack of removal of inhibiting molecules might interfere with the Table 1 CSF preparation prior to nucleic acid amplification Principle

Method (examples) CSF cell lysis Heating to 95 8C, freezing thawing CSF cell lysis-protein digestion Detergents (SDS), proteases (protease K), chaotropioc agents (guanidiniun thiocyanate) a Nuclic acid concentration Ultracentrifugation Ethanol precipitation of nucleic acids Nucleic acid extraction Phenol Á/chloroform, spin column, silicate absorption, magnetic separation Methods for cell lysis, and concentration and extraction of nucleic acids can variably be combined. Required time varies from 10 min (e.g., by mechanical cell lysis) to E/1 h (e.g., protease K digestion and/or complex nucleic acid procedures, e.g., phenol Á/chloroform). Required volume varies from 2-5 ml (e.g., mechanical cell lysis) to E/1 ml (use of CSF concentration procedures). enzymes used for amplification. NAs may also be concentrated and/or purified from CSF, by a number of in-house procedures or commercial kits (Table 1) . Although some extraction methods seem to perform better than others in comparative studies (Casas et al., 1995; Fahle and Fischer, 2000) , none of the known protocols has been shown to be clearly superior. Practically, the choice of one CSF preparation method is supported by a number of considerations, including the type of NA target and the amplification protocol employed, as well as the individual laboratory experience.

NA amplification techniques enable the amplification of small quantities of target NA molecules to considerably larger amounts (over 10 6 copies), which can be visualised by means of common laboratory procedures. This results in a very high sensitivity, which is the main advantage of these techniques. PCR is the most popular amplification method, but a number of other techniques have been described, including the ligase chain reaction, the strand displacement assay, the transcription mediated amplification, the nucleic acid sequence based amplification (NASBA), the branched DNA technique, and the hybrid capture assay .

PCR has been widely used for detection in CSF of both DNA and RNA viruses. In the case of RNA viruses, a complementary DNA (cDNA) needs to be generated from RNA prior to amplification, by means of a reverse transcriptase (RT). A variant of the classical procedure, largely used for analysis of CSF and other biological fluids containing few viral particles, is the 'nested' PCR. This consists of two separate amplification reactions using two primer sets, the second of which is located between the first one, thus increasing substantially both sensitivity and specificity of detection .

NA amplification techniques other than PCR that have been applied to detect viral genomes in CSF include the NASBA and the branched DNA assays. NASBA, like PCR, is based on target NA amplification, but the synthesis of new molecules occurs through an isothermal reaction and re-quires three different enzymes. Furthermore, the template consists of RNA (Kievits et al., 1991) . Examples of NASBA studies of CSF include those performed to detect the CMV pp67 late gene transcripts in HIV-infected patients with CMV encephalitis; enterovirus, West Nile (WN) or St. Louis encephalitis virus RNA in patients with aseptic meningitis or encephalitis (Zhang et al., 2000; Bestetti et al., 2001; Lanciotti and Kerst, 2001; Heim and Schumann, 2002; Fox et al., 2002) , and to assess HIV-1 RNA levels in patients with HIV infection at different disease stages (McArthur et al., 1997; Shepard et al., 2000) . The branched DNA assay is based on signal amplification rather than target-amplification (Urdea, 1994) . This assay has been used to measure CMV DNA and HIV-1 RNA levels in the CSF of HIV-infected patients (Flood et al., 1997; Stingele et al., 2001) .

There are different procedures to detect the products of NA amplification. The simplest technique is based on the visualisation of DNA bands of the expected size by agarose gel electrophoresis after staining with ethidium-bromide. Alternative or supportive methods include hybridisation with DNA probes complementary to the target DNA, following DNA transfer to a filter, tubes, or microplates. Probes are labelled with enzymes or other molecules that lead to signal detection on appropriate stimulation. Colorimetric enzymelinked immunosorbent assays (ELISA), in which the amplified products is captured by a probe coated to a microplate, have proved to be very practical. For this reason colorimetric ELISAs have largely been adapted to commercial kits and used for CSF analysis for a number of viruses (Rotbart et al., 1994; Ellis et al., 1997; .

Since similar neurological pictures may result from different CNS infections, it may be practical to use PCR assays that detect more that one virus or infectious agent in the same reaction. The most obvious advantage of this approach is that the number of tests are reduced, with substantial time and cost savings. Two main strategies are used for this purpose: multiplex PCR and PCR with consensus primers.

Multiplex PCR enables the identification of more than one DNA sequence by means of two or more primer pairs, each specific for one sequence (Fig. 1 ). An important requirement of this approach is that the amplification conditions, i.e. reagent mixture composition and thermocycling profile, are similar for all the primer pairs, in order not to compromise amplification efficiency for each primer pair. Developments of multiplex PCR might lead to universal diagnostic protocols, based on the use of several primer pairs with fixed thermocycle programs and reagent composition (Kuno, 1998) . Multiplex PCR assays are largely employed in diagnostic neurovirology, e.g. for simultaneous detection of HSV-1 and HSV-2 (Kimura et al., 1990; Cassinotti et al., 1996; Cinque et al., 1998a) , or of a larger number of herpesviruses (Tenorio et al., 1993; Baron et al., 1996; Pozo and Tenorio, 1999; Quereda et al., 2000; Markoulatos et al., 2001) . Protocols have also been designed for simultaneous amplification of viruses causing similar clinical pictures. These include assays for herpesvirus and enterovirus (Read and Kurtz, 1999; Casas et al., 1999) , for measles, rubella and parvovirus B19 (del Mar Mosquera et al., 2002) or for different combinations of mosquito-transmitted viruses (Lee et al., 2002) . A duplex PCR protocol for the amplifica-tion of Epstein Á/Barr virus (EBV) and Toxoplasma gondii has also been proposed in AIDS patients to help distinguish CNS lymphoma from toxoplasmosis (Roberts and Storch, 1997) .

PCR with consensus primers is used to amplify conserved sequences in common to different viruses, but belonging to the same family. Following amplification, the product may be identified by DNA sequencing, by hybridisation with specific probes or by restriction-enzyme analysis (Fig. 2) , or, when possible, by visualisation of bands of different size on agarose gel. This strategy has widely and successfully been used with herpesviruses: to amplify simultaneously HSV-1, HSV-2, CMV and EBV by means of primers targeting conserved region of the herpesvirus DNA polymerase gene (Rozenberg and Lebon, 1991) , to detect all the known human herpesviruses in two PCR assays or most of them in a single reaction through the use of 'stair primers' (Minjolle et al., 1999; Bouquillon et al., 2000) . Further examples include protocols designed to detect most enterovirus strains using primers that recognise conserved sequences within the 5? noncoding region of the picornavirus family (Kammerer et al., 1994) , the three human polyomaviruses JC virus (JCV), BK virus and SV40 (Arthur et al., 1989; Fedele et al., 1999) or different flaviviruses (Harris et al., 1998; Scaramozzino et al., 2001 ; Mousavi-Jazi and Lundkvist, unpublished observation). Fig. 1 . Example of multiplex PCR. Three unrelated sequences of HSV-1, HSV-2 and VZV, are amplified simultaneously in the same test tube by using three different primer pairs, specific for each virus. The amplification products can be differentiated on agarose gel if the amplified fragments yield bands of different size (M: 100 bp DNA ladder marker). Alternatively, amplification products can be identified through an additional step, by hybridization with specific probes, restrictions enzyme analysis, nested PCR with specific internal primers, or DNA sequencing.

The possibility of generating false positive results by sample contamination is a major risk of NA amplification techniques. Clinical specimens or products of previous amplifications (carry-over) are the most common source of contamination. In order to minimize this risk, it is recommended to maintain CSF sterility in all the pre-laboratory and laboratory steps and to carry out the different laboratory steps in separate areas (Persing, 1991) . In addition, some laboratories use the enzyme uracil N -glycosilase (UNG), which degrades products from previous amplifications but not native NA templates, to prevent carry-over of amplification products. This is accomplished by substituting dUTP for dTTP in the amplification mixture, and pretreating all subsequent mixtures with UNG prior to amplification (Longo et al., 1990) . More in general, the use of negative controls, usually water or known negative samples tested in parallel with the CSF specimens throughout the whole procedure, and analysis of samples in duplicate, are useful to recognise false positive results.

False negative results can be caused by the presence of inhibitors, i.e. molecules affecting the correct functioning of enzymes. Inhibition of amplification has been reported in 1 Á/5% of CSF specimens . In order to reveal the presence of inhibition, it may be useful to amplify 'internal standard' molecules together with the target. In addition, amplification of Rozenberg and Lebon, 1991) . Conserved DNA sequence from the polymerase genes of HSV-1, HSV-2, EBV and CMV are amplified simultaneously in the same tube by a consensus primer pair targeting regions in common to these viruses. Following agarose gel electrophoresis, the amplified products have similar length (top) but each virus can be distinguished by using restriction enzymes (bottom) (a, Sma I and b, Bam HI; M: DNA marker). Alternatively, viruses can be differentiated following hybridization with virus-specific probes or DNA sequencing. both strong and weak positive controls in parallel with CSF samples can further help monitoring amplification efficiency of the whole reaction.

Since viable virus is not necessary for NA amplification-based procedures, NAs can be found in CSF samples kept stored for long periods. DNA of HSV can be recovered in CSF samples maintained for up to 30 days at (/20 8C, 2 Á/8 8C, and even at room temperature (Wiedbrauk and Cunningham, 1996) . Actually, it has been recovered following storage at (/20 8C for several years (personal observation). From a practical point of view, it is considered safe to send CSF specimens for the search of DNA viruses to the laboratory at room temperature. However, it is preferable that CSF samples are kept at 4 8C even for short-term storage, or frozen if they cannot be delivered or examined within 1 day (Cinque et al., 1996a) .

On the other hand, RNA is regarded to be less stable than DNA, and its detection in plasma seems to be affected by factors such as, the type of anticoagulant used for specimen collection and storage temperature (Moudgil and Daar, 1993; Holodniy et al., 1995) . However, recovery of enterovirus RNA in CSF seems not to be reduced by storage at 4 8C or room temperature for 96 h after sampling (Rotbart et al., 1985) . Similarly, no significant decay of HIV-1 RNA load was observed after up to 96 h of storage at 4 8C (Ahmad et al., 1999) . Theoretically, storage at 4 8C might actually be advantageous in the case of enveloped RNA viruses, because freezing may destroy the envelope, and thus make the RNA vulnerable to nucleases. Tables 2 and 3 show an overview of NA amplification use in diagnostics, in immunocompetent and immunocompromised patients, respectively. Some of the most relevant examples will be briefly emphasised.

The detection of HSV-1 DNA in CSF is one of the most convincing examples of the diagnostic use of molecular analysis. This approach has now largely replaced the identification of HSV in brain tissue biopsies as the method of choice for the etiological diagnosis of HSE (Linde et al., 1997; . A number of retrospective and prospective studies have demonstrated the reliability of PCR methods, reporting a sensitivity higher than 90% and a virtually 100% specificity (Aurelius et al., 1991; Lakeman and Whitley, 1995; Linde et al., 1997) . Furthermore, NA amplification techniques enable the diagnosis of uncommon forms of HSV CNS infection that might otherwise go unrecognised (Schlesinger et al., 1995b; DeVincenzo and Thorne, 1994; Domingues et al., 1997; Fodor et al., 1998) . Most importantly, CSF analysis by these techniques is rapid, making the laboratory diagnosis useful for management decisions. However, it is important that the PCR results are interpreted cautiously in relation to the clinical presentation and the duration of antiviral therapy. Initially negative PCR results have been observed very early after onset of HSE symptoms, likely to reflect a still limited virus replication (Rozenberg and Lebon, 1991; Puchhammer-Stö ckl et al., 2001) . On the other hand, the likelihood of finding a positive CSF PCR result is reduced following a few days of acyclovir treatment, as well as in untreated patients from whom CSF is obtained late after onset of neurological symptoms (Rozenberg and Lebon, 1991; Aurelius et al., 1991; Lakeman and Whitley, 1995) .

Although enteroviruses are frequently isolated from CSF of patients with enteroviral meningitis by cell culture, molecular techniques have significantly improved the detection rate of these infections (Jeffery et al., 1997) . Protocols have been designed with primers that recognise almost all of the enterovius serotypes, including those that can not be isolated in cell systems. Exceptions are Echo viruses 22 and 23 that diverge extremely from the other serotypes (Oberste et al., 1998) . Because of the enhanced sensitivity of NA amplification methods, the virus can also be detected in CSF samples obtained a few days after onset of symptoms, where isolation of virus is usually infrequent (Yerly et al., 1996) . Furthermore, the Aurelius et al. (1991) , Kimura et al. (1991) , Lakeman and Whitley (1995) , Linde et al. (1997) , Kimberlin et al. (1996) HSV-2 Aseptic mealaigitis, recurrent meningitis, neonatal infection 

Parvoviridae (ssDNA)

Aseptic meningitis Etiological characterization and diagnosis of parvovirus B19 meningitis Cassinotti et al. (1993) , Okumura and Ichikawa (1993) , Druschky et al. (2000), Barah et al. (2001) Picronaviridae ( Diagnostic potential Detection of cell-associated DNA Kompoliti et al. (1996) , Furuya et al. (1998) , Cavrois et al. (2000) NAs of other viruses have also been found in the CSF, but without clear association with CNS disease, e.g., hepatitis C virus (HCV), TTV, coronavirus, SV40 (Maggi et al., 1999; Dessau et al., 1999; Cristallo et al., 1997; Maggi et al., 2001; Tognon et al., 2001 HCV RNA has also been found in the CSF of HIV-infected patients, but without clear association with CNS disease (Maggi et al., 1999; Morsica et al., 1997; Gazzola et al., 2001) . PCNSL, primary CNS lymphoma. a See Table 2 , footnotes.

use of these techniques has reduced the time for diagnosis from 4 Á/10 days to 1 day. The diagnostic reliability of both in-house and commercial NA amplification assays has been extensively evaluated, showing a /90% sensitivity and 48Á/89% specificity when compared to viral isolation. The low specificity probably reflects enterovirus detection in culture-negative CSF samples from patients with true enterovirus meningitis (Glimaker et al., 1993; Lina et al., 1996; Yerly et al., 1996; Muir and van Loon, 1997; Romero, 1999) .

Although highly sensitive methods have been developed for detection of viral genomes in patient samples for most of the important arbo-and rodent-borne viruses, these methods are in many cases of limited value in the routine diagnostics. The major reason is that the time period for the viremia is often very short, i.e. the virus is usually no longer detectable at the onset of systemic or CNS disease. A typical example is tick-borne encephalitis (TBE), where attempts to use RT-PCR to tract TBE virus RNA in acute phase CSF or serum have to large extent been unsuccessful, with only a few samples having been found positive in the sero-negative, or in the IgM-positive but IgG-negative, phases (Gü nther, 1997; Puchhammer-Stö ckl et al., 1995; Lundkvist et al., unpublished observation) . Other examples where detection of viral genomes is of limited use, and the diagnostics have to be based, or at least supplemented, by serology, are Japanese encephalitis (Igarachi et al., 1994) , encephalitis caused by the Western, Eastern and Venezuelan equine encephalitis, and the Dengue viruses. On the other hand, the use of RT-PCR in CSF has rendered more positive results for other flaviviruses, such as West Nile virus, or Bunyaviruses, e.g. La Crosse, Jamestown Canyon or Toscana viruses. Real-time PCR analysis of CSF samples collected from patients with serologically confirmed West Nile encephalitis during the 1999 epidemics in the New York area, revealed a 57% sensitivity and 100% specificity, respectively (Lanciotti et al., 2000) . Although a correlation was observed between positivity rate and survival, this observation re-mains to be confirmed (Briese et al., 2000) . Toscana virus, a phlebotomus-transmitted virus causing an endemic infection in the Tuscany area of Italy is the responsible of benign forms of meningitis. By RT-PCR, Toscana virus sequences have been identified in CSF from as many as in 30% of patients with acute meningitis who were hospitalised in this geographic area. These findings not only indicate NA amplification analysis as a valid diagnostic support, but also as a means for estimating the relevance of this disease in the population (Valassina et al., 2000) .

Neurological complications is one of the major problems in patients with HIV infection. Although their frequency has significantly declined in the developed world following the advent of highly active anti-retroviral therapies (HAART), they still represent a major diagnostic and therapeutic challenge. CNS diseases caused by viruses include encephalitis, meningitis, myelitis or mixed pictures caused by herpesviruses, and progressive multifocal leukoencephalopathy (PML), associated with JCV infection of the CNS. In this field, the impact of diagnostic molecular techniques has been remarkable (Cinque et al., 1997a) . Detection of CMV DNA in CSF has shown to be highly sensitive and specific for the diagnosis of CMV encephalitis, a disease reported in as many as one third of AIDS patients (Cinque et al., 1992; Gozlan et al., 1992; Wolf and Spector, 1992; Clifford et al., 1993; Fox et al., 1995; Cinque et al., 1998b) . The identification of HSV-1, HSV-2 and varicella-zoster virus DNA in CSF has largely contributed to identify and clinically characterise the CNS complications induced by these viruses, and also provided a useful tool for their diagnosis and clinical management (Tan et al., 1993; Miller et al., 1995; Burke et al., 1997; Cinque et al., 1997a Cinque et al., , 1998a Iten et al., 1999) . CSF PCR for JCV has partly replaced the practice of brain biopsy as diagnostic method of choice for PML, though JCV sequences are demonstrated by PCR in only two thirds of the patients, with higher rates of detection in the more advanced stages of disease (Weber et al., 1994; Fong et al., 1995; McGuire et al., 1995; Cinque et al., 1996a; de Luca et al., 1996) . Furthermore, clearance of JCV DNA from CSF has frequently been observed in patients receiving HAART, in association with PML stabilisation (Miralles et al., 1998; Giudici et al., 2000) , suggesting that the rate of JCV DNA detection among PML patients might further decrease as a consequence of anti-HIV therapy. Another virusrelated CNS disease in HIV-infected patients is primary CNS lymphoma (PCNSL), which is almost always associated with the presence of EBV in the tumour cells (MacMahon et al., 1991) . Studies in patients with histologically proven PCNSL or CNS localization of systemic non-Hodgkin lymphomas have reported a striking association between the presence of these complications and EBV DNA detection in CSF. In some patients, EBV-DNA could even be detected days or months before the lymphoma manifested itself clinically (Cinque et al., 1993; Arribas et al., 1995a; de Luca et al., 1995; Cinque et al., 1996b; Cingolani et al., 2000) .

Measuring the amount of NAs in clinical specimens is a successful development of basic diagnostic molecular techniques. Both PCR and other NA amplification techniques have been proven to be reliable for this purpose, and a variety of semiquantitative and quantitative PCR methods are described (Clementi et al., 1996; Hodinka, 1998; Preiser et al., 2000) . Semi-quantitative techniques include methods based on limiting dilution of samples before amplification, or comparison of the extent of amplification between samples and 'external' standards at known NA concentration. The main disadvantage of these procedures is that they do not take into account the possible differences in amplification efficiency between the different samples and/or standards. Quantitative techniques allow a more accurate estimate of the NA levels, through co-amplification in the same tube of target NA and an 'internal' standard at a known concentration, which enables control of the amplification efficiency.

New automated procedures based on real-time detection of NAs are becoming popular in diagnostic neurovirology. The real-time PCR is based on detection and quantification of a fluorescent reporter. Fluorescence emission is recorded at each cycle making it possible to monitor the PCR reaction during its exponential phase, where the amount of PCR product correlates to the initial quantity of template. Compared to classical methods where the amounts of DNA are measured at the end of amplification, when the amplification efficiency is reduced, real-time PCR is more accurate and expands the dynamic range of quantification. It also eliminates post-PCR processing of amplification products, resulting in reduced risk of contamination and increased speed (Bustin, 2000) . There are two general methods for NA quantitation by real-time PCR: those based on the use of DNA-binding dyes, e.g. syber green, or of fluorescence-labelled probes. The latter are employed in the TaqMan (Fig. 3) (Holland et al., 1991; Higuchi et al., 1993; Heid et al., 1996) and LightCycler (Wittwer et al., 1997a,b) technology, that have both been described for quantitation of viral DNA in CSF (Kessler et al., 2000; Verstrepen et al., 2001; Gunther et al., 2001; Nagai et al., 2001; Read et al., 2001; Gautheret-Dejean et al., 2002; Aberle and Puchhammer-Stö ckl, 2002 ) (Table 4). Real-time PCR also allows simultaneous quantification, in the same tube, of different genomes (Read et al., 2001) , as well as virus genotyping and mutational analysis.

Quantification of viral genomes in the CSF can be important at the time of diagnosis of viral encephalitis or meningitis, in order to obtain diagnostic or prognostic information, as well as for subsequent patient management, e.g. during antiviral therapy. Table 4 summarises the most significant clinical applications of quantitative molecular techniques in viral CNS infections.

CMV and HIV infections of the CNS are good examples where quantitative methods are most useful. In CMV encephalitis, the measurement of CSF CMV DNA levels at the time of diagnosis is useful in order to distinguish extensive from mild infections (Arribas et al., 1995b; Bestetti et al., 2001) . In patients with CMV encephalitis or polyradiculomyelitis, the CMV DNA levels tend to decrease following antiviral therapy with ganciclovir or foscarnet, although the persistence of significant levels is frequent and it is associated with lack of clinical improvement (Cinque et al., 1995; Flood et al., 1997) .

Commercial assays, including PCR, NASBA and bDNA assays, have been used for HIV-1 RNA quantification in CSF . As a consequence of early virus invasion of the CNS, HIV-1 RNA can be detected in CSF at any stage of HIV infection and irrespective of the presence of neurological symptoms. However, CSF viral load is usually higher in patients with productive HIV infection of brain cells, i.e. HIVassociated dementia or encephalitis (McArthur et al., 1997; Brew et al., 1997; Ellis et al., 1997; Cinque et al., 1998c) . Current anti-HIV treatments induce substantial decreases of CSF RNA levels and quantitative molecular techniques can thus be useful to monitor the local response to treatment (Gisslen et al., 1997; Foudraine et al., 1998; Staprans et al., 1999; Gisolf et al., 2000; Price et al., 2001) .

Besides their use for direct diagnosis, NA amplification techniques constitute the base for genomic analysis. These techniques can yield high amounts of genomic material, which can be analysed for different purposes. These include virus characterisation for epidemiologic and phylogenetic studies, detection of viral mutations, e.g. those associated with antiviral drug resistance, neurotropism or neurovirulence. In addition, genotyping of viruses in CSF may be used to recognise unusual viral strains or to characterise new viral pathogens involved in CNS disease. There are a Fig. 3 . Example of a standard curve consisting of 5 Á/50 000 EBV DNA genomes/reaction generated by automated real-time PCR using the TaqMan technology. The fluorescence, proportional to the amount of amplified products, is acquired at each PCR cycle by an automated fluorometer. A threshold cycle (C T ) defines the cycle number at which the fluorescence passes a fixed threshold. Quantification of the amount of target in unknown samples is accomplished by measuring the C T and comparing this value to the C T values of the standard curve. number of post-amplification methods described, recently reviewed elsewhere (Arens, 1999) . Among these, DNA sequencing, restriction fragment length polymorphism (RFLP) and hybridisationbased techniques have all been used in neurovirology.

Nucleotide sequencing is the most accurate method to collect information on genome composition. Automated procedures have been developed during recent years, making sequencing relatively easy to perform (Fig. 4) . By RFLP, restriction enzymes are used to digest NAs into fragments of different size, which can be visualised by gel electrophoresis. This technique can be used after CSF PCR with consensus primers to distinguish individual viruses or viral strains (Fig. 2 ) (Rozenberg and Lebon, 1991; Arthur et al., 1989) . Hybridisation-based techniques include classical procedures, such as the Southern Blot, as well as modern high stringency hybridisation. The latter identifies minimal variations in the genome composition, such as single mutations. An example is the reverse hybridisation technique, incorporated into the commercial Line Probe Assay (LIPA), that has been used to detect HIV resistance mutations in CSF and plasma pairs (Cunningham et al., 2000) . DNA microarrays, or 'DNA chip' technology might become an additional tool for the identification and genomic analysis of virus sequences amplified in the CSF (Pease et al., 1994; Lockhart and Winzeler, 2000; McGlennen, 2001) . Despite high costs and current limited availability of technology and instrumentation, the DNA chip technology is in rapid development in virology, especially in the field of research, e.g. for measuring viral gene expression (Chambers et al., 1999; Jenner et al., 2001) . Sequences from plasma or other clinical samples have initially been tested for epidemiological or diagnostic purposes, such as screening for multiple HIV-1 drug resistance mutations (Wilson et al., 2000) . Table 5 summarises some of the most significant applications of genotypic analysis in neurovirology. The identification of enterovirus strains is an example of virus genotyping for both epidemiological and clinical purposes. Molecular typing of enteroviruses may be useful in epidemiological Fig. 4 . DNA sequencing from paired CSF and plasma specimens. An example of nucleotide sequencing from paired CSF and plasma samples using cycle-sequencing with dye-labeled oligonucleotides. Amplified products are obtained from paired CSF and plasma specimens following nucleic acid extraction, RNA retrotranscription and PCR amplification of a fragment from the HIV-1 reverse transcriptase (RT) gene. The amplified DNA is purified from unincorporated primers and nucleotides and added to the sequencing reaction mixture. The products of the sequencing reaction are subected to automated electrophoresis and recognized by a laser scanner. A four-color electropherogram is produced, which is translated into a linear nucleotide sequence by a computer software. The final sequence is compared to reference sequences, e.g., HXB2 for HIV-1. Three nucleotide mutations, resulting in two aminoacid substitutions at codons 215 (treonin0/phenylalanin) and 219 (lysin0/glutamin) are found in plasma but not in SCF (arrows). Such mutations are associated with resistance to the RT inhibitor drug zidovudine. Rozenberg and Lebon (1996) Tymidine kinase DNA sequencing Possible determinants for neurovirulence not found surveillance programmes, but also has diagnostic and prognostic significance: to correctly identify polioviruses, new enterovirus types or variants, enteroviruses responsible of severe infections, or those causing infections in neonates or immunodeficient (Muir et al., 1998) . Molecular typing systems for enteroviruses might become a rapid alternative to traditional serotyping, which is timeconsuming, labour-intensive and requires virus isolation in cell culture (Muir et al., 1998; Oberste et al., 1999) . DNA sequencing in the CSF has enabled the diagnosis of CNS diseases caused by attenuate vaccine strains rather than wild-type viruses. Examples are the meningitis cases caused by the Urabe vaccine against mumps in the end of the 80s (Forsey et al., 1990; Brown et al., 1991) , or the more recently reported vaccine-induced yellow fever cases (Martin et al., 2001) . Novel CNS pathogens have been identified following their isolation and/or amplification in the CSF. Recent examples are the two paramyxoviruses Hendra virus, transmitted by horses and causing meningitis and encephalitis in humans (O'Sullivan et al., 1997) , and Nipah virus, identified in patients with encephalitis during the 1998 and 1999 outbreaks in Malaysia and Singapore (Chua et al., 2000) . Similarly, a newly described B adenovirus was unexpectedly identified by molecular techniques during the 1997 epidemic of enterovirus 71-associated encephalitis in Malaysia (Cardosa et al., 1999) .

Another field of application of post-amplification analyses is pharmacogenomics, a term that indicates the study of genome for treatment management. One of the most significant examples in neurovirology is the study of the HIV genome for mutations selected by anti-HIV drugs (Schinazi et al., 1997; Hirsch et al., 2000) . Drug resistant viral mutants can be identified in CSF and these not infrequently show mutation profiles differing from those found in plasma or other body sites (Fig. 4) (Cunningham et al., 2000; Venturi et al., 2000; Cinque et al., 2001; Stingele et al., 2001) . These findings might provide information concerning viral dynamics in different body compartments, and might also be useful for clinical management of CNS HIV-induced complications. Steuler et al. (1992) , Keys et al. (1993) , Kuiken et al. (1995) env DNA sequencing Identification of polymorphisms possibly associated with ADC Power et al. (1994 ), Di Stefano et al. (1996 RFLP, restriction fragment length polymorphism; DGGE: denaturing gradient gel electrophoresis; LIPA, line probe assay; ADC, AIDS dementia complex; PML, progressive multifocal leukoencephalopathy; YF, yellow fever.

It is clear that the use of molecular techniques has tremendously improved the diagnosis and clinical management of viral CNS infections. On the other hand, potential problems related to interpretation of NA amplification results, costs and standardisation of these techniques deserve particular consideration.

Although the diagnostic value of NA amplification techniques is well established in a number of viral CNS infections such as HSE, enterovirus meningitis or opportunistic diseases in HIV-infected patients, it is still unclear in less frequently encountered infections. Examples are encephalitis caused by certain zoonotic viruses, or complicating viral exanthemas like measles or rubella (Table 2 ). Viral genomes are occasionally found in the CSF of patients with these infections by PCR or other amplification techniques. However, because of the paucity of cases studied, the rate of detection in diseased patients or controls and their clinical significance are not well known, and the potential of NA amplification methods remains in many cases uncertain.

It is not infrequent that viral NAs are found in the CSF in patients with infectious or noninfectious CNS diseases, but without a clear causative association with the disease itself. This is more frequently observed with viruses that may be latent in circulating blood cells, brain or other body sites. An example is the detection of EBV in CSF of patients diagnosed with other CNS infections, such as HSE or HIV-related opportunistic infections (Cinque et al., 1996b; Tang et al., 1997; Studahl et al., 1998; Portolani et al., 1998) . Theoretically, this finding might result from sliding of virus or latently infected lymphocytes through an impaired blood-CSF barrier, but also from reactivation of EBV infection in the CNS (Bossolasco et al., 2002) . Viral genomes have also been found in the CSF of patients with non-infectious CNS diseases. For instance, both JCV and HHV-6 genomes have been demonstrated in patients with multiple sclerosis, though their etiologic role in this disease has never been confirmed (Ferrante et al., 1998; Liedtke et al., 1995) .

In other instances, viral NAs can be detected in CSF prior to the onset of clinically relevant neurological symptoms, which can be advantageous in order to allow for an early diagnosis. This has been observed in AIDS patients, in whom viral agents, e.g. CMV or EBV */not yet causing clinical disease */may occasionally be identified in CSF in patients with another neurological complication (Cinque et al., 1996b) .

These examples are the consequence of the extreme sensitivity of NA amplification techniques, and underline the importance of careful interpretation of NA amplification findings in CSF within the individual clinical contexts. It is likely that quantitative molecular techniques might be of help to discriminate a clinically significant infection, characterised by viral replication and high viral loads, from incidental CSF findings.

Elevated cost is a potential disadvantage of CSF examination by NA amplification techniques. Taking into account only expenses for technical equipment, reagents, and disposables, the cost per sample of a basic PCR usually varies between approximately 20 and 200 US$ or a. In-house developed assays are the cheapest to perform and costs can be further reduced by avoiding, when possible, expensive procedures for CSF preparation and NA detection, or by using assays for simultaneous examination of multiple viruses. Commercial assays have some advantages, including standardisation and, sometimes, automation (Jungkind, 2001) , but are much more expensive. In general, the savings of establishing a rapid diagnosis often overcome the costs of NA amplification techniques (Ross, 1999) . In the diagnosis of HSE, the CSF PCR approach is evidently much cheaper than brain biopsy, but it seems costeffective also when compared to empirical initia-tion of antiviral therapy. Using a decision analysis model, the use of CSF PCR was associated with better outcome, and, on the other hand, with significant savings of acyclovir, resulting from higher rate of correct drug discontinuation in PCR-negative patients (Tebas et al., 1998) . In aseptic meningitis, cost savings seem to be increased by adopting a PCR testing procedure, as compared to standard practice, especially during the year season characterised by higher enterovirus infection prevalence. Early demonstration of an enterovirus as causative agent is associated with reduced requests for other diagnostic examinations, duration of empirical antibiotic treatments and periods of hospitalisation (Swingler et al., 1994; Rice et al., 1995; Marshall et al., 1997; Ramers et al., 2000) .

A major drawback of NA amplification techniques is their limited standardisation. Different protocols are in use for each virus in the different laboratories and reference standards for the evaluation of assay sensitivity are often lacking, making it difficult to compare results among laboratories (Saldanha, 2001) . Furthermore, testing of CSF might be subjected to laboratory errors, due to inaccurate test validation, quality of reagents and equipment or staff training (Garrett, 2001) . To help obviate these problems, quality control (QC) programmes are being carried out for viruses and other infectious agents. QC assessments for viruses responsible of CNS infections, including enteroviruses, HSV-1, HSV-2 and JCV, have been performed as a part of European Union sponsored QC in virology programmes (van Vliet et al., 1998; Muir et al., 1999; van Loon et al., 1999; Weber et al., 1997; Schloss et al., 2001) . These are based on the use of panels consisting of coded samples containing known amounts of NA molecules and control samples, which are distributed to participant laboratories and therein tested blindly. Analysis of reported results has commonly revealed substantial different reports between laboratories, especially with samples containing low amounts of NA. Furthermore, a significant rate of false positive results has been observed (Muir et al., 1999; Schloss et al., 2001) . On the other hand, little or no relationship is generally found between performance and the use of in-house rather than commercial techniques.

An array of NA amplification techniques is nowadays applicable to the CSF in order to establish an etiological diagnosis of viral infections of the CNS. Over the last 10 years, the spectrum of clinical conditions that can be recognised has largely expanded and diagnostic reliability significantly improved. Quantitative methods have provided a valuable additional tool for clinical management of these diseases, whereas post-amplification techniques have enabled precise characterisation of viral genomes following their recovery in the CSF. Current efforts are aiming at improvement of the diagnostic efficiency of molecular techniques, in both frequent and less common infections. They also will increase the diagnostic speed and standardisation. 

Infantile convulsions with mild gastroenteritis

Puchhammer-Stö ckl E. Diagnosis of herpesvirus infections of the central nervous system

Amplification of the complete polyomavirus JC genome from brain, cerebrospinal fluid and urine using pre-PCR restriction enzyme digestion

Asian genotypes of JC virus in Native Americans and in a Pacific Island population: markers of viral evolution and human migration

Cerebrospinal fluid and plasma HIV-1 RNA stability at 4 degrees C

Quantitative analysis of herpes simplex virus DNA in cerebrospinal fluid of children with herpes simplex encephalitis

Methods for subtyping and molecular comparison of human viral genomes

Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis

Detection of Epstein-Barr virus DNA in cerebrospinal fluid for diagnosis of AIDS-related central nervous system lymphoma

Level of cytomegalovirus (CMV) DNA in cerebrospinal fluid of subjects with AIDS and CMV infection of the central nervous system

Cytomegalovirus encephalitis

Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction

Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid

Encephalitis in immunocompetent patients due herpes simplex virus type 1 or 2 as determined by type-specific polymerase chain reaction and antibody assays of cerebrospinal fluid

Association of human parvovirus B19 infection with acute meningoencephalitis

Evaluation of a new general primer pair for rapid detection and differentiation of HSV-1, HSV-2, and VZV by polymerase chain reaction

Comparison of three nucleic acid amplification assays of cerebrospinal fluid for diagnosis of cytomegalovirus encephalitis

Epstein-Barr virus DNA load in cerebrospinal fluid and plasma of patients with AIDS-related lymphoma

Simultaneous detection of 6 human herpesviruses in cerebrospinal fluid and aqueous fluid by a single PCR using stair primers

BK virus as the cause of meningoencephalitis, retinitis and nephritis in a patient with AIDS

Levels of human immunodeficiency virus type 1 RNA in cerebrospinal fluid correlate with AIDS dementia stage

Detection of West Nile virus sequences in cerebrospinal fluid

Nucleotide sequence analysis of Urabe mumps vaccine strain that caused meningitis in vaccine recipients

Polymerase chain reaction detection and clinical significance of varicella-zoster virus in cerebrospinal fluid from human immunodeficiency virus-infected patients

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

A polymerase chain reaction-based epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants 90 days and younger

Prospective case-control study of encephalopathy in children with dengue hemorrhagic fever

Isolation of subgenus B adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in

New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay

Viral diagnosis of neurological infection by RT multiplex PCR: a search for entero-and herpesviruses in a prospective study

Neuroinvasion and persistence of human herpesvirus 6 in children

Persistent human parvovirus B19 infection following an acute infection with meningitis in an immunocompetent patient

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

Common human T cell leukemia virus type 1 (HTLV-1) integration sites in cerebrospinal fluid and blood lymphocytes of patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis indicate that HTLV-1 crosses the blood-brain barrier via clonal HTLV-1-infected cells

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression

Nipah virus: a recently emergent deadly paramyxovirus

Archetypal and rearranged sequences of human polyomavirus JC transcription control region in peripheral blood leukocytes and in cerebrospinal fluid

Epstein-Barr virus infection is predictive of CNS involvement in systemic AIDS-related non-Hodgkin's lymphomas

Cytomegalovirus infection of the central nervous system in patients with AIDS: diagnosis by DNA amplification from cerebrospinal fluid

Epstein-Barr virus DNA in cerebrospinal fluid from patients with AIDS-related primary lymphoma of the central nervous system

Ganciclovir therapy for cytomegalovirus (CMV) infection of the central nervous system in AIDS patients: monitoring by CMV DNA detection in cerebrospinal fluid

The role of laboratory investigation in the diagnosis and management of patients with suspected herpes simplex encephalitis: a consensus report

Polymerase chain reaction on cerebrospinal fluid for diagnosis of virus-associated opportunistic diseases of the central nervous system in HIV-infected patients

Diagnosis of central nervous system complications in HIV-infected patients: cerebrospinal fluid analysis by the polymerase chain reaction

Varicella-zoster virus (VZV) DNA in cerebrospinal fluid of patients infected with human immunodeficiency virus: VZV disease of the central nervous system or subclinical reactivation of VZV infection?

Herpes simplex virus infections of the central nervous system in human immunodeficiency virusinfected patients: clinical management by polymerase chain reaction assay of cerebrospinal fluid

Cerebrospinal fluid HIV-1 RNA levels: correlation with HIV encephalitis

Molecular analysis of cerebrospinal fluid: potential for the study of HIV-1 infection of the central nervous system

Effect of genotypic resistance on the virological response to highly active antiretroviral therapy in cerebrospinal fluid

Clinical use of quantitative molecular methods in studying human immunodeficiency virus type 1 infection

Use of polymerase chain reaction to demonstrate cytomegalovirus DNA in CSF of patients with human immunodeficiency virus infection

Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid

Human coronavirus polyadenylated RNA sequences in cerebrospinal fluid from multiple sclerosis patients

Evidence for independent development of resistance to HIV-1 reverse transcriptase inhibitors in the cerebrospinal fluid

Clinical, serological and PCR evidence of cytomegalovirus infection in the central nervous system in infancy and childhood

The polymerase chain reaction: application to nervous system disease

A case of rubella encephalitis: rubella virus genome was detected in the cerebrospinal fluid by polymerase chain reaction

Evaluation of cerebrospinal fluid EBV-DNA and IL-10 as markers for in vivo diagnosis of AIDS-related primary central nervous system lymphoma

Improved detection of JC virus DNA in cerebrospinal fluid for diagnosis of AIDS-related progresisolated from vaccine-associated paralytic poliomyelitis

HTLV-1-associated myelopathy associated with multi-organ inflammatory disease: a case report

Dual qualitative-quantitative nested PCR for detection of JC virus in cerebrospinal fluid: high potential for evaluation and monitoring of progressive multifocal leukoencephalopathy in AIDS patients receiving highly active antiretroviral therapy

Development of a realtime polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients

Possible hepatitis C virus involvement in acute meningoradiculitis/polyradiculitis of HIV-1-co-infected patients

Cerebrospinal fluid HIV-1 RNA during treatment with ritonavir/saquinavir or ritonavir/saquinavir/stavudine

The effect on human immunodeficiency virus type 1 RNA levels in cerebrospinal fluid after initiation of zidovudine or didanosine

Highly active antiretroviral therapy and progressive multifocal leukoencephalopathy: effects on cerebrospinal fluid markers of JC virus replication and immune response

Detection of enteroviral RNA by polymerase chain reaction in cerebrospinal fluid from patients with aseptic meningitis

Rapid detection of cytomegalovirus DNA in cerebrospinal fluid of AIDS patients with neurologic disorders

Tick-borne encephalitis-on pathogenesis and prognosis (Thesis), Division of Infectious Diseases

Lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum

CSF and MRI findings in patients with acute herpes zoster

Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A

Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR

Real time quantitative PCR

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples

Kinetic PCR analysis: real-time monitoring of DNA amplification reactions

Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel

The clinical utility of viral quantitation using molecular methods

Detection of specific polymerase chain reaction product by utilizing the 5? */3? exonuclease activity of Thermus aquaticus DNA polymerase

Comparative stabilities of quantitative human immunodeficiency virus RNA in plasma from samples collected in VACUTAINER CPT, VACUTAINER PPT, and standard VACUTAINER tubes

Diagnosis of Jamestown Canyon encephalitis by polymerase chain reaction

Diagnosis of viral infections of the central nervous system

Detection of west Nile and Japanese Encephalitis viral genome sequences in cerospinal fluid from acute encephalitis cases in Karachi

Epstein-Barr virus genomic sequences and specific antibodies in cerebrospinal fluid in children with neurologic complications of acute and reactivated EBV infections

Impact of cerebrospinal fluid PCR on the management of HIV-infected patients with varicella-zoster virus infection of the central nervous system

Detection of influenza virus RNA by reverse transcription-PCR and proinflammatory cytokines in influenza-virus-associated encephalopathy

Diagnosis of viral infections of the central nervous system: clinical interpretation of PCR results

Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays

A classification scheme for human polyomavirus JCV variants based on the nucleotide sequence of the noncoding regulatory region

Comprehensive PCR-based assay for detection and species identification of human herpesviruses

Automation of laboratory testing for infectious diseases using the polymerase chain reaction */our past, our present, our future

Nested PCR for specific detection and rapid identification of human picornaviruses

Molecular epidemiology and changing distribution of genotypes of measles virus field strains in Japan

Rotavirus encephalopathy: evidence of central nervous system involvement during rotavirus infection

Detection of Herpes simplex virus DNA by realtime PCR

V3 sequences of paired HIV-1 isolates from blood and cerebrospinal fluid cluster according to host and show variation related to the clinical stage of disease

NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection

Application of the polymerase chain reaction to the diagnosis and management of neonatal herpes simplex virus disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group

Detection and direct typing of herpes simplex virus by polymerase chain reaction

Detection o viral DNA in neonatal herpes simplex virus infections: frequent and prolonged presence in serum and cerebrospinal fluid

Fulminant human herpesvirus six encephalitis in a human immunodeficiency virus-infected infant

Detection of human herpesvirus 7 DNA in the cerebrospinal fluid of a child with exanthem subitum

Human T-cell lymphotropic virus type 1-associated myelopathy, Sjogren syndrome, and lymphocytic pneumonitis

Association of human herpesvirus 6 infection of the central nervous system with recurrence of febrile convulsions

JC virus DNA load in patients with and without progressive multifical leukoencephalopathy

Partial amplification of the measles virus nucleocapsid gene from stored sera and cerebrospinal fluids for molecular epidemiological studies

Differences in human immunodeficiency virus type 1 V3 sequences from patients with and without AIDS dementia complex

Universal diagnostic RT-PCR protocol for arboviruses

Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay

Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses

Diagnosis of Epstein-Barr virusinduced central nervous system infections by DNA amplification from cerebrospinal fluid

) with a single-tube multiplex reverse transcriptase polymerase chain reaction assay

Role of genotypic analysis of the thymidine kinase gene of herpes simplex virus for determination of neurovirulence and resistance to acyclovir

Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome

Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis

Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens

Specific diagnostic methods for herpesvirus infections of the central nervous system: a consensus review by the European Union Concerted Action on Virus Meningitis and Encephalitis

Genomics, gene expression and DNA arrays

Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions

Dengue encephalitis: a true entity?

Epstein-Barr virus in AIDS-related primary central nervous system lymphoma

Detection and quasispecies analysis of hepatitis C virus in the cerebrospinal fluid of infected patients

Low prevalence of TT virus in the cerebrospinal fluid of viremic patients with centra nervous system disorders

Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay

Potential cost savings through rapid diagnosis of enteroviral meningitis

Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases

Quantitation of enteroviral RNA by competitive polymerase chain reaction

Detection of measles virus from clinical samples using the polymerase chain reaction

Relationship between human immunodeficiency virus-associated dementia and viral load in cerebrospinal fluid and brain

Human herpesvirus 6 is associated with focal encephalitis

Influenza B virus encephalitis

Miniaturization technologies for molecular diagnostics

JC virus DNA in cerebrospinal fluid of human immunodeficiency virus-infected patients: predictive value for progressive multifocal leukoencephalopathy

Herpes simplex virus type 2 encephalitis and concomitant cytomegalovirus infection in a patient with AIDS: detection of virus-specific DNA in CSF by nested polymerase chain reaction

Amplification of the six major human herpesviruses from cerebrospinal fluid by a single PCR

Treatment of AIDSassociated progressive multifocal leukoencephalopathy with highly active antiretroviral therapy

Detection of hepatitis C virus genomic sequences in the cerebrospinal fluid of HIV-infected patients

Infectious decay of human immunodeficiency virus type 1 in plasma

Molecular typing of enteroviruses: current status and future requirements

Multicenter quality assessment of PCR methods for detection of enteroviruses

Increased HTLV-1 proviral load and preferential expansion of HTLV-1 tax-specific CD8'/ T cells in cerebrospinal fluid from patients with HAM/TSP

Detection of measles virus genome directly from clinical samples by reverse transcriptasepolymerase chain reaction and genetic variability

Detection of rotavirus in cerebrospinal fluid and blood of patients with convulsions and gastroenteritis by means of the reverse transcription polymerase chain reaction

Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses

Typing of human enteroviruses by partial sequencing of VP1

Aseptic meningitis caused by human parvovirus B19

Fatal encephalitis due to novel paramyxovirus transmitted from horses

Outbreak of Nipah-virus infection among abattoir workers in Singapore

Light-generated oligonucleotide arrays for rapid DNA sequence analysis

Polymerase chain reaction: trenches to benches

JC virus regulatory region tandem repeats in plasma and central nervous system isolates correlate with poor clinical outcome in patients with progressive multifocal leukoencephalopathy

Nested PCR for rapid detection of mumps virus in cerebrospinal fluid from patients with neurological diseases

Human herpes virus type 7 DNA in the cerebrospinal fluid of children with central nervous system diseases

Epstein-Barr virus DNA in cerebrospinal fluid from an immunocompetent man with herpes simplex virus encephalitis

Demented and nondemented patients with AIDS differ in brain-derived human immunodeficiency virus type 1 envelope sequences

Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction

Quantitative molecular virology in patient management

Cerebrospinal fluid response to structured treatment interruption after virological failure

Detection of varicella-zoster virus DNA by polymerase chain reaction in the cerebrospinal fluid of patients suffering from neurological complications associated with chicken pox or herpes zoster

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay

Screening for possible failure of herpes simplex virus PCR in cerebrospinal fluid for the diagnosis of herpes simplex encephalitis

Diagnostic utility of a multiplex herpesvirus PCR assay performed with cerebrospinal fluid from human immunodeficiency virus-infected patients with neurological disorders

Impact of a diagnostic cerebrospinal fluid enterovirus polymerase chain reaction test on patient management

Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay

LightCycler multiplex PCR for the laboratory diagnosis of common viral infections of the central nervous system

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

Clinical characteristics, management strategies, and cost implications of a statewide outbreak of enterovirus meningitis

Multiplex PCR for diagnosis of AIDS-related central nervous system lymphoma and toxoplasmosis

Reverse-transcription polymerase chain reaction detection of the enteroviruses

Financial determinants of outcomes in molecular testing

Factors affecting the detection of enteroviruses in cerebrospinal fluid with coxsackievirus B3 and poliovirus 1 cDNA probes

Diagnosis of enteroviral meningitis with the polymerase chain reaction

Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay

Amplification and characterization of herpesvirus DNA in cerebrospinal fluid from patients with acute encephalitis

Analysis of herpes simplex virus type 1 glycoprotein D nucleotide sequence in human herpes simplex encephalitis

Detection of viruses in spinal fluid

Validation and standardisation of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products

Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences

Resistance table: mutations in retroviral genes associated with drug resistance

Expanded spectrum of herpes simplex encephalitis in childhood

Herpes simplex virus type 2 meningitis in the absence of genital lesions: improved recognition with use of the polymerase chain reaction

European panels for quality control of nucleic acid amplification of herpes simplex virus (HSV)

Fifth Annual Meeting of the European Society for Clinical Virology

Quantitation of human immunodeficiency virus type 1 RNA in different biological compartments

Quantitation of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid by competitive PCR in AIDS patients with different HCMV central nervous system diseases

Encephalitis caused by human herpesvirus-6 in transplant recipients: relevance of a novel neurotropic virus

Time course of cerebrospinal fluid responses to antiretroviral therapy: evidence for variable compartmentalization of infection

Distinct populations of human immunodeficiency virus type 1 in blood and cerebrospinal fluid

Independent HIV replication in paired CSF and blood viral isolates during antiretroviral therapy

BK virus regulatory region rearrangements in brain and cerebrospinal fluid from a leukemia patient with tubulointerstitial nephritis and meningoencephalitis

Detection of cytomegalovirus DNA in cerebrospinal fluid in immunocompetent patients as a sign of active infection

Acute viral encephalitis in adults */a prospective study

Clinical and virological analyses of 21 infants with exanthem subitum (roseola infantum) and central nervous system complications

Typing of urinary JC virus DNA offers a novel means of tracing human migrations

An audit of the use of antibiotics in presumed viral meningitis in children

Diagnosis of horizontal enterovirus infections in neonates by nested PCR and direct sequence analysis

Molecular evidence and clinical significance of herpesvirus coinfection in the central nervous system

Molecular detection and identification of microorganisms

Molecular diagnosis of herpes simplex virus infections in the central nervous system

Prognostic value of JC virus load in cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy

Use of the polymerase chain reaction in the diagnosis of herpes simplex encephalitis: a decision analysis model

Herpes simplex virus infection as a cause of benign recurrent lymphocytic meningitis

Detection and typing of human herpesviruses by multiplex polymerase chain reaction

Epidemiology of influenza-associated encephalitis-encephalopathy in Hokkaido, the northernmost island of Japan

Investigation of the simian polymavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients

Combined treatment with interferon-alpha and ribavirin for subacute sclerosing panencephalitis

Human herpesvirus 7 infection associated with central nervous system manifestations

Polymerase chain reaction to detect cytomegalovirus DNA in the cerebrospinal fluid of neonates with congenital infection

Polymerase chain reaction and the diagnosis of viral central nervous system diseases

Branched DNA signal amplification

Detection and sequencing of rotavirus VP7 gene from human materials (stools, sera, cerebrospinal fluids, and throat swabs) by reverse transcription and PCR

Rapid identification of Toscana virus by nested PCR during an outbreak in the Siena area of Italy

Neuroinvasion by human herpesvirus type 7 in a case of exanthem subitum with severe neurologic manifestations

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis

Analysis of the transcriptional control region in progressive multifocal leukoencephalopathy

Antiretroviral resistance mutations in human immunodeficiency virus type 1 reverse transcriptase and protease from paired cerebrospinal fluid and plasma samples

Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube realtime reverse transcription-PCR assay

BK virus encephalitis in an immunocompetent patient

Nucleic-acid sequence based amplification in the rapid diagnosis of rabies

Human herpesvirus 6 DNA in cerebrospinal fluid specimens from allogeneic bone marrow transplant patients: does it have clinica significance?

Specific diagnosis of progressive multifocal leukoencephalopathy by polymerase chain reaction

Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system

Polymerase chain reaction for detection of JC virus DNA in cerebrospinal fluid: a quality control study

Stability of herpes simplex virus DNA in cerebrospinal fluid specimens

Comparative evaluation of three human immunodeficiency virus genotyping systems: the HIV-GenotypR method, the HIV PRT GeneChip assay, and the HIV-1 RT line probe assay

Diagnosis of human cytomegalovirus central nervous system disease in AIDS patients by DNA amplification from cerebrospinal fluid

Detection of human cytomegalovirus mutations associated with ganciclovir resistance in cerebrospinal fluid of AIDS patients with central nervous system disease

Rapid and sensitive detection of enteroviruses in specimens from patients with aseptic meningitis

Relation of JC virus DNA in the cerebrospinal fluid to survival in acquired immunodeficiency syndrome patients with biopsy-proven progressive multifocal leukoencephalopathy

Invasion by human herpesvirus 6 and human herpesvirus 7 of the central nervous system in patients with neurological signs and symptoms

Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification