Application of competitive PCR to cerebrospinal fluid samples from patients with herpes simplex encephalitis

Abstract

The purpose of the present study was to determine if the quantity of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of patients with herpes encephalitis would be useful in establishing the prognosis of the disease and to determine the effect of antiviral therapy on the clearance of viral DNA from the CSF. Quantitation of HSV DNA was done by constructing an internal standard (IS) from the glycoprotein B amplicon which had a 25-bp deletion between primer annealing sites. Each CSF specimen was coamplified with the IS and the ratio of the amount of HSV/amount of IS was compared to the ratios on a standard curve constructed with the same IS plus known amounts of HSV DNA. CSF specimens were available from 16 patients who were treated with intravenous acyclovir, and the amount of HSV DNA ranged from < 25 to 18,000 copies per microliter in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with > 100 copies of HSV DNA per microliter were older, were found by computed tomography to have lesions, and had poorer outcomes than patients with < 100 copies. Follow-up CSF specimens were available from seven patients. In six of these seven patients, the HSV DNA levels decreased during therapy. One patient had a twofold increase in HSV DNA levels after 1 week of therapy and died on day 8. The application of this assay may be helpful in establishing the prognosis and in the monitoring of patients with herpes simplex encephalitis.

FIG. 1

Procedure used to construct IS. The top line depicts the locations of the sequences used to construct the IS. The normal glycoprotein B primer is shown at the left; the IS primer was composed of the last 12 bases of this primer plus 17 bases located downstream, as shown in the box. The resulting primer shown in the second line loops out 25 bases resulting in a 115-bp product. This product was reamplified to reconstruct the original primer site and produce a 123-bp amplicon. BP, base pairs.

FIG. 2

Construction of a standard curve for quantitation of HSV-1. (A) Agarose-ethidium bromide gel; (B) image analysis of the gel in panel A; (C) standard curve plotted from the data in the table at the bottom. In the gels, the first band is 250 copies of the 123-bp IS. The next six lanes contain 100, 200, 500, 1,000, 2,000, and 5,000 copies of HSV DNA, respectively, plus 250 copies of IS. The last lane in the agarose gel contains a molecular size marker (1,000, 700, 500, 400, 300, 200, 100, and 50 bp). The table shows the pixel intensities of the amount of HSV/amount of IS. The double bands are the 148-bp product of HSV DNA and the 123-bp product of the IS and are seen in the lanes containing 100, 200, 500, and 1,000 copies of HSV DNA.

FIG. 3

Quantity of HSV-1 DNA per microliter of CSF from patients (Pt) 1, 2, 3, 4, 6, and 8 according to duration of acyclovir therapy.