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

Abstract

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

FIG. 1

Specificity of the multiplex PCR to detect HSV-1, EBV, HSV-2, CMV, and VZV. Detection and typing of clinical isolates by multiplex PCR. Amplification was performed with 0.8 mM dNTPs. All primer pairs were used at the concentration of 10 pmol. Amplification conditions were as follows: 40 cycles of 30 s at 94°C, 40 s at 60°C, and 50 s at 72°C. The amplified products were subjected to a 3.5% agarose gel electrophoresis containing 1 μg of ethidium bromide/ml in Tris-borate-EDTA buffer. Lane M, molecular weight markers corresponding to a HinfI digest of pBR322 DNA (Minotech, Crete, Greece). Shown in lanes 1 to 5 are amplifications performed on 10 log PCRD₅₀ of HSV-1/ml, 160 ag of EBV, 7 log PCRD₅₀ of HSV-2/ml, 18 ag of CMV, and 14 ag of VZV in the presence of 1 μg DNA of pooled CSF samples. Lanes 1, 2, 3, 4, and 5, amplifications with primer pairs H₁P₃₂/H₁M₃₂, EP₅/EM₃, H₂M₄₀/H₂P₄, CP₁₅/CM₃, and VP₂₂/VM₂₀, respectively; lane 6, positive multiplex PCR amplification with the five sets of primers of the above five herpesviruses (product size marker used in the screening of the clinical specimens). Shown in lanes 7 to 11 are amplifications performed with α-tubulin primers and the above set of five primers on clinical samples. Lane 7, negative CSF sample; lanes 8, 9, 10, and 11, CSF samples containing HSV-1, HSV-2, CMV, and VZV, respectively. The predicted size of each amplimer is shown on the right.

FIG. 2

Restriction enzyme digestion pattern for HSV-1, EBV, HSV-2, CMV, and VZV by HpaII (lanes 1 to 5) and Tru9I (lanes 6 to 10). Lane M₁, molecular weight marker, corresponding to a HinfI digest of pBR322 DNA (Minotech, Crete, Greece); lane M₂, molecular weight marker, 50-bp DNA ladder (MBI, Vilnius, Lithuania). Fragments smaller than 30 bp are not visible on the ethidium bromide-stained agarose gel. Lane 1, HSV-1 digest (58- and 62-bp fragments; the 27-bp fragment is not visible); lane 2, EBV, no digestion; lane 3, HSV-2 digest (61-, 82-, and 84-bp fragments); lane 4, CMV digest (235-bp fragment; the 21-bp fragment is not visible); lane 5, VZV, no digestion; lane 6, HSV-1, no digestion; lane 7, EBV digest (49- and 133-bp fragments); lane 8, HSV-2, no digestion; lane 9, CMV digest (60- and 171-bp fragments; the 25-bp fragment is not visible); lane 10, VZV digest (75-, 78-, and 122-bp fragments).