INTRODUCTION

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Human herpesviruses, particularly herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6), are major agents of central nervous system infections. The clinical signs produced by the six viruses are not viral taxon specific. Accurate etiological diagnosis is essential now that effective and virus-specific therapy (for example, acyclovir, gancyclovir, and foscarnet) is available. Isolation of these viruses by culture of cerebrospinal fluid (CSF) gives poor results, and PCR is now the method of choice for virus detection (3, 10). Generally, a series of independent PCRs in which each PCR detects a single virus is performed. However, human herpesvirus DNA polymerase genes contain highly conserved spans of nucleotides that encode the same amino acids (7, 17). Since 1990 (16), several PCR methods for the amplification of these regions have been developed. In 1991, an amplification system became available for HSV-1, HSV-2, CMV, and EBV. This system uses a single pair of consensus primers whose sequences correspond to a conserved region of the DNA polymerase gene (11). Multiplex PCR was described in 1993 for the detection of HSV-1, HSV-2, CMV, EBV, and VZV (13). Consensus primers and virus-specific probes were designed for HSV-1, HSV-2, and VZV in 1994 (1). Degenerate primers have been described for 22 species of herpesviruses (human and animal viruses), but they have not been used for clinical diagnosis (14).

We have described a new type of primer, the stair primer, which is designed to overcome the problems encountered with standard primers if there are mutations in the sequence of the priming region (4). We used this approach to amplify a well-conserved region of the DNA polymerase gene and simultaneously detected the six major human herpesviruses in a single reaction (9). Here we report the results of a preliminary study that was performed to evaluate the feasibility of the technique for CSF samples.

	MATERIALS AND METHODS

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Biological samples. We studied all available CSF samples sent to the laboratory in 1995 for herpesvirus testing, irrespective of the clinical reasons for sample collection. The samples had thus already been tested by targeted PCR at the request of the clinicians involved. All samples were stored frozen at 80°C.

Sample preparation. DNA was extracted from 90-µl aliquots of CSF. Ten microliters of 10× lysis buffer (1 M Tris HCl [pH 7.4], 0.1 M EDTA, 5% sodium dodecyl sulfate, 2 mg of proteinase K per ml) was added. The samples were incubated for 1 h at 37°C and were then extracted twice with phenol-chloroform-isoamyl alcohol (25/24/1; vol/vol). The DNA was precipitated with absolute alcohol in the presence of 0.3 M sodium acetate. The DNA pellets were washed with 70% alcohol, dried, and suspended in 30 µl of sterile distilled water. The resulting DNA preparations were stored at 80°C. For reference viruses, the infected cells were lysed in 2× lysis buffer, and DNA was prepared as described above. DNA preparations were stored at 80°C. Negative controls were prepared by repeating the DNA preparation protocol, but CSF was replaced with distilled water and infected cells were replaced with uninfected cells.

Amplification with herpesvirus consensus primers. The pair of stair primers (PolyHer1 and PolyHer2) were produced by Argène Biosoft, Varilhes, France (Hybridowell Herpes Consensus kit). The consensus sequences were selected from within the DNA polymerase gene and were 76 to 86% identical to the genomic sequences of the six herpesviruses. Each stair primer used comprised an equimolar mixture of 11 oligonucleotides corresponding to a consensus sequence: all primers had the same 5' end but extended for 20 to 30 nucleotides in the 3' direction. The amplified fragment corresponded to positions 2163 to 2481 of the CMV DNA polymerase gene (GenBank accession no. X17403). The primers were used at a final concentration of 2 µM per reaction mixture, with each oligonucleotide contributing 1/11 to the final molarity.

Amplification by specific primers. The samples sent to the laboratory had been tested for one or several herpesviruses by targeted PCR, as requested by the clinician. The primers used were those described by Griffais et al. (5). The sequences of these primers correspond to those of different genes in the six viral genomes. DNA was extracted as described above, and the DNA pellets were suspended in 30 to 75 µl of water, according to the number of specific viral infections requested to be diagnosed for a patient's sample. The samples were amplified in a 50-µl reaction mixture containing 15 µl of the DNA preparation, 10 mM Tris HCl (pH 8.4), 50 mM KCl, 0.01% gelatin, 2 mM MgCl₂, 200 µM (each) deoxynucleotide triphosphate (Pharmacia, Orsay, France), and 2 U of Taq polymerase (Pharmacia). This reaction mixture was overlaid with 100 µl of paraffin oil. The amplification protocol (Thermocycler, as described above) consisted of 3 min of denaturation at 92°C and then 35 cycles of 30 s of denaturation at 92°C, 1 min and 15 s of annealing at 55°C, and 1 min and 15 s of elongation at 72°C (increments of 1 s/cycle). The amplification products were analyzed by Southern blotting and hybridization with the specific probes described by Griffais et al. (6) and were obtained by amplification with internal primers labeled by incorporation of digdUTP (Boehringer Mannheim, Meylan, France).

Statistical methods. Fisher's exact test and the ² test with Yates' correction were used, where appropriate, for comparison of the results obtained by the two PCR methods.

	RESULTS

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In 1995, our laboratory received 164 CSF samples for herpesvirus screening. One hundred forty-two samples were available for retesting by the herpesvirus consensus PCR. Four hundred one PCRs had been performed with these 142 samples to test for HSV-1, HSV-2, CMV, and VZV; tests for EBV and HHV-6 were not prescribed by the clinicians for any sample. Eighteen samples (12.7%) tested positive by the targeted PCR tests and 37 (26%) tested positive by the consensus PCR tests, including three samples that had coinfections (CMV, VZV, and HSV-2, VZV and HSV-2, and CMV and HHV-6), such that the number of individual virus detections was 41. The numbers of positive amplifications obtained by the two approaches were significantly different (P < 0.006). Of the 142 CSF samples, 103 were classified as negative by both the targeted and the consensus PCRs.

We present the results in more detail in Tables 1 and 2. The consensus PCR test detected a virus not tested for by the targeted PCR in 16 samples: 13 samples classified as negative and 3 coinfected samples (1 classified as false negative and 2 classified as positive). The targeted PCR detected HSV-1 in 3 samples (3 of 125; 2.4%), HSV-2 in 1 sample (1 of 120; 0.8%), CMV in 9 samples (9 of 90; 10%), and VZV in 5 samples (5 of 66; 7.5%). The consensus PCR detected HSV-1 in 4 of the 142 samples (2.8%) HSV-2 in 5 samples (3.5%), CMV in 11 samples (7.7%), EBV in 8 samples (5.6%), VZV in 11 samples (7.7%), and HHV-6 in 2 samples (1.4%). The four viruses for which tests were prescribed were detected in 18 samples by the targeted PCR and in 24 samples by the consensus PCR, giving sensitivities of 69 and 92%, respectively. Two samples identified as positive by the targeted PCR were classified as negative by the consensus PCR, and eight samples identified as negative by the targeted PCR were positive by the consensus PCR (Table 2, group B).

The specificity of amplification was verified for all samples: the positive controls (with reference strains) gave no hybridization signal with any probe other than that expected. The negative controls gave no hybridization signal with any of the six probes. This specificity was strongly suggested by the two EBV-positive samples (samples 2 and 14), both of which were shown to be from the same patient. Testing for EBV was not initially prescribed, and the two samples were handled in independent series. The same applied to the two samples (samples 12 and 20) positive for VZV.

	DISCUSSION

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The storage of CSF samples at 80°C did not appear to impair their suitability for the consensus PCR. The discordant results in this study involving a retrospective comparison could not be checked by retesting because many of the samples were completely used up. Nevertheless, it is clear that the consensus PCR was specific. The sensitivity of the consensus PCR was higher than that of the standard, targeted PCR, although the difference was not significant in this small series. The precautions taken (the use of testing in three geographically different locations) and the checking of negative and positive controls for each series made it possible to rule out false-positive results due to contamination of the samples. There are several possible explanations for this higher sensitivity: more amplification cycles were used for the consensus PCR than for the targeted PCR, more DNA was used in the PCR because a single reaction was performed, so the subdivision of the sample into several aliquots was not necessary, and the enzyme-linked immunosorbent assay may be a more sensitive detection method than Southern blotting with a nonradioactive probe (15). Industrially produced optimized products appear to perform better than our homemade reagents. The problems of standardization have been highlighted by the European Union Concerted Action on Virus Meningitis and Encephalitis (8) and must be resolved if PCR is to become the reference method for virological diagnosis of neurological herpesvirus infections.

The PCR technique described by Rozenberg and Lebon (11) does not detect VZV or HHV-6. The approach described here is much simpler and quicker than analysis involving gel electrophoresis and determination of the sizes of restriction fragments. Multiplex PCR may be less sensitive and, as described by Tenorio et al. (13), requires a second, nested amplification for virus identification. The diagnostic assay developed by Aono et al. (1) is useful for the detection of only three herpesviruses.

We detected viruses for which tests were not requested by the clinician in several cases, showing that the same clinical syndrome may be produced by different herpesviruses and that specific detection of these viruses in CSF on the basis of clinical signs is unsatisfactory. Our findings of mixed viral infection are consistent with those of Tang et al. (12), who reported coinfections with two herpesviruses in the central nervous system. In one sample, we detected three different herpesviruses. The proportion of samples testing positive for EBV by the consensus PCR was consistent with recent reports (2).

The herpesvirus consensus PCR described here appears to be reliable for use with clinical samples. It is the first system to detect six different herpesviruses in a single amplification by a technique suitable for routine use. There are clear advantages to a method that involves only one reaction: less sample material, reagents, and time are required. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of simultaneous detection. A sensitivity study that includes a rigorous parallel comparison of these PolyHer stair primers with the standard tests for individual viruses is also necessary.