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 β-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.

Varicella zoster virus infection

“…There were 957 confirmed evaluable cases of zoster (315 in vaccine recipients and 642 in placebo recipients). In both groups, >93% of the subjects with zoster were positive for wild-type VZV DNA by PCR and none had vOka DNA 22,104 .…”

“…For laboratory diagnosis of VZV infection, the following approaches are currently most useful: PCR on material from skin vesicles (submitted as swabs, fluid or scabs [102][103][104] ), saliva 90,102,103,[105][106][107] and cerebrospinal fluid if neurological symptoms or signs are present 91,92,108,109 . Detection of VZV antigens by direct immunofluorescence from vesicles is also rapid and specific, although less sensitive than PCR 110 .…”

“…When symptoms develop in vaccinated individuals that suggest VZV infection, such as rash or meningitis (even without rash), it is important to identify VZV by PCR, and it can be useful to determine whether the virus is wild-type VZV or vOka 104,[112][113][114] . In the United States, testing can be performed free of charge at either the National Virus Laboratory at the Centers for Disease Control and Prevention (CDC) or the Columbia University VZV Identification Program of the Worldwide Adverse Experience System of Merck & Co.…”

“…The exact mechanisms for the attenuation of VZV in live vaccines remain unknown; mutations in ORF62 were identified in vOka 122 and mutations at positions 106262, 107252 and 108111 in ORF62 are important 112 , as is a stop codon mutation at position 560 in ORF0 (REF 123). These mutations, particularly the unique mutations at positions 106262 and 108111 in ORF62, can be used to differentiate parental Oka and other wild-type strains of VZV from vOka 104,112 .…”

“…Rates of serious adverse events, systemic adverse events, hospitalizations and deaths in the SPS were low in vaccine recipients and comparable with those in placebo recipients 22,151,154 . During the first 42 days after vaccination, 24 cases of zoster in placebo recipients were reported and 7 cases in the vaccination group, none of which were caused by vOka 104 . In contrast to prophylactic vaccines, such as those against varicella and measles, the zoster vaccine is a therapeutic vaccine intended to prevent reactivation and replication of latent VZV with which the recipient is already infected before vaccination and to which the recipient already has substantial immunity.…”