A real-time PCR assay to identify and discriminate among wild-type and vaccine strains of varicella-zoster virus and herpes simplex virus in clinical specimens, and comparison with the clinical diagnoses

Abstract

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human beta-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results.

Fig. 1

Shingles Prevention Study PCR assay specificity for VZV-WT and VZV-Oka DNA. A: VZV amplification plots for a typical VZV-WT positive clinical specimen: The specimen was assayed in duplicate using VZV-WT (left) and VZV-Oka (right) primer-probe sets. With VZV-WT primers and probe, the fluorescence signal crosses the threshold at approximately cycle 13. With VZV-Oka primers and probe, the fluorescence signal crosses the threshold at approximately cycle 26. B: Difference in the average of replicate C_t values for mixtures of purified VZV-WT and VZV-Oka viral DNA with the following VZV-WT to VZV-Oka viral DNA ratios: 100/0, 98/2, 95/5, 90/10, 75/25, 50/50, 25/75, 10/90, 5/95, 2/98, and 0/100. These data indicate that when the C_t for VZV-WT is 5 cycles lower than the C_t for VZV-Oka, there is 97.5% confidence that ≥95.9% of the VZV DNA present is VZV-WT. Conversely, when the C_t for VZV-Oka is 5 cycles lower than the C_t for VZV-WT, there is 97.5% confidence that ≥93.9% of the VZV DNA present is VZV-Oka.

Fig. 2

Shingles Prevention Study PCR assay results. A: PCR Assay Results for Suspected Cases of HZ in the Shingles Prevention Study. Of a total of 1,308* suspected cases of HZ, 1,156 (88.4%) were diagnosed by PCR assay. Nine hundred eighteen cases (79%) were positive for VZV-WT, 46 (4%) positive for HSV DNA and 192 (17%) were negative for both VZV and HSV DNA. No cases were positive for VZV-Oka DNA or of indeterminate VZV strain. (B) PCR Assay Results for Shingles Prevention Study Specimens. A total of 1,479 clinical specimens from 1,220 of the 1,308* suspected cases of HZ in the Shingles Prevention Study were assayed for viral and cellular DNA. Nine hundred seventy-one (66%) of the clinical specimens were positive for VZV-WT and 49 (3%) were positive for HSV DNA. One specimen (0.1%) was positive for both VZV-WT and HSV DNA. 325 (22%) of the clinical specimens were negative for virus DNA and 133 (9%) were inadequate specimens (negative for viral and β-globin DNA). No specimens were positive for VZV-Oka DNA or of indeterminate VZV strain. *Includes two PCR-positive cases unrecognized clinically.

Fig. 3

Average C_t values for virus positive clinical specimens. The VZV-WT and HSV positive Shingles Prevention Study specimens were grouped according to mean C_t value. Low virus positive specimens are defined as those with C_t values between the cutoff of 36.35 and 32. There were only 13 specimens that tested low positive for VZV-WT DNA and 1 specimen that tested low positive for HSV DNA.